Microbiology

🧭 Overview

🧠 One-sentence thesis

Microbiology studies relatively simple, often microscopic organisms that lack complex cellular differentiation, and the field emerged once microscopes allowed scientists to observe and isolate these tiny life forms.

📌 Key points (3–5)

• What defines a microbe: not just size (some are visible), but simplicity and lack of differentiation—even multicellular microbes have cells that can act independently.
• Size scales matter: cellular microbes (bacteria, protists) are measured in micrometers; acellular microbes (viruses) are measured in nanometers, requiring different microscopes.
• Historical discovery: Robert Hooke and Antony van Leeuwenhoek provided the first proof of microbes in the 1600s using compound and simple microscopes, respectively.
• Three domains of life: all organisms fall into Bacteria, Archaea, or Eukarya (based on ribosomal RNA sequences); viruses are classified separately because they lack ribosomes.
• Common confusion: don't confuse genus and species—both are needed for a scientific name, and the genus can change with new information while the species name is permanent.

🔬 Defining microbes beyond size

🔬 The traditional size criterion and its limits

• Traditional definition: microbes are organisms so small you need a microscope to see them.
• Problem: some microbes are visible to the naked eye (not well, but visible).
• These visible organisms still "look/act/perform like other well-studied microbes" in all other respects, so they cannot be dismissed.

🧩 The modified definition: simplicity and lack of differentiation

Microbes are fairly simple agents/organisms that are not highly differentiated, meaning even multicellular microbes are composed of cells that can act independently—there is no set division of labor.

• What "not differentiated" means: cells can function independently; no fixed division of labor.
• Example: chop half the cells off a giant fungus → the remaining cells continue to function unimpeded.
• Contrast: chop half the cells off a human → "that would be a problem" (because human cells are highly specialized and interdependent).
• Multicellular microbes, even with billions of cells, are relatively simple in design, usually composed of branching filaments.

🧪 Common techniques in microbiology

Because microbes are so small and numerous, research requires:

• Sterilization: to prevent unwanted contamination.
• Observation: to confirm full isolation of the microbe you want to study.
• These methods are necessary to isolate the one type of microbe you are interested in from the many around.

📏 Microbe size scales

📏 Cellular microbes: micrometers (μm)

Micrometers (μm): the unit used to measure cellular microbes; there are 1000 μm in every millimeter.

Microbe type	Example	Typical size
Bacterial cell	E. coli	~1 μm wide × 4 μm long
Protozoal cell	Paramecium	~25 μm wide × 100 μm long

• This scale shows why it is difficult to see most microbes without assistance.
• Exception: multicellular microbes (e.g., fungi)—if you get enough cells together in one place, you can definitely see them without a microscope.

📏 Acellular microbes: nanometers (nm)

Nanometers (nm): the unit used to measure acellular microbes (viruses); there are 1000 nm in every micrometer.

• A typical virus (e.g., influenza virus) has a diameter of about 100 nm.
• This scale shows why you need a more powerful microscope to see a virus.
• Proportional analogy: if a typical bacterium (E. coli) were inflated to the size of the Statue of Liberty, a typical virus (influenza) would be the size of an adult human.

🔭 Historical discovery of microbes

🔭 Why discovery was delayed

• The small size of microbes hindered their discovery.
• Hard to convince people that their skin is covered with billions of small creatures if you cannot show them.
• "Seeing is believing"—proof required microscopes.

🔬 Robert Hooke (1635–1703)

• What he used: a compound microscope (microscope with two lenses in tandem).
• What he did: observed many different objects and made detailed drawings, publishing them in the scientific literature.
• Key contribution: credited with publishing the first drawings of microorganisms.
• In 1665 he published Micrographia, with drawings of microbes such as fungi, as well as other organisms and cell structures.
• Limitation: his microscopes were restricted in their resolution (clarity), which limited what microbes he could observe.

🔬 Antony van Leeuwenhoek (1632–1723)

• Who he was: a Dutch cloth merchant who dabbled in microscopes.
• What he used: a simple microscope (single lens) held between two silver plates.
• What he observed: microbes from many sample types—pond water, fecal material, teeth scrapings, etc.
• What he did: made detailed drawings and notes, sending them to the Royal Society of London (the scientific organization of that time).
• Key contribution: his invaluable record clearly indicates he saw both bacteria and a wide variety of protists.
• Title: some microbiologists refer to van Leeuwenhoek as the "Father of Microbiology" because of his contributions to the field.

🔍 Don't confuse: compound vs. simple microscopes

• Compound microscope (Hooke): two lenses in tandem; better for larger microbes but limited resolution.
• Simple microscope (van Leeuwenhoek): single lens; surprisingly effective for observing bacteria and protists.

🌳 The Three Domain Classification

🌳 Basis and origin

Three Domain Classification: a system that groups all organisms into Bacteria, Archaea, or Eukarya, based on ribosomal RNA (rRNA) sequences.

• First proposed by Carl Woese in the 1970s.
• Widely accepted by scientists today as the most accurate current portrayal of organism relatedness.
• Classification continually changes as we acquire new information and new tools.

🦠 Bacteria domain

Characteristic	Description
Cellular organization	Most are unicellular (but not all)
Nucleus/organelles	Lack a nucleus or any other organelle
Cell wall	Have a cell wall with peptidoglycan (not found anywhere else but in bacteria)
Ribosomes	70S ribosomes
Habitat	Common in soil, water, foods, and human bodies
Microbe status	All Bacteria are considered microbes

• Best known microbial examples include E. coli.

🦠 Archaea domain

• Why it's a separate domain: used to be grouped with bacteria, but rRNA sequences showed they are not closely related to Bacteria at all.
• Similarities to Bacteria: mostly (but not all) unicellular, cells lack a nucleus or any other organelle, have 70S ribosomes, and all Archaea are microbes.
• Key difference: completely different cell walls that can vary markedly in composition—notably lack peptidoglycan and might have pseudomurien instead.

🦠 Eukarya domain

Characteristic	Description
Cell type	Eukaryotic: has a nucleus and many organelles (mitochondria, endoplasmic reticulum, etc.)
Ribosomes	80S ribosomes
Organization	Commonly unicellular or multicellular
Members	Includes many non-microbes (animals, plants) and numerous microbial examples (fungi, protists, slime molds, water molds)

🦠 Don't confuse: Bacteria vs. Archaea

• Both lack a nucleus and have 70S ribosomes, but:
  • Bacteria: have peptidoglycan in cell walls.
  • Archaea: lack peptidoglycan; may have pseudomurien; rRNA sequences show they are not closely related to Bacteria.

🦠 Viruses: outside the three domains

🦠 Why viruses are classified separately

• Viruses lack ribosomes and therefore lack rRNA sequences for comparison.
• They are not part of the Three Domain Classification.
• Classified separately, using characteristics specific to viruses.

🦠 Basic characteristics

Obligate intracellular parasites: viruses have a strict requirement for a host cell in order to replicate or increase in number.

• These acellular entities are often agents of disease, a result of their cell invasion.

📛 Naming organisms: binomial nomenclature

📛 Taxonomic ranks overview

Taxonomic ranks: a way for scientists to organize information about organisms by determining relatedness.

• Domains are the largest grouping, followed by numerous smaller groupings.
• Each smaller grouping consists of organisms that share specific features in common.
• Each level becomes more and more restrictive as to who can be a member.
• Eventually we get down to genus and species, the groupings used for formation of a scientific name.
• This is the binomial nomenclature devised by Carl Linnaeus in the 1750s.

📛 How scientific names work

• A scientific name is composed of a genus (generic name) and a species (specific name).
• Species name: once assigned, is permanent for the organism.
• Genus name: can change if new information becomes available.
• Example: the bacterium previously known as Streptococcus faecalis is now Enterococcus faecalis because sequencing information indicates it is more closely related to the Enterococcus genus.

📛 Rules for writing scientific names

1. Genus: always capitalized.
2. Species: always lowercase.
3. Both: italicized (if typewritten) or underlined (if handwritten).
4. Abbreviation: the genus may be shortened to its starting letter, but only if the name has been referred to in full at least once first (exception: E. coli, due to its commonality).
5. Never use species alone: it is inappropriate to refer to an organism by the species alone (e.g., never refer to E. coli as "coli" alone—other bacteria can have the species "coli" as well).

📛 Don't confuse: genus vs. species

• Genus: generic grouping; can change with new information; always capitalized.
• Species: specific identifier; permanent; always lowercase.
• Both are required for a complete scientific name.

🧭 Overview

🧠 One-sentence thesis

Microscopes enable the visualization of microorganisms by magnifying them, and different microscope types—from simple light microscopes to electron and scanning probe microscopes—offer varying levels of resolution and magnification suited to observing different microbes, from bacteria to viruses and even atoms.

📌 Key points (3–5)

• Why microscopes matter: Microbes are extremely small (counting microbes in 1 gram of soil at 1/second would take over 33 years), so magnification is essential to visualize them and study them.
• Historical foundation: Robert Hooke used a compound microscope (two lens sets) in 1665, while Antonie van Leeuwenhoek used a simple microscope (single lens) to discover single-celled life forms.
• Resolution vs magnification: Resolution is the ability to distinguish two close objects as separate; it depends on wavelength, numerical aperture, and refractive index (Abbé equation), not just magnification.
• Light microscopes have limits: Maximum magnification ~1500×, resolution ~0.2 μm; they work for eukaryotic microbes and stained bacteria but not for viruses.
• Common confusion: Different microscope types (bright-field, dark-field, phase contrast, fluorescence, electron, scanning probe) are not interchangeable—each is suited to specific specimens and purposes (e.g., living vs stained cells, bacteria vs viruses vs atoms).

🔬 Early microscopy and magnification principles

🔬 Compound vs simple microscopes (1600s)

• Robert Hooke (1665) observed microbes using a compound microscope:

  Compound microscope: contains two sets of lenses for magnification—the ocular lens (next to the eye) and the objective lens (next to the specimen).

  • Total magnification = ocular magnification × objective magnification.
  • Example: 10× ocular and 50× objective → 500× total magnification.
  • Published observations in Micrographia.

• Antonie van Leeuwenhoek ("Father of Microbiology") used a simple microscope:

  Simple microscope: a microscope with just a single lens (essentially a magnifying glass).

  • He was a cloth merchant, not a scientist, likely inspired by Hooke's work.
  • Produced extremely high-quality lenses and kept detailed notes.
  • Credited with discovering single-celled life forms.

📐 Resolution and the Abbé equation

Resolution: the ability of a lens to distinguish two objects that are close together as individual objects.

• The Abbé equation defines the minimal distance (d) at which two objects can be resolved.

• Three factors affect resolution:

  1. Wavelength of illumination: shorter wavelength → smaller d (better resolution).
  2. Numerical aperture: a function of the objective lens's ability to gather light.
     • Defined by n (refractive index of the medium) and sin θ (cone of light entering the objective).
  3. Refractive index: air = 1.00, oil = 1.25; oil directs more light into the objective.

• Oil immersion objective: uses oil to increase light collection.

  • Maximum magnification for visual light microscopes: ~1500× (e.g., 15× oculars + 100× oil immersion objective).
  • Highest resolution: ~0.2 μm; objects closer than this cannot be distinguished as separate.

• Don't confuse: Resolution ≠ magnification. High magnification without good resolution just makes a blurry image bigger.

💡 Light microscopes: six types

💡 Bright-field microscope

• Standard microscope (can be purchased at a toy store).
• Light source at the base illuminates the specimen → magnified by objective lens → magnified again by ocular lens.
• Specimen visualized by contrast differences between specimen and surroundings.
• Limitation: Unstained bacteria have very little contrast (almost invisible) unless naturally pigmented.
  • Staining is essential for bacteria.
  • Works reasonably well for larger eukaryotic microbes (protozoa, algae) without stain.
• Stained bacteria appear dark against a bright background.

🌑 Dark-field microscope

• Modified bright-field microscope using a dark-field stop (opaque disk).
• Blocks light directly underneath the specimen; light reaches specimen from the sides.
• Only light reflected or refracted by the specimen is collected by the objective.
• Cells appear bright against a dark background.
• Use: Observation of living, unstained cells; useful for observing motility or eukaryotic organelles.

🌀 Phase-contrast microscope

• Another modified bright-field microscope, more complex and expensive.
• Uses an opaque ring (annular stop) with a transparent ring releasing light in a hollow cone.
• Principle: Cells have a different refractive index than surroundings → light differs slightly in phase.
• Phase differences amplified by a phase ring in a special phase objective → translated into brightness differences.
• Results in a dark image against a bright background.
• Use: Observation of living, unstained cells (motility, organelles).

🔀 Differential interference contrast (DIC) microscope

• Operates on the same principle as phase-contrast (refractive index differences).
• Uses polarized light split into two beams by a prism:
  • One beam passes through the specimen.
  • One beam passes through the surrounding area.
• Beams combined via a second prism; they "interfere" with each other (out of phase).
• Resulting images have an almost 3D effect.
• Use: Observing living, unstained cells.

🌟 Fluorescence microscope

• Utilizes light emitted from a specimen, not light passing through it.
• Mercury-arc lamp generates intense light → filtered to produce a specific wavelength → directed at specimen by a dichromatic mirror (reflects short wavelengths, transmits longer wavelengths).
• Naturally fluorescent organisms: absorb short wavelengths, emit fluorescent light with longer wavelength → passes through dichromatic mirror → visualized.
• Fluorochromes: fluorescent dyes that bind to specific cell components; can be attached to antibodies to highlight specific structures or organisms.
• Use: Organisms with natural fluorescence or those stained with fluorochromes.

🎯 Confocal scanning laser microscope (CSLM)

• Uses a laser for illumination (high intensity).
• Light directed at dichromatic mirrors that move, "scanning" the specimen.
• Longer wavelengths emitted by fluorescently stained specimen pass back through mirrors, through a pinhole, measured by a detector.
• Pinhole: conjugates the focal point of the lens (hence "confocal") → allows complete focus of a given point.
• Entire specimen scanned in x-z planes (all three axes) → computer compiles data → creates a single 3D image entirely in focus.
• Use: Viewing complex structures such as biofilms.

🎨 Staining techniques

🎨 Why staining matters

• Most microbes (especially unicellular) are not apparent without staining.
• Provides contrast between microorganism and background.

🖌️ Simple stain

• Uses a single dye.
• Two types:
  • Direct stain: stains the cells directly.
  • Negative stain: stains the background surrounding the cells.
• Provides basic information: cell size, morphology (shape), and cell arrangement.

🧪 Differential stains

• Combine stains to allow differentiation of organisms based on characteristics.
• Gram stain (developed 1884): most common differential stain.
  • Separates bacteria based on cell wall type:
    • Gram-positive bacteria → stain purple.
    • Gram-negative bacteria → stain pink.
• Acid-fast stain: for bacteria with specialized cell walls.
  • Acid-fast bacteria → stain red.
  • Non-acid-fast bacteria → stain blue.
• Other differential stains target specific structures: endospores, capsules, flagella.

⚡ Electron microscopes: beyond light

⚡ Why electron microscopes?

• Light microscopes cannot visualize viruses: resolution limit 0.2 μm (200 nm); most viruses are smaller.
• Electron microscopes replace light with electrons.
  • Electron wavelength: 1.23 nm (vs 530 nm for blue-green light).
  • Resolution increases to ~0.5 nm, magnifications over 150,000×.

⚠️ Drawbacks

• Electrons must be contained in a vacuum → cannot work with live cells.
• Extensive sample preparation may distort specimen or cause artifacts.

📡 Transmission electron microscope (TEM)

• Electron beam directed at specimen using electromagnets.
• Dense areas scatter electrons → dark areas on image.
• Less dense areas allow electrons to "transmit" through → brighter sections.
• Image generated on a fluorescent screen, then captured.
• Sample preparation: specimens must be sliced to 20–100 nm thickness (embedded in plastic, cut with diamond knife).
• Resulting pictures represent one slice or plane of the specimen.

🔍 Scanning electron microscope (SEM)

• Also uses an electron beam, but image formed from secondary electrons released from specimen surface, collected by a detector.
• More electrons released from raised areas; fewer from sunken areas.
• Electron beam scanned over specimen surface → produces a 3D image of external features.

Microscope	Image source	Sample preparation	Result
TEM	Electrons transmitted through specimen	Sliced to 20–100 nm	One slice/plane
SEM	Secondary electrons from surface	Surface scanning	3D external features

🔬 Scanning probe microscopes: atomic-level visualization

🔬 21st-century microscopy

• Scanning probe microscopes: move a probe over specimen surface in x-z planes → computers generate extremely detailed 3D images.
• Resolution: 0.1 nm; magnification: 100,000,000×.
• Probe size much smaller than wavelength of visible light or electrons.
• Can study objects in liquid → examination of biological molecules.
• Used in microbiology but more often in other fields (chemicals, metals, magnetic samples, nanoparticles).

🔌 Scanning tunneling microscope (STM)

• Extremely sharp probe (1 atom thick) maintains constant voltage with specimen surface → electrons travel between them ("tunneling current").
• Probe raised and lowered to sustain constant height above sample.
• Motion tracked by computer → generates final image.

🎸 Atomic force microscope (AFM)

• Developed as alternative to STM for samples that do not conduct electricity well.
• Uses a cantilever with an extremely sharp probe tip maintaining constant height above specimen (typically by direct contact).
• Movement of cantilever deflects a laser beam → translates into an image.
• Computers generate the image.

Microscope	Probe mechanism	Sample requirement	Use case
STM	Tunneling current (voltage)	Conducts electricity	Conductive samples
AFM	Cantilever deflects laser	Does not need to conduct	Non-conductive samples

📊 Comparison: microscope capabilities

Microscope type	Magnification	Resolution	Main use	Live cells?
Light (general)	~1500×	~0.2 μm	Eukaryotic microbes, stained bacteria	Some types
Bright-field	~1500×	~0.2 μm	Stained bacteria	No (staining kills)
Dark-field	~1500×	~0.2 μm	Unstained bacteria, motility	Yes
Phase contrast	~1500×	~0.2 μm	Unstained cells, organelles	Yes
DIC	~1500×	~0.2 μm	Unstained cells, 3D effect	Yes
Fluorescence	~1500×	~0.2 μm	Fluorescent organisms, antibody tagging	Depends on stain
CSLM	~1500×	Better than standard light	3D structures (biofilms)	No (fluorescent stain)
TEM	>150,000×	~0.5 nm	Viruses, internal structures (slices)	No (vacuum)
SEM	>150,000×	~0.5 nm	Viruses, surface features (3D)	No (vacuum)
STM	100,000,000×	~0.1 nm	Atoms, conductive samples	Can work in liquid
AFM	100,000,000×	~0.1 nm	Atoms, non-conductive samples	Can work in liquid

Don't confuse: Light microscopes are limited by wavelength of light (~200 nm resolution); electron microscopes overcome this with electrons (~0.5 nm); scanning probe microscopes use physical probes (~0.1 nm). Each step up requires more complex sample preparation and loses the ability to view live cells (except scanning probe in liquid).

🧭 Overview

🧠 One-sentence thesis

Cells are divided into bacteria, archaea, and eukaryotes based on structural differences—especially the presence or absence of a nucleus—and these differences in size, shape, and components determine how cells grow, reproduce, and function.

📌 Key points (3–5)

• Prokaryotic vs eukaryotic distinction: bacteria and archaea lack a nucleus and organelles, while eukaryotes have a true nucleus and membrane-bound organelles.
• Cell morphology and size matter: shape dictates growth, reproduction, nutrient uptake, and movement; smaller cells grow and reproduce faster due to a higher surface-to-volume ratio.
• Common confusion: "prokaryote" lumps bacteria and archaea together, but they are not closely related genetically—modern microbiology prefers to refer to them as separate groups.
• All cells share four components: cytoplasm, DNA, ribosomes, and a cell membrane, though their organization differs between bacteria/archaea and eukaryotes.
• Plasma membrane structure: bacteria and eukaryotes share a phospholipid bilayer structure (fluid-mosaic model), while archaea have marked differences.

🔬 Cell classification and naming

🔬 Prokaryotic vs eukaryotic terminology

• Traditionally, cells were divided into prokaryotic (bacteria and archaea) or eukaryotic based on cell type.
• The name difference comes from Greek: "karyose" = nut/center (nucleus), "pro" = before, "eu" = true.
• Prokaryotes lack a nucleus ("before a nucleus"); eukaryotes have a true nucleus.

🧬 Why microbiologists are moving away from "prokaryote"

• The term lumps bacteria and archaea into the same category, but they are not closely related genetically.
• Both lack a nucleus and organelles (mitochondria, Golgi apparatus, endoplasmic reticulum, etc.), but this is a shared absence, not a sign of close relationship.
• Modern approach: refer to the three groups separately—archaea, bacteria, and eukaryotes—rather than using "prokaryotic."

🔍 General differences

Feature	Bacteria/Archaea	Eukaryotes
Size	Smaller	Larger (about 10× larger)
Complexity	Simpler, less "stuff"	More complex, more cluttered
Nucleus	Absent	Present (true nucleus)
Organelles	Absent	Present (membrane-bound)

🔷 Cell morphology

🔷 What morphology means

Cell morphology: a reference to the shape of a cell.

• Not trivial—shape dictates how a cell grows, reproduces, obtains nutrients, and moves.
• Cells must maintain their shape to function properly.
• Can be used to help identify microbes, but cells with the same morphology are not necessarily related.

🔷 Common bacterial morphologies

• Coccus (pl. cocci): spherically shaped cell.
• Bacillus (pl. bacilli): rod-shaped cell.
• Curved rods: rods with curvature, including three sub-categories:
  • Vibrio: rods with a single curve.
  • Spirilla / spirochetes: rods that form spiral shapes; differentiated by motility type (hard to separate unless viewing a wet mount).
• Pleomorphic: organisms that exhibit variability in their shape.

🌟 Morphology in other groups

• Bacteria tend to display the most representative morphologies.
• Archaea show an even wider array of shapes, including star or square shapes.
• Eukaryotic microbes (especially protozoa that lack a cell wall) also exhibit a wide array of shapes.

📏 Cell size and surface-to-volume ratio

📏 Why size matters

• Cell size is not trivial; there are reasons why bacterial/archaeal cells are much smaller than eukaryotic cells.
• Advantages of being small relate to the surface-to-volume ratio (S/V ratio).

Surface-to-volume ratio: the ratio of the external cellular layer in contact with the environment compared to the liquid inside.

• This ratio changes as a cell increases in size.

📐 How the ratio changes with size

• Example comparison: a 2 μm cell vs. a 4 μm cell (twice as large):
  • Smaller cell (radius = 1 μm): surface area = 12.6 μm², volume = 4.2 μm³, S/V ratio = 3.
  • Larger cell (radius = 2 μm): surface area = 50.3 μm², volume = 33.5 μm³, S/V ratio = 1.5.
• As the cell increases in size, the S/V ratio decreases.

⚖️ Surface vs volume trade-offs

• Surface area = ability to bring in nutrients and let out waste products; larger surface area = more possibilities.
• Volume = what the cell has to support.
• As S/V ratio decreases, the larger cell struggles to bring in nutrients rapidly enough to support activities like growth and reproduction, despite having a larger surface area.
• Result: small cells grow and reproduce faster than larger cells, even though they have smaller surface area.
• Small cells also evolve faster over time, giving them more opportunities to adapt to environments.

🦠 Exceptions: monster bacteria

• Typical bacterial/archaeal cell: a few micrometers; typical eukaryotic cell: about 10× larger.
• Some bacteria fall outside the norm and are very large but still grow and reproduce quickly.
• Example: Thiomargarita namibiensis can measure 100–750 μm in length (compared to typical 4 μm for E. coli).
  • Maintains rapid reproduction by producing very large vacuoles (bubbles) that occupy a large portion of the cell, reducing volume and increasing S/V ratio.
• Other large bacteria use a ruffled membrane for their outer surface layer, increasing surface area and thus S/V ratio.

🧱 Cell components shared by all cells

🧱 Four common components

All cells (bacterial, archaeal, eukaryotic) share these four components:

💧 Cytoplasm

Cytoplasm: the gel-like fluid that fills each cell, providing an aqueous environment for chemical reactions.

• Composed mostly of water, with some salts and proteins.

🧬 DNA

DNA (deoxyribonucleic acid): the genetic material of the cell, the instructions for the cell's abilities and characteristics.

• The complete set of genes is called the genome.
• In bacteria and archaea: localized in an irregularly-shaped region called the nucleoid.
• In eukaryotes: enclosed in a membrane-bound nucleus.

🏭 Ribosomes

Ribosomes: the protein-making factories of the cell.

• Composed of both RNA and protein.
• Distinct differences between bacteria/archaea and eukaryotes in size and location.
• Bacteria/archaea: ribosomes float in the cytoplasm; measured at 70S (Svedberg unit, which corresponds to sedimentation rate when centrifuged).
• Eukaryotes: many ribosomes organized along the endoplasmic reticulum (an organelle); measured at 80S, indicating larger size and mass.

Svedberg unit: measurement corresponding to the rate of sedimentation when centrifuged.

🧫 Cell membrane

Cell membrane (also called plasma membrane when referring to the cell boundary): one of the outer boundaries of every cell.

• Separates the cell's inner contents from the surrounding environment.
• Not a strong layer, but participates in several crucial processes, especially for bacteria and archaea (which typically only have one membrane):
  • Semi-permeable barrier: allows entrance and exit of select molecules; lets in nutrients, excretes waste, possibly keeps out toxins or antibiotics.
  • Metabolic processes: participates in converting light or chemical energy into ATP (readily useable form); involves developing a proton motive force (PMF) based on charge separation across the membrane, like a battery.
  • "Communication" with environment: binds or takes in small molecules that act as signals, providing information about nutrients, toxins, or other organisms.

🧩 Eukaryotic organelles

• Eukaryotes have numerous additional components called organelles: nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, etc.
• These are membrane-bound compartments that house different activities.
• Each organelle has its own plasma membrane, providing multiple locations for membranous functions.

🧪 Plasma membrane structure

🧪 Fluid-mosaic model

Fluid-mosaic model: describes the plasma membrane structure, accounting for the movement of various components within the membrane itself.

• Bacteria and eukaryotes share the same basic structure; archaea have marked differences (covered in the archaea chapter).

🧱 Phospholipid bilayer

• The membrane is typically composed of two layers (a bilayer) of phospholipids, which form the basic structure.

Phospholipid: composed of a polar region that is hydrophilic ("water loving") and a non-polar region that is hydrophobic ("water fearing").

• Phospholipids spontaneously assemble to keep polar regions in contact with the aqueous environment (outside the cell and cytoplasm inside), while non-polar regions are sequestered in the middle—like jelly in a sandwich.

🔬 Phospholipid components

• Polar head: negatively-charged phosphate group (hydrophilic).
• Connected by a glycerol linkage to two fatty acid tails (hydrophobic).
• The membrane is not particularly strong, but is strengthened by additional lipid components:
  • Steroids in eukaryotes.
  • Sterol-like hopanoids in bacteria.

🧩 Membrane proteins

• Embedded and associated with the phospholipid bilayer are various proteins with many functions.
• Integral proteins: embedded within the bilayer itself; dominant type, representing about 70–80% of membrane proteins.
• Peripheral proteins: associate on the outside of the membrane; represent 20–30% of membrane proteins.
  • Some are anchored via a lipid tail.
  • Many associate with specific integral proteins to fulfill cellular functions.

📊 Protein to phospholipid ratio

Cell type	Protein:phospholipid ratio	Notes
Bacteria (cell membrane)	2.5:1	Very high protein content
Eukaryotes (cell membrane)	1:1	Lower protein content
Eukaryotes (mitochondrial membrane)	2.5:1	Same as bacterial membrane

• The mitochondrial membrane ratio (2.5:1) matches the bacterial plasma membrane ratio, providing additional evidence that eukaryotes evolved from a bacterial ancestor.

🔍 Don't confuse

• Plasma membrane can refer to any membrane (e.g., the membrane around the eukaryotic nucleus).
• Cell membrane refers specifically to the boundary of the cell proper.

🧭 Overview

🧠 One-sentence thesis

Bacterial cell walls—predominantly gram positive or gram negative—provide structural strength, shape maintenance, and osmotic protection through peptidoglycan and additional components, with their differences detectable by the Gram stain and critical to bacterial function and pathogenicity.

📌 Key points (3–5)

• What most bacteria have: about 90% of bacteria possess a cell wall, typically either gram positive or gram negative.
• Core ingredient: peptidoglycan (murein) is unique to bacterial cell walls and found nowhere else on Earth; it forms a cross-linked lattice structure.
• Two distinct types: gram positive walls are thick peptidoglycan layers with teichoic acid; gram negative walls have thin peptidoglycan plus an outer membrane with lipopolysaccharide (LPS).
• Common confusion: gram positive vs. gram negative—the Gram stain reveals purple (positive) vs. pink (negative), correlating with thick vs. thin peptidoglycan and presence/absence of an outer membrane.
• Why it matters: the cell wall maintains cell shape, protects from osmotic lysis (internal pressure similar to a fully inflated car tire), blocks toxins, and contributes to disease-causing ability in pathogens.

🧱 What the bacterial cell wall does

🧱 Multiple functions beyond strength

The bacterial cell wall is an additional semi-rigid layer outside the cell membrane. It performs several roles:

• Structural strength: the cell membrane alone is relatively weak; the cell wall reinforces it.
• Shape maintenance: important for growth, reproduction, nutrient uptake, and movement.
• Osmotic protection: prevents osmotic lysis as the cell moves between environments or transports nutrients.
  • Water freely crosses both membrane and wall, risking osmotic imbalance.
  • Internal pressure can equal that of a fully inflated car tire—too much for the plasma membrane alone.
• Selective barrier: keeps out certain molecules (e.g., toxins), especially in gram negative bacteria.
• Pathogenicity: contributes to disease-causing ability in some bacterial pathogens.

🔬 Detection by Gram stain

Gram stain: a differential stain developed in 1884 that reliably separates bacteria into two groups based on cell wall structure.

• After staining, gram positive bacteria appear purple; gram negative bacteria appear pink.
• Originally the mechanism was unknown; electron microscopy in the 1940s revealed the staining difference correlated with cell wall differences.
• The excerpt links to a website showing the actual steps (not reproduced here).

🧬 Peptidoglycan: the shared ingredient

🧬 What peptidoglycan is

Peptidoglycan (murein): a polysaccharide unique to bacterial cell walls, made of alternating glucose derivatives N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) in long chains.

• This substance has not been found anywhere else on Earth.
• Both gram positive and gram negative walls contain it, though in different amounts.
• Eukaryotic microbe cell walls are simpler—typically a single ingredient (cellulose in algae, chitin in fungi).

🔗 Structure and cross-linking

Peptidoglycan forms a lattice-like structure:

• Chains: NAG and NAM alternate in long chains.
• Tetrapeptide: a four-amino-acid chain extends off each NAM unit.
  • Composition: L-alanine, D-glutamine, L-lysine (or meso-diaminopimelic acid, DPA), and D-alanine.
  • Cells normally use only L-amino acids, but the mirror-image D-amino acids protect against proteases that would otherwise break down the peptidoglycan.
• Cross-linking: tetrapeptides link chains to one another.
  • Direct cross-link: D-alanine on one tetrapeptide binds to L-lysine/DPA on another.
  • Peptide interbridge: in many gram positive bacteria, a bridge of five amino acids (e.g., glycine) connects tetrapeptides.
  • Complete cross-linking: every tetrapeptide is bound to a tetrapeptide on another chain, maximizing strength.

Recent research (past ten years) suggests peptidoglycan is synthesized as a cylinder with coiled substructure, each coil cross-linked to the next, creating even greater strength.

🧩 Why the structure matters

• More cross-linking → stronger cell wall.
• D-amino acids → resistance to enzymatic attack.
• The lattice allows some porosity (important for nutrient passage) while maintaining integrity.

🟣 Gram positive cell walls

🟣 Composition and structure

• Peptidoglycan dominance: up to 90% of the cell wall is peptidoglycan.
• Multiple layers: layer after layer forms around the cell membrane.
• Cross-linking: NAM tetrapeptides typically use a peptide interbridge; complete cross-linking is common.
• Result: an incredibly strong cell wall.

🧷 Teichoic acid

Teichoic acid: a glycopolymer embedded within the peptidoglycan layers of gram positive bacteria.

Proposed roles:

Function	How it works
Net negative charge	Essential for generating a proton motive force
Rigidity	Contributes to overall cell wall rigidity, maintaining cell shape (especially in rod-shaped bacteria)
Cell division	Interacts with peptidoglycan biosynthesis machinery
Resistance	Helps resist high temperatures, high salt, and β-lactam antibiotics

Two types:

• Wall teichoic acids (WTA): covalently linked to peptidoglycan.
• Lipoteichoic acid: connected to the cell membrane via a lipid anchor.

🚪 Nutrient passage and exoenzymes

• Peptidoglycan is relatively porous, so most substances pass through easily.
• Large nutrients require breakdown by exoenzymes:
  • Made in the cytoplasm.
  • Secreted past the cell membrane and through the cell wall.
  • Function outside the cell to break down large macromolecules into smaller components.

🔴 Gram negative cell walls

🔴 Composition and complexity

Gram negative cell walls are more complex than gram positive:

• Thin peptidoglycan: only a couple of layers, representing 5–10% of the total cell wall.
• Outer membrane: a second plasma membrane located outside the peptidoglycan, making up the bulk of the wall.
  • Composed of a lipid bilayer similar to the cell membrane (polar heads, fatty acid tails, integral proteins).
  • Key difference: presence of lipopolysaccharide (LPS).

🧪 Lipopolysaccharide (LPS)

Lipopolysaccharide (LPS): large molecules anchored into the outer membrane, projecting from the cell into the environment.

Three components:

1. O-antigen (O-polysaccharide): outermost part.
2. Core polysaccharide: middle section.
3. Lipid A: anchors the LPS into the outer membrane.

Functions:

Function	Mechanism
Net negative charge	Contributes to the cell's overall charge
Membrane stability	Helps stabilize the outer membrane
Chemical protection	Physically blocks access to other cell wall parts
Immune response	O-antigen triggers host antibody production (e.g., E. coli O157:H7)
Endotoxin	Lipid A acts as a toxin, causing fever and diarrhea; large amounts can trigger life-threatening endotoxic shock

Don't confuse: The O-antigen triggers protective immunity, while lipid A causes toxic symptoms—different parts, different effects.

🚰 Periplasm and nutrient transport

The outer membrane is a barrier to both harmful and beneficial molecules. Gram negative bacteria solve this with specialized structures:

Periplasm: the space between the outer surface of the cell membrane and the inner surface of the outer membrane; contains the peptidoglycan layers.

Periplasmic enzymes:

• Stored in the periplasm (not secreted outside like exoenzymes).
• Break down large nutrients into smaller molecules that can pass the LPS.

Porins:

Porins: transmembrane proteins in the outer membrane, composed of a trimer of three subunits forming a pore.

• Non-specific porins: transport any molecule that fits.
• Specific porins: recognize and transport only certain substances via a binding site.

Transport pathway:

1. Periplasmic enzymes break down large molecules.
2. Smaller molecules pass through porins across the outer membrane.
3. Molecules move through porous peptidoglycan.
4. Integral proteins transport them across the cell membrane into the cytoplasm.

🔗 Braun's lipoprotein

Braun's lipoprotein: a lipoprotein linking the peptidoglycan to the outer membrane.

• One end: covalently bound to peptidoglycan.
• Other end: embedded in the outer membrane via its polar head.
• Provides additional structural integrity and strength.

🧬 Gram positive vs. gram negative comparison

Feature	Gram Positive	Gram Negative
Gram stain color	Purple	Pink
Peptidoglycan thickness	Thick (up to 90% of wall)	Thin (5–10% of wall)
Layers	Many peptidoglycan layers	Few peptidoglycan layers
Cross-linking	Peptide interbridge common; complete cross-linking	Direct cross-links more common
Additional components	Teichoic acid (WTA, lipoteichoic acid)	Outer membrane with LPS
Periplasm	Absent	Present (between membranes)
Enzymes for large nutrients	Exoenzymes (secreted outside)	Periplasmic enzymes (in periplasm)
Porins	Not needed	Required for transport across outer membrane
Structural linkage	N/A	Braun's lipoprotein links peptidoglycan to outer membrane

🦠 Exceptions: unusual and wall-less bacteria

🦠 Chlamydiae: no peptidoglycan

• Bacteria in phylum Chlamydiae appear to lack peptidoglycan.
• Cell walls have a gram negative structure otherwise (outer membrane, LPS, porins, etc.).
• Likely use a protein layer functioning like peptidoglycan.
• Advantage: resistance to β-lactam antibiotics (e.g., penicillin), which attack peptidoglycan.

🦠 Tenericutes: no cell wall

• Bacteria in phylum Tenericutes lack a cell wall altogether.
• Extremely susceptible to osmotic changes.
• Often strengthen the cell membrane by adding sterols (usually associated with eukaryotic membranes).
• Many are pathogens, hiding within the protective environment of a host.

Don't confuse: Chlamydiae have a wall but no peptidoglycan; Tenericutes have no wall at all.

🧭 Overview

🧠 One-sentence thesis

Bacteria possess a variety of internal structures beyond the basic cytoplasm, nucleoid, and ribosomes—including cytoskeletal proteins, storage inclusions, specialized metabolic compartments, plasmids, and endospores—that expand their capabilities and survival strategies.

📌 Key points (3–5)

• Beyond the basics: bacteria were once thought to lack organelles and cytoskeletons, but recent discoveries reveal protein filaments analogous to eukaryotic cytoskeletal proteins and membrane-bound compartments that rival eukaryotic organelles.
• Inclusions serve multiple roles: most store metabolic reserves (carbon, phosphate, sulfur) when substances are in excess, but some enable motility, orientation, or specialized metabolism.
• Microcompartments vs simple inclusions: bacterial microcompartments (BMCs) have protein shells and house enzymes for specific metabolic activities, not just storage.
• Endospores are survival transformations: a few gram-positive bacteria can convert from metabolically active vegetative cells into dormant, highly resistant endospores when threatened, then revert when conditions improve.
• Common confusion: inclusions range from simple chemical aggregates to complex membrane-bound structures; the anammoxosome and chlorosome have true membranes, while carboxysomes have only protein shells.

🦴 Cytoskeleton: structural proteins

🦴 Discovery and roles

• Originally, bacteria were thought to lack a cytoskeleton (a hallmark of eukaryotic cells).
• In the last 20 years, scientists discovered bacterial filaments made of proteins that are analogues to eukaryotic cytoskeletal proteins.
• Roles: cell shape, cell division, and integrity of the cell wall.

🔵 FtsZ (tubulin analogue)

FtsZ: homologous to the eukaryotic protein tubulin; forms a ring structure in the middle of the cell during cell division.

• Attracts other proteins to construct a septum that will eventually separate the two daughter cells.
• Example: during division, FtsZ assembles at mid-cell to orchestrate splitting.

🧬 MreB (actin analogue)

MreB: homologous to the eukaryotic protein actin; found in bacillus and spiral-shaped bacteria.

• Plays an essential role in cell shape formation.
• Assumes a helical configuration running the length of the cell.
• Dictates the activities of the peptidoglycan-synthesis machinery, ensuring a non-spherical shape.

🌀 Crescentin (lamin/keratin analogue)

Crescentin: homologous to the eukaryotic proteins lamin and keratin; found in spiral-shaped bacteria with a single curve.

• Assembles lengthwise in the inner curvature of the cell.
• Bends the cell into its final curved shape.

🗂️ Inclusions: storage and specialized structures

🗂️ What inclusions are

Inclusions: distinct structures located within the cytoplasm or periplasm; range from simple chemical compilations (crystals) to complex structures with membranous external layers.

• Often store components as metabolic reserves when a substance is in excess.
• Can also play roles in motility and metabolic functions.

🍬 Carbon storage

Glycogen:

• One of the simplest and most common inclusions.
• Glucose units linked together in a multi-branching polysaccharide structure.
• Formed when there is excess carbon; broken down later for both carbon and energy.

Poly-β-hydroxybutyrate (PHB):

• A granule formed when β-hydroxybutyric acid units aggregate.
• Very plastic-like in composition; scientists investigate using it as biodegradable plastic.
• Has a shell composed of both protein and a small amount of phospholipid.
• Formed in excess carbon; broken down for carbon and energy.

⚛️ Inorganic storage

Polyphosphate granules:

• Accumulate inorganic phosphate (PO₄³⁻).
• Phosphate used to make nucleic acid (sugar-phosphate backbone) or ATP (adenosine triphosphate).

Sulfur globules:

• Store excess sulfur as a source of electrons for metabolism.
• Result when the cell oxidizes hydrogen sulfide (H₂S) to elemental sulfur (S⁰).
• Form refractile inclusions.

🎈 Non-storage functions

Gas vacuoles:

• Used to control buoyancy in a water column.
• Provide limited motility on the vertical axis only.
• Composed of conglomerations of gas vesicles: cylindrical, hollow, rigid structures.
• Freely permeable to all gases by passive diffusion; can be quickly constructed or collapsed to ascend or descend.

Magnetosomes:

• Contain long chains of magnetite (Fe₃O₄).
• Used as a compass in geomagnetic fields for orientation.
• Magnetotactic bacteria are typically microaerophilic (prefer lower oxygen than atmosphere).
• Help cells locate the optimum depth for growth.
• Have a true lipid bilayer (invagination of the plasma membrane modified with specific proteins), reminiscent of eukaryotic organelles.

🏗️ Microcompartments and specialized organelles

🏗️ Bacterial microcompartments (BMCs)

Bacterial microcompartments (BMCs): unique inclusions with icosahedral shape and a protein shell made of BMC family proteins.

• Differ from other inclusions by structure and functionality.
• Provide both a location and the substances (usually enzymes) necessary for particular metabolic activities.
• Go beyond simple storage.

🌱 Carboxysome (CO₂ fixation)

• Best-studied example of a BMC.
• Found in many CO₂-fixing bacteria.
• Contain the enzyme ribulose-1,5-bisphosphate carboxylase (RubisCO), crucial for converting CO₂ into sugar.
• Also play a role in concentrating CO₂, ensuring all components for CO₂-fixation are in the same place at the same time.

🔄 Anammoxosome (nitrogen metabolism)

Anammoxosome: a large membrane-bound compartment in bacterial cells capable of the anammox reaction (anaerobic ammonium oxidation).

• Converts ammonium (NH₄⁺) and nitrite (NO₂⁻) into dinitrogen gas (N₂).
• The cell gets energy by using ammonium as an electron donor and nitrite as an electron acceptor.
• Important for the nitrogen cycle.

🌞 Chlorosome (light harvesting)

• Found in some phototrophic bacteria.
• Highly efficient structure for capturing low light intensities.
• Lines the inside perimeter of the cell membrane.
• Each can contain up to 250,000 bacteriochlorophyll molecules arranged in dense arrays.
• Harvested light is transferred to reaction centers in the cell membrane, converting light energy to chemical energy (ATP).
• Bounded by a lipid monolayer.

🧬 Plasmids: extrachromosomal DNA

🧬 What plasmids are

Plasmid: an extrachromosomal piece of DNA that some bacteria have, in addition to the genetic material in the nucleoid.

• Composed of double-stranded DNA, typically circular (although linear plasmids exist).
• Described as "non-essential": the cell can function normally in their absence.
• Have only a few genes but can confer important capabilities, such as antibiotic resistance.

🔁 Replication and loss

• Replicate independently of the cell.
• Can be lost (curing), either spontaneously or due to adverse conditions (UV light, thymine starvation, growth above optimal conditions).

🔗 Episomes

Episomes: plasmids that can be integrated into the cell chromosome.

• Genes will be replicated during cell division when integrated.

🛡️ Endospores: dormant survival form

🛡️ What endospores are

Endospore: a conversion of the cell into an alternate, dormant form; formed within the vegetative cell and released when the vegetative cell lyses.

• Only formed by a few gram-positive genera.
• Provide resistance to a wide variety of harsh conditions: starvation, extremes in temperature, desiccation, UV light, chemicals, enzymes, and radiation.
• The vegetative cell is the active form (growing, metabolizing); the endospore is dormant (survival only, no growth or reproduction).

🏗️ Endospore structure (layers)

Layer	Description	Function
Core	Nucleoid, ribosomes, cytoplasm in extremely dehydrated form (25% water of vegetative cell)	Increases heat resistance; DNA protected by small acid-soluble proteins (SASPs) and dipicolinic acid complexed with calcium (Ca-DPA)
Inner membrane	Permeability barrier	Protects from chemicals
Cortex	Thick peptidoglycan layer with less cross-linking than vegetative cell	Structural protection
Outer membrane	Additional membrane layer	Further barrier
Spore coats	Several layers made of protein	Protection from environmental stress (chemicals, enzymes)

• SASPs stabilize DNA and protect it from degradation.
• Ca-DPA inserts between DNA bases, increasing stabilization.

🔄 Sporulation: vegetative → endospore

Sporulation: the conversion of a vegetative cell into an endospore; typically occurs when the cell's survival is threatened.

• Very complex process; takes several hours.
• Steps:
  1. Cell replicates its DNA (as if about to divide).
  2. A septum forms asymmetrically, sequestering one chromosome copy at one end (the forespore).
  3. Synthesis of endospore-specific substances; forespore develops endospore layers and dehydrates.
  4. "Mother cell" lyses, releasing the mature endospore.

🌱 Germination: endospore → vegetative

• Endospore remains dormant until environmental conditions improve.
• Chemical change initiates gene expression.
• Three stages:
  1. Activation: preparation step; can be initiated by heat.
  2. Germination: endospore becomes metabolically active; begins to take on water.
  3. Outgrowth: vegetative cell fully emerges from the endospore shell.

🔍 Key distinctions

🔍 Inclusions vs microcompartments

• Simple inclusions: aggregates of chemicals (glycogen, PHB, polyphosphate, sulfur); may have protein/phospholipid shells but primarily for storage.
• Microcompartments (BMCs): protein-shell icosahedral structures housing enzymes; participate in metabolic activities beyond storage.
• Membrane-bound compartments: anammoxosome and chlorosome have true lipid membranes (bilayer or monolayer); magnetosome is an invagination of the plasma membrane.

🔍 Vegetative cell vs endospore

• Vegetative cell: active, metabolizing, reproducing; normal bacterial form.
• Endospore: dormant, highly resistant, no metabolism or reproduction; survival form only.
• Don't confuse: the endospore is not a reproductive structure; it is a one-to-one conversion for survival.

🧭 Overview

🧠 One-sentence thesis

Bacteria possess multiple optional surface layers and appendages outside the cell wall—capsules, slime layers, S-layers, fimbriae, pili, and flagella—that enable attachment, protection, motility, and directed movement toward favorable environments.

📌 Key points (3–5)

• Layers outside the cell wall: capsule (tightly packed polysaccharide), slime layer (loose polysaccharide), and S-layer (protein matrix) provide protection, adhesion, and structural integrity.
• Attachment structures: fimbriae and pili are thin filamentous appendages made of pilin proteins; some pili also enable DNA transfer (conjugative pili) or twitching motility (type IV pili).
• Flagella drive motility: bacterial flagella are rigid, propeller-like structures powered by the proton motive force, not ATP like eukaryotic flagella.
• Common confusion: fimbriae vs. pili—both are pilin-based filaments for attachment, but pili are longer, fewer per cell, and some types perform additional functions (conjugation, twitching).
• Chemotaxis enables directed movement: bacteria use chemoreceptors and temporal sensing to bias their random walk, moving toward attractants and away from repellants by adjusting run and tumble frequencies.

🛡️ Protective layers outside the cell wall

🧬 Capsule

A bacterial capsule is a polysaccharide layer that completely envelopes the cell, well organized and tightly packed, resistant to staining.

• Protection from: desiccation, hydrophobic toxins (detergents), bacterial viruses.
• Pathogenicity: enhances disease-causing ability, protects from phagocytosis (engulfment by white blood cells).
• Attachment: helps bacteria adhere to surfaces.
• The tight packing and firm integration around the cell wall distinguish it from the slime layer.

🧪 Slime layer

A bacterial slime layer is typically composed of polysaccharides, completely surrounds the cell, but is a loose, unorganized layer easily stripped from the cell.

• Protection from: desiccation, antibiotics.
• Attachment: allows adherence to surfaces.
• Don't confuse with capsule: slime layer is loose and easily removed; capsule is tightly packed and firmly integrated.

🧱 S-layer

A highly organized S-layer is made of secreted proteins or glycoproteins that self-assemble into a matrix on the outer part of the cell wall, anchored but not officially part of the cell wall.

• Structural roles: maintains rigidity of cell wall and surface layers, preserves cell shape (important for reproduction).
• Protection from: ion/pH changes, osmotic stress, harmful enzymes, bacterial viruses, predator bacteria.
• Pathogenicity: provides protection from phagocytosis.
• Attachment: enables adhesion to other cells or surfaces.

🔗 Attachment structures

🧵 Fimbriae

Fimbriae are thin filamentous appendages that extend from the cell, often in the tens or hundreds, composed of pilin proteins.

• Function: attach to surfaces.
• Pathogenicity: particularly important for pathogenic bacteria to attach to host tissues.

🧷 Pili

Pili are very similar to fimbriae—thin filamentous appendages made of pilin proteins—but typically longer with only 1–2 present per cell.

• Basic function: attachment to surfaces and host cells.
• Example: Neisseria gonorrhea uses pili to grab onto sperm cells for passage to the next human host.
• Why differentiate from fimbriae? A few specific pili perform functions beyond attachment:

Pilus type	Function
Conjugative pili	Participate in conjugation, transferring a small piece of DNA from donor to recipient cell
Type IV pili	Enable twitching motility, where the pilus attaches to a solid surface, contracts, and pulls the bacterium forward in a jerky motion

🚀 Flagella and bacterial motility

⚙️ Flagellar structure

Bacterial flagella provide motility; the bacterial flagellum is rigid, operates like a propeller, and is powered by the proton motive force (not ATP like eukaryotic flagella).

Three main components:

1. Filament: long thin appendage extending from the cell surface, composed of flagellin protein, hollow.

   • Grows from its tip (not the base).
   • Flagellin units are transcribed in the cytoplasm, transported across membrane and cell wall, and guided into place by a protein cap.

2. Hook: curved coupler attaching the filament to the motor.

3. Motor: rotary motor spanning cell membrane and cell wall (plus outer membrane in gram negatives).

   • Basal body: provides rotation; central shaft surrounded by protein rings (2 in gram positive, 4 in gram negative).
   • Stator: provides torque; consists of Mot proteins surrounding the ring(s) in the cell membrane.

🏊 Swimming motility

• Energy source: proton motive force—protons accumulate outside the cell membrane, driven through pores in Mot proteins, interact with charges in ring proteins, causing basal body rotation.
• Speed: 200–1000 rpm, up to 60 cell lengths/second (cheetah: 25 body lengths/second).
• Rotation direction matters:
  • Counterclockwise (CCW): bacterium moves forward in a run.
  • Clockwise (CW): bacterium reorients randomly in a tumble.

🌀 Corkscrew motility

Spirochetes (spiral-shaped gram negative bacteria) use corkscrew-motility with specialized endoflagella.

• Endoflagella attach to one end of the cell, extend back through the periplasm, and attach to the other end.
• Rotation puts torsion on the entire cell, causing a flexing motion effective for burrowing through viscous liquids.

🐌 Gliding motility

Gliding motility is a slower, more graceful movement exhibited by certain filamentous or bacillus bacteria, does not require flagella.

• Requires contact with a solid surface.
• Mechanisms:
  • Slime propulsion: secreted slime propels the cell forward.
  • Surface layer proteins: pull the cell forward.

🧭 Chemotaxis and directed movement

🧪 What is chemotaxis?

Chemotaxis refers to the movement of an organism toward or away from a chemical; phototaxis is response to light.

• Attractant: favorable substance (e.g., nutrient).
• Repellant: substance with adverse effect (e.g., toxin).
• Random walk: in the absence of attractant or repellant, the cell alternates between tumbles and runs, going nowhere in particular.
• Biased random walk: in the presence of a gradient, movements become biased, resulting over time in movement toward attractants and away from repellants.

🧠 How bacteria sense direction

• Chemoreceptors: protein receptors embedded in the membrane that bind specific molecules.
• Binding triggers methylation or phosphorylation, activating a protein pathway that impacts flagellar motor rotation.
• Temporal sensing: bacteria compare the current concentration of a substance with the concentration from a few seconds (or microseconds) earlier, gathering information about the gradient orientation.

🎯 How chemotaxis works

• As the bacterium moves closer to higher concentrations of an attractant:
  • Runs (CCW rotation) become longer.
  • Tumbling (CW rotation) decreases.
• The cell may still head in the wrong direction after a tumble (random reorientation), but won't continue long.
• Result: biased random walk allows the cell to quickly move up the gradient of an attractant (or down the gradient of a repellant).

Movement type	Flagellar rotation	Result
Run	Counterclockwise (CCW)	Forward movement
Tumble	Clockwise (CW)	Random reorientation
Biased random walk	Longer runs toward attractant, shorter tumbles	Net movement toward favorable environment

🧭 Overview

🧠 One-sentence thesis

Archaea, originally mistaken for bacteria due to physical similarities, constitute their own domain with unique membrane chemistry, cell wall composition, and molecular structures that distinguish them from both Bacteria and Eukaryotes.

📌 Key points (3–5)

• Domain status: Archaea earned their own domain in the Three Domain Classification (alongside Bacteria and Eukarya) after genetic analysis revealed they are distinct from bacteria despite physical similarities.
• Prokaryotic features shared with bacteria: lack of nucleus or membrane-bound organelles, 70S ribosomes, unicellular, asexual reproduction, and similar structures (plasmids, flagella, pili).
• Unique membrane chemistry: L-isomeric glycerol linkage (vs. D-isomeric in bacteria/eukaryotes), ether-linkages (vs. ester-linkages), isoprenoid chains with branching (vs. unbranched fatty acids), and lipid monolayers (vs. bilayers only).
• Cell wall diversity: lack peptidoglycan (found in bacteria); instead have S-layers, pseudomurein, methanochondroitin, or protein sheaths—some lack cell walls entirely.
• Common confusion: Archaea were initially called "archaeabacteria" and isolated from extreme environments, but are now found everywhere bacteria are found (soil, water, human skin).

🧬 Why Archaea were confused with Bacteria

🔬 Physical similarities to bacteria

Archaea share many prokaryotic traits with bacteria:

• No nucleus or membrane-bound organelles (prokaryotic category)
• Unicellular organisms
• 70S-sized ribosomes
• Typically a few micrometers in size
• Reproduce asexually only
• Possess similar structures: plasmids, inclusions, flagella, pili
• Capsules and slime layers exist but are rare

🌍 Habitat misconception

• Originally isolated from extreme environments (high acid, salt, or heat) → earned the name "extremophiles"
• More recently isolated from all bacteria-rich environments: surface water, ocean, human skin, soil
• Don't confuse: "extremophile" label was based on early sampling bias; archaea are not limited to extreme environments.

🧪 Unique membrane chemistry

🔗 Glycerol linkage chirality

L-isomeric form: the chirality of the glycerol linkage in archaeal membranes, as opposed to the D-isomeric form found in bacteria and eukaryotes.

• This difference in molecular "handedness" is a fundamental chemical distinction.

⚗️ Ether vs. ester linkages

Ether-linkage: the bond between glycerol and side chains in archaeal membranes, providing more chemical stability than the ester-linked lipids found in bacteria and eukaryotes.

• Ether bonds are chemically more stable than ester bonds.
• This contributes to archaeal survival in harsh environments.

🌿 Isoprenoid chains with branching

Isoprenoid chains: the side chains in archaeal membranes, which can have branching side chains, unlike the unbranched fatty acids in bacteria and eukaryotes.

• The branching structure adds to membrane stability and flexibility.

🧱 Monolayer vs. bilayer structure

Monolayers: membrane structures where isoprene chains of one phospholipid connect with isoprene chains of a phospholipid on the opposite side of the membrane.

• Bacteria and eukaryotes only have lipid bilayers, where the two sides remain separated.
• Monolayers provide additional stability in extreme conditions.

Membrane feature	Archaea	Bacteria & Eukaryotes
Glycerol chirality	L-isomeric	D-isomeric
Linkage type	Ether-linkage	Ester-linkage
Side chains	Isoprenoid (branched)	Unbranched fatty acids
Layer structure	Monolayers or bilayers	Bilayers only

🧱 Cell wall diversity

🛡️ Absence of peptidoglycan

• Bacterial cell walls typically contain peptidoglycan.
• Archaea lack peptidoglycan entirely.
• Instead, archaea display a wide variety of cell wall types adapted to their environment.
• Some archaea lack a cell wall altogether.

🔲 S-layer (surface layer)

S-layer: a proteinaceous layer considered part of the cell wall itself in many archaea (unlike in bacteria, where it is an additional structure).

• Can be made of protein or glycoprotein, often anchored into the plasma membrane.
• Forms a two-dimensional crystalline array with a smooth outer surface.
• For some archaea, the S-layer is the only cell wall component.
• A few S-layers are composed of two different S-layer proteins.

🧩 Pseudomurein

Pseudomurein: a substance with chemical structure similar to peptidoglycan, containing N-acetylalosaminuronic acid (NAT) linked to NAG, with peptide interbridges for strength.

• Found in a few archaea as a peptidoglycan alternative.

🧬 Methanochondroitin

Methanochondroitin: a cell wall polymer found in some archaeal cells, similar in composition to chondroitin (a connective tissue component in vertebrates).

🕸️ Protein sheath

Protein sheath: a lattice structure similar to an S-layer, but enclosing an entire filamentous chain of cells rather than individual cells.

• Found in archaea that form filamentous chains.

🧬 Ribosomes: similarity with a key difference

📏 Size similarity

• Archaea have 70S ribosomes, the same size as bacteria.

🧬 rRNA differences prove separate domain

• rRNA nucleotide differences provided conclusive evidence that archaea deserved a separate domain from bacteria.
• This genetic analysis overturned the original "archaeabacteria" classification.

🔬 Shape and protein differences

• Archaeal ribosomes have a different shape than bacterial ribosomes.
• Contain proteins unique to archaea.
• Result: resistance to antibiotics that inhibit bacterial ribosomal function.

Don't confuse: same ribosome size (70S) does not mean same structure or function—genetic and protein differences are key.

🏗️ Unique archaeal structures

🔗 Cannulae

Cannulae: hollow tube-like structures unique to archaea, discovered in some marine strains.

• Appear to connect cells after division.
• Eventually lead to a dense network of numerous cells and tubes.
• Possible role: anchoring a community of cells to a surface.

🪝 Hami

Hamus (plural: hami): a long helical tube with three hooks at the far end, unique to archaea.

• Allow cells to attach to one another and to surfaces.
• Encourage formation of a community.

📌 Pili

Pili: tube-like structures in archaea composed of proteins most likely modified from bacterial pilin.

• Used for attachment to surfaces.
• Different from bacterial pili: archaeal flagellum proteins are similar to bacterial pili proteins, not bacterial flagellum proteins.

🚩 Flagella (proposed name: "archaellum")

Archaeal flagellum (proposed name: archaellum): a rigid filament used for motility, so different from bacterial flagella that a distinct name has been proposed.

Similarities to bacterial flagella:

• Both used for movement.
• Cell propelled by rotation of a rigid filament extending from the cell.

Differences from bacterial flagella:

Feature	Archaeal flagellum	Bacterial flagellum
Energy source	ATP	Proton motive force
Protein similarity	Similar to bacterial pili proteins	Unique flagellin proteins
Filament structure	Not hollow	Hollow
Growth mechanism	Flagellin inserted at base	Added to the end
Flagellin types	Several different types	One type
Rotation direction	Clockwise = forward; counterclockwise = backward	Opposite
Movement pattern	No runs and tumbles observed	Alternation of runs and tumbles

Example: An archaeal cell rotates its flagellum clockwise to move forward (powered by ATP), whereas a bacterial cell uses proton flow and different rotation mechanics.

🌳 Classification

📂 Recognized phyla

Currently two recognized phyla:

1. Euryarchaeota
2. Proteoarchaeota

🔬 Proposed phyla (not yet official)

Several additional phyla proposed but not officially recognized:

• Nanoarchaeota
• Korarchaeota
• Aigarchaeota
• Lokiarchaeota

Reason for lack of recognition: evidence comes from environmental sequences only (not cultured organisms).

🧭 Overview

🧠 One-sentence thesis

Viruses are obligate intracellular parasites that require host cells to replicate, and they employ diverse genome types, structures, and replication strategies to infect all types of cells.

📌 Key points (3–5)

• What viruses are: acellular infectious agents that cannot multiply without a host cell; they infect all cell types (humans, animals, plants, bacteria, archaea, protozoa).
• Structural diversity: viruses range from simple (nucleic acid + protein capsid) to complex (with envelopes, enzymes, and unusual shapes).
• Genome variety: unlike cells (which use dsDNA), viruses can have dsDNA, ssDNA, dsRNA, +ssRNA, or -ssRNA genomes.
• Common confusion—bacteriophage replication: temperate phage can choose between lytic cycle (immediate lysis) and lysogenic cycle (integration into host DNA), whereas virulent phage always lyse the host.
• Why it matters: understanding viral replication cycles explains infection mechanisms, viral persistence, and how some viruses (oncoviruses) cause cancer.

🦠 What viruses are and their basic components

🦠 Obligate intracellular parasites

Viruses: obligate intracellular parasites, acellular infectious agents that require the presence of a host cell in order to multiply.

• Viruses are not cells—they are inert (metabolically inactive) outside a host.
• They have been found to infect all types of cells: humans, animals, plants, bacteria, yeast, archaea, protozoa.
• Some scientists claim viruses can even infect other viruses, but only with cellular help.

🧱 Core structural components

The simplest virus consists of:

Component	Definition	Notes
Capsid	Protein coat surrounding nucleic acid	Composed of smaller protein subunits called capsomers
Nucleocapsid	Capsid + genome combination	The core infectious unit
Envelope	Additional membranous layer around nucleocapsid	Acquired from host cell nuclear or plasma membrane; modified with viral proteins called peplomers
Viral enzymes	Enzymes necessary for host infection	Coded within the viral genome
Virion	Complete virus with all components needed for infection	Fully assembled infectious particle

• Don't confuse: nucleocapsid (capsid + genome) vs. virion (complete infectious particle, which may include envelope and enzymes).

🧬 Viral genome diversity

🧬 Types of viral genomes

Unlike cells, which contain double-stranded DNA, viruses are not limited to this form.

Viral genomes can be:

• dsDNA: double-stranded DNA
• ssDNA: single-stranded DNA
• dsRNA: double-stranded RNA
• +ssRNA: positive-sense single-stranded RNA (can transcribe a message, like mRNA)
• -ssRNA: negative-sense single-stranded RNA (complementary to mRNA)

🔄 Genome conversion

Some viruses start with one form of nucleic acid in the nucleocapsid and then convert it to a different form during replication.

Example: A virus might enter with RNA but convert it to DNA during the replication process inside the host cell.

🔷 Viral structure and shapes

🔷 Two basic nucleocapsid shapes

Shape	Description	Structure
Helical viruses	Elongated tube-like structure	Capsomers arranged helically around the coiled genome
Icosahedral viruses	Spherical shape	Icosahedral symmetry with 20 triangular faces; simplest form has 3 capsomers per face = 60 total capsomers

🧩 Complex viruses

Complex viruses: viruses that do not fit neatly into helical or icosahedral categories due to unusual design or components.

Examples:

• Poxvirus: brick-shaped exterior with complicated internal structure
• Bacteriophage: tail fibers attached to an icosahedral head

Don't confuse: the overall appearance can be altered by the presence of an envelope, even if the underlying nucleocapsid is helical or icosahedral.

🔁 General viral replication cycle

🔁 Five-step pattern

While replication varies from virus to virus, there is a general pattern:

1. Attachment: virion attaches to the correct host cell
2. Penetration or Viral Entry: virus or viral nucleic acid gains entrance into the cell
3. Synthesis: viral proteins and nucleic acid copies are manufactured by the cell's machinery
4. Assembly: viruses are produced from the viral components
5. Release: newly formed virions are released from the cell

🎯 Attachment

• Outside their host cell, viruses are inert (metabolically inactive).
• Encounter between virion and host cell is a random event.
• Attachment is highly specific: between molecules on the virus surface and receptors on the host cell surface.
• This specificity accounts for why viruses only infect particular cell types or particular hosts.

🚪 Penetration or viral entry

Unenveloped (naked) viruses:

• Many inject their nucleic acid into the host cell, leaving an empty capsid outside (common with bacteriophage).
• Eukaryotic naked viruses often gain entry via endocytosis: host cell engulfs the capsid, creating an endocytic vesicle.

Enveloped viruses:

• Gain entrance through membrane fusion: viral envelope fuses with host cell membrane, pushing the nucleocapsid past the membrane.

Uncoating:

• If the entire nucleocapsid enters the cell, there is an uncoating process to strip away the capsid and release the viral genome.

🏭 Synthesis

• Largely dictated by the type of viral genome.
• Genomes that differ from the cell's dsDNA can involve intricate viral strategies.
• Viral-specific enzymes (e.g., RNA-dependent RNA polymerases) might be necessary.
• Protein production is tightly controlled to ensure components are made at the right time.

🔧 Assembly

Complexity depends on the virus:

• Simplest virus: capsid composed of 3 different protein types; self-assembles easily.
• Most complex virus: composed of over 60 different proteins; must come together in specific order.
• Complex viruses often employ multiple assembly lines and use scaffolding proteins to organize all components.

🚀 Release

Method	Description	Common in
Lysis	Host cell lyses at end of replication; all newly formed virions released	Majority of viruses
Budding	One virus released at a time; nucleocapsid pushes out through modified membrane portion to acquire envelope	Enveloped viruses

🦠 Bacteriophage replication strategies

🦠 Virulent phage and the lytic cycle

Virulent phage: phage that always lyses the host cell at the end of replication.

• Follows the five steps of replication (attachment, penetration, synthesis, assembly, release).
• This is called the lytic cycle of replication.
• Results in host cell destruction and release of many new virions.

🔀 Temperate phage: two options

Temperate phage: viruses that have two options regarding their replication.

Option 1—Lytic cycle:

• Mimic virulent phage: follow five steps and lyse the host cell.

Option 2—Lysogenic cycle:

• Remain within the host cell without destroying it.
• Process known as lysogeny or the lysogenic cycle.

🧬 Lysogeny mechanism

How it works:

1. Phage undergoes attachment and penetration.
2. Viral DNA integrates with host DNA, forming a prophage.
3. Infected bacterium is called a lysogen or lysogenic bacterium.
4. Virus does not interfere with host metabolism or reproduction.
5. Host cell gains immunity from reinfection by the same virus.

Induction:

• Exposure to stressful conditions (e.g., UV irradiation) causes induction.
• Viral DNA excises from host DNA.
• Triggers remaining steps of lytic cycle (synthesis, assembly, release), leading to lysis.

🎲 What dictates replication type?

Condition	Likely replication choice	Reason
Plenty of host cells	Lytic cycle	Large increase in viral production
Host cells scarce	Lysogeny	Viral survival until host numbers increase
Phage greatly outnumber hosts	Lysogeny	Allows host cell numbers to rebound, ensuring long-term viral survival

🎁 Lysogenic conversion

Lysogenic conversion: development of a prophage leads to a change in the host's phenotype.

Example: Corynebacterium diphtheriae (causative agent of diphtheria)

• The diphtheria toxin that causes disease is encoded within the phage genome.
• Only C. diphtheriae lysogens cause diphtheria.
• Non-lysogenic C. diphtheriae do not produce the toxin.

Don't confuse: lysogeny (viral integration) vs. lysogenic conversion (phenotypic change in host due to prophage genes).

🧫 Eukaryotic virus infection outcomes

🧫 Four possible outcomes

Eukaryotic viruses can cause one of four different outcomes for their host cell:

Outcome	Description	Similar to
Virulent infection	Host cell lysis	Lytic cycle in phage
Latent infection	Viral DNA inserted into host genome; co-exist peacefully for long periods	Lysogeny in phage
Persistent infection	Enveloped viruses released one at a time via budding	Budding release mechanism
Transformation	Host cell transforms into malignant or cancerous cell	Unique to eukaryotic viruses

🧬 Latent infection

• Viral DNA integrates into host cell genome.
• Allows virus to co-exist peacefully with host for long periods.
• Much like temperate phage during lysogeny.

🔄 Persistent infection

• Certain enveloped eukaryotic viruses can be released one at a time from infected host cell.
• Uses a budding process.
• Host cell may survive longer than in lytic infection.

⚠️ Transformation

• Certain eukaryotic viruses cause host cell to transform into a malignant or cancerous cell.
• Mechanism known as transformation.

🎗️ Viruses and cancer

🎗️ Oncoviruses

Oncoviruses: viruses that have a known association with the development of cancer.

• There are many causes of cancer (unregulated cell growth and reproduction): chemicals, UV light, and certain viruses.

🛡️ Mechanism: inactivating tumor suppressor proteins

Tumor suppressor proteins: host proteins that function to regulate cell growth and initiate programmed cell death if needed.

How oncoviruses cause cancer:

1. Oncoviruses produce proteins that bind to host tumor suppressor proteins.
2. Viral proteins inactivate the tumor suppressor proteins.
3. Cells grow out of control.
4. Leads to development of tumors and metastasis (cells spread throughout the body).

Example: An oncovirus infects a cell and produces a protein that binds to a tumor suppressor protein, preventing it from stopping cell division. The cell divides uncontrollably, forming a tumor.

Don't confuse: not all viruses cause cancer—only oncoviruses have this association, and they do so by specifically targeting tumor suppressor proteins.

🧭 Overview

🧠 One-sentence thesis

Microbial growth—measured by population increase rather than individual size—follows a predictable four-phase curve in closed systems, and understanding this pattern allows us to predict and control microbial behavior under specific conditions.

📌 Key points (3–5)

• What microbial growth means: increase in population (cell number or total mass), not increase in size of a single organism.
• How bacteria reproduce: bacteria and archaea reproduce only asexually (most commonly by binary fission), while eukaryotic microbes can reproduce sexually or asexually.
• The four-phase growth curve: in a closed system, bacteria grow through lag phase (adaptation), exponential/log phase (rapid doubling), stationary phase (growth stops), and death/decline phase (cell death).
• Common confusion: the growth curve pattern (four phases) is consistent across organisms, but the details (number of cells, length of each phase, speed) vary by organism and conditions.
• Why it matters: knowledge of growth phases helps predict microbial behavior, calculate generation time, and choose optimal cells for experiments (exponential phase cells are healthiest).

🔬 Bacterial reproduction mechanisms

🔬 Binary fission process

Binary fission: a process where a single cell splits into two equally sized cells.

• This is the most common reproduction method for bacteria and archaea.
• Other less common processes include multiple fission, budding, and spore production.

Steps of binary fission:

1. Cell elongation: the cell membrane and cell wall enlarge carefully; cell volume increases.
2. DNA replication: the cell replicates its DNA to prepare two copies of its chromosome (one for each daughter cell).
3. Septum formation: the protein FtsZ forms a ring in the middle of the elongated cell, creating a septum (dividing structure).
4. Nucleoid segregation: the nucleoids (DNA regions) move to each end of the elongated cell.
5. Division completion: the septum finishes forming, dividing the elongated cell into two equally sized daughter cells.

• The entire cell cycle can take as little as 20 minutes for an active culture of E. coli.

🧬 Reproductive differences

Organism type	Reproductive methods
Bacteria and archaea	Asexual only
Eukaryotic microbes	Sexual or asexual

📈 The four-phase growth curve

📈 What a growth curve shows

Growth curve: the predictable pattern of bacterial growth in a closed system or batch culture (no food added, no wastes removed), composed of four distinct phases.

• A closed system/batch culture means nutrients are not replenished and wastes are not removed.
• The growth curve can also yield generation time (g): the amount of time it takes for the population to double.
• Don't confuse: the pattern of four phases is consistent, but the details (cell numbers, phase lengths, growth speed, total time) vary by organism and conditions.

🕐 Lag phase (adaptation period)

Lag phase: an adaptation period where bacteria adjust to their new conditions.

What influences lag phase length:

• How different the new conditions are from the old conditions.
• The condition of the bacterial cells themselves.
• Actively growing cells transferred to the same media and conditions → shortest lag period.
• Damaged cells → long lag period (they must repair before reproducing).

What cells do during lag phase:

• Synthesize RNA, enzymes, and essential metabolites missing from the new environment (e.g., growth factors, macromolecules).
• Adjust to environmental changes (temperature, pH, oxygen availability).
• Repair injured cells if necessary.

Example: Cells moved from one growth medium to a completely different medium will spend more time in lag phase than cells moved to identical medium.

📊 Exponential or log phase (rapid growth)

Exponential or log phase: the phase marked by predictable doublings of the population (1 cell → 2 → 4 → 8, etc.).

Characteristics:

• Optimal conditions → very rapid growth (steeper slope on the growth curve).
• Less than ideal conditions → slower growth.
• Cells in this phase are the healthiest and most uniform, which is why most experiments use cells from exponential phase.

Why plot on a semilog scale:

• The relationship between time and cell number is exponential, not linear.
• Plotting cell concentration on a semilog scale standardizes the data, making it appear linear and easier to analyze.

🧮 Generation time calculation

Generation time (g): the time it takes for the bacterial population to double in number.

The formula relationship:

• N = final cell concentration
• N₀ = initial cell concentration
• n = number of generations that occurred during the time period
• t = the specified period of time (in minutes, hours, days, or months)
• Generation time g = t/n

How to calculate:

1. Know the cell concentration at the start of exponential phase (N₀).
2. Know the cell concentration after some period of exponential growth (N).
3. Calculate the number of generations (n).
4. Use the time period (t) to calculate g.

Example: If you know a population started with 100 cells and grew to 1,600 cells in 2 hours, you can calculate how many generations occurred (4 doublings: 100→200→400→800→1,600) and find that generation time = 2 hours / 4 generations = 30 minutes per generation.

⏸️ Stationary phase (growth plateau)

Stationary phase: the phase where the number of new cells produced equals the number of cells dying, or growth has entirely ceased, resulting in a flattening of the growth curve.

Why growth stops:

• The population runs out of an essential nutrient/chemical.
• Growth is inhibited by the population's own waste products (remember, it's a closed container).
• Lack of physical space.

Physiological changes in stationary phase:

• New cells produced are smaller; bacilli (rod-shaped bacteria) become almost spherical.
• Plasma membrane becomes less fluid and permeable, with more hydrophobic molecules on the surface (promotes cell adhesion and aggregation).
• Nucleoid condenses; DNA becomes bound with DNA-binding proteins from starved cells (DPS) to protect DNA from damage.
• These changes help cells survive longer in adverse conditions while waiting for better conditions.

Special activities during stationary phase:

• Cells produce secondary metabolites (metabolites produced after active growth), such as antibiotics.
• Cells capable of making endospores activate sporulation genes during this stage.

🧫 Oligotrophic environments

Oligotrophic: low-nutrient environments.

• Cells in oligotrophic environments use the same survival strategies as stationary-phase cells.
• Hypothesis: cells in nature typically exist for long periods in oligotrophic environments, with only sporadic nutrient infusions that briefly return them to exponential growth.

☠️ Death or decline phase (cell death)

Death or decline phase: the phase where the number of viable cells decreases in a predictable (exponential) fashion.

Characteristics:

• The steepness of the slope shows how fast cells are losing viability.
• Culture conditions have deteriorated so much that cells are irreparably harmed.
• Cells collected from this phase fail to show growth when transferred to fresh medium.

Important measurement note:

• If turbidity (cloudiness) is used to measure cell density, measurements might not decrease during this phase because cells could still be intact even though they are dead.

🔄 Alternative explanations for the death phase

Viable but nonculturable (VBNC):

Viable but nonculturable (VBNC): a state where cells thought to be dead might be revived under specific conditions; cells enter very low metabolism and lack cellular division, only to resume growth later when conditions improve.

• This state might be important for pathogens.

Mutation and survival:

• 100% cell death is unlikely for any cell population because cells mutate to adapt to harsh environmental conditions.
• Often a "tailing effect" is observed: a small population of cells cannot be killed off.
• These surviving cells might benefit from nutrients released when fellow cells lyse (burst) and release their cellular contents.

🌍 Environmental factors affecting growth

💧 Osmotic pressure and water movement

Osmotic pressure: pressure changes cells experience because the plasma membrane is freely permeable to water (a process known as passive diffusion).

• Water moves to try to equilibrate the cell's solute concentration with the environment's solute concentration.

💧 Hypotonic environments

Hypotonic: the environment's solute concentration is lower than the cell's internal solute concentration.

What happens:

• Water passes into the cell.
• The cell swells and internal pressure increases.
• If not corrected, the cell will eventually burst from lysis of the plasma membrane.

Cell response:

• Cells need to reduce the osmotic concentration of their cytoplasm.
• Sometimes cells use inclusions to chemically change their solutes, reducing molarity.

💧 Hypertonic environments

Hypertonic: the environment's solute concentration is higher than the cell's internal solute concentration.

What happens:

• Water leaves the cell.
• The cell dehydrates.
• Extended dehydration causes permanent damage to the plasma membrane.

Comparison table:

Environment type	Solute concentration	Water movement	Effect on cell
Hypotonic	Lower outside than inside	Water enters cell	Cell swells, may burst
Hypertonic	Higher outside than inside	Water leaves cell	Cell dehydrates, membrane damage

🧭 Overview

🧠 One-sentence thesis

Microbes that adapt to harsh physical environments—extreme osmolarity, pH, temperature, oxygen levels, pressure, or radiation—face less competition and can thrive where others cannot.

📌 Key points (3–5)

• Core idea: Physical environmental conditions (temperature, oxygen, pH, osmolarity, pressure, radiation) shape where microbes can grow; adaptation to extremes reduces competition.
• Osmolarity: Water moves across membranes to balance solute concentration; hypotonic environments risk cell lysis, hypertonic environments risk dehydration.
• Temperature, pH, oxygen: Microbes are classified by their optima (psychrophiles, mesophiles, thermophiles; acidophiles, neutrophiles, alkaliphiles; aerobes, anaerobes) and require specific adaptations (enzymes, membrane composition, protective proteins).
• Common confusion: "Aerotolerant anaerobe" vs "facultative anaerobe"—both grow with or without oxygen, but facultatives prefer oxygen and switch metabolism; aerotolerants show no preference.
• Why it matters: Understanding environmental tolerances explains microbial distribution, pathogen habitats, food safety, and extreme-environment survival.

💧 Osmolarity and water balance

💧 Osmotic pressure and passive diffusion

Osmotic pressure: the pressure exerted by water movement across a freely permeable plasma membrane to equilibrate solute concentrations inside and outside the cell.

• Water moves by passive diffusion (no energy required) toward the side with higher solute concentration.
• The cell cannot control water movement directly; it must manage internal solute levels.

🔻 Hypotonic environments (low external solute)

Hypotonic: the environment has a lower solute concentration than the cell's interior.

• Water flows into the cell → cell swells → internal pressure increases.
• If uncorrected, the plasma membrane lyses and the cell bursts.
• Adaptation: Cells reduce internal osmotic concentration by chemically changing solutes (using inclusions) or opening mechanosensitive (MS) channels in the membrane to release solutes and lower pressure.

🔺 Hypertonic environments (high external solute)

Hypertonic: the environment has a higher solute concentration than the cell's interior.

• Water flows out of the cell → cell dehydrates.
• Extended dehydration causes permanent plasma membrane damage.
• Adaptation: Cells take up solutes from the environment, but must choose compatible solutes (sugars, amino acids) that do not interfere with cellular processes.

🧂 Halophiles (extreme salt adaptation)

Halophiles: microbes that require NaCl concentrations above 0.2 M to grow.

• They live in extreme hypertonic (high-salt) environments.
• They take up both potassium and chloride ions to offset the external salt.
• Their ribosomes, enzymes, transport proteins, cell wall, and plasma membrane have evolved to require high K⁺ and Cl⁻ concentrations to function.
• Example: An organism adapted to a salt lake cannot survive in freshwater because its cellular machinery depends on high ion levels.

🧪 pH and hydrogen ion concentration

🧪 What pH measures

pH: the negative logarithm of the hydrogen ion concentration (in molarity); ranges from 0 (extremely acidic, 1.0 M H⁺) to 14 (extremely alkaline, 1.0 × 10⁻¹⁴ M H⁺).

• Each pH unit represents a tenfold change in H⁺ concentration.
• Example: pH 3 is 10× more acidic than pH 4.

😐 Neutrophiles (neutral pH preference)

Neutrophiles: microbes preferring pH 5.5–8.0, close to the cytoplasmic pH of ~7.2.

• Most microbes are neutrophiles because their internal environment is near neutral.

🍋 Acidophiles (acid lovers)

Acidophiles: microbes preferring environmental pH 0–5.5.

• Must maintain internal pH in an acceptable range despite external acidity.
• Adaptations:
  • Transport cations (e.g., K⁺) into the cell to decrease H⁺ entry.
  • Use proton pumps to actively pump H⁺ out.
  • Preserve plasma membrane stability.

🧼 Alkaliphiles (alkaline lovers)

Alkaliphiles: microbes preferring environmental pH 8.0–11.5.

• Must pump protons in to maintain cytoplasmic pH.
• Adaptation: Employ antiporters that pump H⁺ in and Na⁺ out.

🌡️ Temperature and enzyme activity

🌡️ Why temperature matters

• Microbes cannot regulate internal temperature; they must adapt to their environment.
• Temperature primarily affects enzyme activity.
• Each microbe has:
  • Optimal temperature: fastest metabolism and growth rate.
  • Minimum and maximum temperatures: growth continues but is slower.
• Typical bacterial growth range: ~30°C span.
• Below optimum → decreased enzyme activity, slower metabolism.
• Above optimum → protein denaturation (enzymes, carrier proteins) → cell death.

❄️ Psychrophiles (cold lovers)

Psychrophiles: microbes with optimum ≤15°C and growth range −20°C to 20°C.

• Found in oceans (often 5°C or colder), Arctic, Antarctic, ice pockets with liquid water.
• Adaptations:
  • Enzymes that function at low temperatures.
  • Plasma membrane with increased unsaturated and shorter-chain fatty acids to keep it semifluid.
  • Cryoprotectants (special proteins or sugars) to prevent ice crystal formation that could damage the cell.
• Psychrotrophs (cold-tolerant): range 0–35°C, optimum ≥16°C.

🧑 Mesophiles (moderate temperature)

Mesophiles: microbes with growth optimum ~37°C and range 20–45°C.

• Almost all human microflora and human pathogens are mesophiles.
• Occupy the same environments as humans: food, surfaces, drinking water, swimming water.

🔥 Thermophiles and hyperthermophiles (heat lovers)

Thermophiles: microbes with range 45–80°C and optimum ~60°C.

Hyperthermophiles: microbes with optimum 88–106°C, minimum 65°C, maximum 120°C.

• Adaptations:
  • Heat-stable enzymes resistant to denaturation and unfolding.
  • Chaperone proteins that protect other proteins from heat damage.
  • Plasma membrane with more saturated fatty acids (higher melting points).

🫁 Oxygen concentration and metabolism

🫁 Oxygen and the electron transport chain

• Oxygen requirement relates to the type of metabolism.
• Energy generation involves the electron transport chain (ETC), where the final electron acceptor can be oxygen or a non-oxygen molecule.

✅ Aerobes (oxygen users)

Aerobic respiration: metabolism using oxygen as the final electron acceptor.

Term	Definition	Oxygen level required
Obligate aerobes	Require atmospheric oxygen (20%) for metabolism	20%
Microaerophiles	Require oxygen, but at lower levels	2–10%

🚫 Anaerobes (oxygen-independent or oxygen-sensitive)

Anaerobes: organisms that can grow in the absence of oxygen.

Term	Growth with O₂?	Growth without O₂?	Preference	Metabolism switch?
Facultative anaerobes	Yes	Yes	Prefer oxygen (aerobic respiration generates more energy)	Yes, switch to match environment
Aerotolerant anaerobes	Yes	Yes	No preference	No switch
Obligate anaerobes	No (toxic)	Yes	Require absence of oxygen	N/A

• Don't confuse: Facultative anaerobes prefer oxygen and switch metabolism; aerotolerant anaerobes grow equally well either way with no preference.

🛡️ Reactive oxygen species (ROS) and protective enzymes

Reactive oxygen species (ROS): toxic oxygen by-products that can damage cells, even those using aerobic respiration.

• Ability to live in oxygenated environments depends on protective enzymes:
  • Superoxide dismutase (SOD): breaks down superoxide radicals.
  • Catalase: breaks down hydrogen peroxide.
• Obligate anaerobes lack both enzymes → little or no protection against ROS → oxygen is toxic.

🏔️ Pressure, radiation, and extreme adaptations

🏔️ Barophiles (pressure lovers)

Barophiles: microbes adapted to prefer or require high hydrostatic pressure (e.g., 600–1,000 atm at ocean bottoms).

• Most microbes live at ~1 atm (land or water surface).
• Adaptations:
  • Increased unsaturated fatty acids in plasma membrane.
  • Shortened fatty acid tails.

☢️ Ionizing radiation

Ionizing radiation (x-rays, gamma rays): causes mutations and destruction of DNA.

• Bacterial endospores are extremely resistant.
• Vegetative cells were thought susceptible, but Deinococcus radiodurans can completely reassemble its DNA after massive radiation doses.

☀️ Ultraviolet (UV) radiation

Ultraviolet (UV) radiation: damages DNA by forming thymine dimers (adjacent thymine bases attach to each other on the DNA strand).

• Thymine dimers inhibit DNA replication and transcription.
• Adaptation: DNA repair mechanisms, such as the enzyme photolyase, which splits apart thymine dimers.

🧭 Overview

🧠 One-sentence thesis

Microbes meet their fundamental needs for carbon, energy, and electrons through diverse nutritional strategies and specialized transport mechanisms that move nutrients across the cell membrane.

📌 Key points (3–5)

• Three fundamental needs: all microbes require carbon, energy, and electrons, with specific terms describing the source of each.
• Nutritional classification: combining terms for carbon source (heterotroph vs autotroph), energy source (phototroph vs chemotroph), and electron source (lithotroph vs organotroph) defines a microbe's nutritional type.
• Macronutrients and growth factors: cells need bulk elements (C, H, O, N, P, S, K, Mg) and some require pre-made organic molecules (amino acids, nucleotides, vitamins).
• Common confusion: passive vs active transport—passive relies on concentration gradients alone; active transport works against gradients and requires energy input.
• Transport diversity: bacteria use multiple mechanisms (passive diffusion, facilitated diffusion, three types of active transport, and specialized iron uptake) to bring nutrients into the cell.

🍽️ Nutritional types and fundamental needs

🍽️ The three essentials

All microbes need:

• Carbon: for macromolecules (proteins, carbohydrates, lipids, nucleic acids)
• Energy: to drive cellular processes
• Electrons: for metabolic reactions

Each need has associated terminology that describes the source.

🌱 Carbon sources

Term	Meaning	Carbon source
Heterotroph	"other eaters"	Reduced, preformed organic substances
Autotroph	"self feeders"	Carbon dioxide (CO₂), which they reduce or "fix" into organic molecules

⚡ Energy sources

Term	Meaning	Energy source
Phototroph	"light eaters"	Light energy from the sun
Chemotroph	"chemical eaters"	Chemical energy from organic or inorganic chemicals
Lithotroph	"rock eaters"	Inorganic chemical sources
Organotroph	"organic eaters"	Organic chemical sources

🔤 Combined terminology

The excerpt emphasizes that these terms can be combined to describe an organism's complete nutritional strategy.

Example: A microbe might be a photolithoautotroph (uses light energy, inorganic electron sources, and CO₂ as carbon) or a chemoorganoheterotroph (uses chemical energy from organic sources, which also provide carbon).

Don't confuse: The energy source and the carbon source are separate—an organism can use light for energy but still need organic carbon, or vice versa.

🧱 Macronutrients and growth factors

🧱 Macronutrients

Macronutrients: elements needed in sufficient quantity, including C, H, O, N, P, S, K, and Mg.

Beyond carbon, hydrogen, and oxygen, cells need:

• Nitrogen: for proteins, nucleic acids, and other cell components
• Phosphorus: crucial for nucleic acids (sugar-phosphate backbone), phospholipids, and ATP
• Sulfur: necessary for some amino acids and vitamins
• Potassium: needed for enzymes
• Magnesium: stabilizes ribosomes and membranes

🧬 Growth factors

Growth factors: organic molecules essential for growth that some microbes cannot synthesize from scratch.

• Some microbes can synthesize all needed organic molecules if given a carbon source and inorganic salts.
• Others require certain organic compounds in their environment.

Three categories of growth factors:

1. Amino acids: building blocks of proteins
2. Purines and pyrimidines: building blocks of nucleic acids
3. Vitamins: enzyme cofactors

🚪 Passive transport mechanisms

🚪 Passive (simple) diffusion

Passive or simple diffusion: passage of simple molecules and gases across the cell membrane without energy input.

• Transports simple molecules and gases: CO₂, O₂, H₂O
• Requires a concentration gradient: higher concentration outside the cell than inside
• As more substance enters, the gradient decreases, slowing the diffusion rate
• No energy required; driven purely by the gradient

🔓 Facilitated diffusion

Facilitated diffusion: transport using a concentration gradient plus carrier proteins (permeases).

• Also relies on concentration gradient (higher outside than inside)
• Uses carrier proteins (permeases) embedded in the cell membrane
• Proteins provide a channel or pore across the membrane for larger molecules
• Each carrier protein exhibits specificity: transports only a particular molecule type or closely related molecules
• Transport stops if the concentration gradient dissipates

Don't confuse with passive diffusion: Facilitated diffusion uses proteins for larger molecules; passive diffusion is for simple molecules only.

🔋 Active transport mechanisms

🔋 Core concept of active transport

Active transport: transport of substances against a concentration gradient using metabolic energy and carrier proteins.

• Works against a concentration gradient (higher concentration inside than outside)
• Requires metabolic energy
• All types use carrier proteins embedded in the membrane
• Three main types: primary active transport, secondary active transport, and group translocation

⚡ Primary active transport

Uses chemical energy (such as ATP) to drive transport.

ABC system example:

ABC system: utilizes ATP-Binding Cassette transporters.

Each ABC transporter has three components:

1. Membrane-spanning proteins: form a pore across the cell membrane (carrier protein)
2. ATP binding region: hydrolyzes ATP, providing energy for passage
3. Substrate-binding protein: peripheral protein that binds the substance and ferries it to the membrane-spanning proteins

Location of substrate-binding protein:

• Gram-negative bacteria: in the periplasm
• Gram-positive bacteria: attached to the outside of the cell membrane

🔄 Secondary active transport

Secondary active transport: utilizes energy from a proton motive force (PMF).

How PMF works:

Proton motive force (PMF): an ion gradient that develops when the cell transports electrons during energy-conserving processes.

• Positively charged protons accumulate outside the negatively charged cell
• Creates a proton gradient between outside and inside

Three transport mechanisms:

Type	Protein	What it does
Uniport	Uniporter	Transports a single substance across the membrane (in or out)
Symport	Symporter	Transports two substances at the same time in the same direction (typically a proton paired with another molecule)
Antiport	Antiporter	Transports two substances in opposite directions (one in, one out)

🔀 Group translocation

Group translocation: active transport using energy from an energy-rich organic compound that is not ATP; the transported substance is chemically modified in the process.

Key difference: Unlike simple transport and ABC transporters, the substance being transported is chemically modified during transport.

PTS system example:

Phosphoenolpyruvate: sugar phosphotransferase system (PTS): uses energy from phosphoenolpyruvate (PEP) to transport sugars into the cell.

• Uses the high-energy molecule phosphoenolpyruvate (PEP)
• A phosphate is transferred from PEP to the incoming sugar during transportation
• One of the best-studied examples of group translocation

🧲 Specialized iron uptake

🧲 Why iron matters

• Iron is required for cytochromes and enzymes
• It is a growth-limiting micronutrient
• Little free iron is available in environments due to its insolubility

🧲 Siderophore mechanism

Siderophores: organic molecules that chelate or bind ferric iron with high affinity.

How the system works:

1. Organism releases siderophores to the surrounding environment
2. Siderophores bind any available ferric iron
3. The iron-siderophore complex binds to a specific receptor on the outside of the cell
4. Iron is transported into the cell

Example: A bacterium in an iron-poor environment secretes siderophores, which scavenge trace iron and deliver it back to the cell via receptor-mediated uptake.

🧭 Overview

🧠 One-sentence thesis

Cells conserve energy by coupling exergonic oxidation-reduction reactions—where electrons flow from donors to acceptors through carrier chains—to the synthesis of ATP, the universal energy currency.

📌 Key points (3–5)

• Free energy and reaction types: Exergonic reactions (negative ΔG°') release energy that cells capture; endergonic reactions (positive ΔG°') require energy input, often from ATP hydrolysis.
• Redox reactions transfer electrons: Oxidation is loss of electrons (donor), reduction is gain of electrons (acceptor); every redox reaction consists of two half-reactions (conjugate redox pairs).
• Standard reduction potential (E'0) predicts electron flow: More negative E'0 means stronger electron donor; larger ΔE'0 (acceptor E'0 minus donor E'0) means more potential energy for the cell.
• Common confusion—electron carriers vs. donors/acceptors: Carriers (NAD+/NADH, FAD, cytochromes) shuttle electrons between donor and acceptor but are never the initial donor or final acceptor; they must be recycled.
• Electron transport chains maximize efficiency: Carriers embedded in membranes in order of E'0 allow stepwise energy capture as electrons "fall" down the redox tower.

⚡ Energy and Work in Cells

⚡ Free energy (G) and ΔG°'

Free energy (G): the energy available to do work (some energy is always lost as heat).

• Cells measure the change in free energy under standard conditions (pH 7, 25°C, 1 atm), denoted ΔG°' (standard free energy change).
• This tells us whether a reaction releases or requires energy.

🔄 Exergonic vs. endergonic reactions

Reaction type	ΔG°' sign	Meaning	Role in the cell
Exergonic	Negative	Releases energy	Energy is conserved (e.g., drives ATP synthesis)
Endergonic	Positive	Requires energy	Coupled with ATP hydrolysis to proceed

• Example: An exergonic reaction releases energy → cell uses it to add orthophosphate (Pᵢ) to ADP, forming ATP.
• Example: An endergonic reaction (e.g., anabolism) couples with ATP → ADP + Pᵢ hydrolysis to get the needed energy.

🏋️ Three types of cellular work

Cells use ATP to perform:

• Chemical work: building complex molecules (anabolism).
• Transport work: moving nutrients into the cell.
• Mechanical work: rotating flagella for movement.

🔋 ATP: The Universal Energy Currency

🔋 Why ATP?

Adenosine triphosphate (ATP): a high-energy molecule used by all cells for energy currency.

• ATP readily donates a phosphoryl group to other molecules.
• Exergonic reactions drive ATP synthesis: ADP + Pᵢ → ATP.
• Endergonic reactions are driven by ATP hydrolysis: ATP → ADP + Pᵢ releases energy.

🧪 Enzymes lower activation energy

Activation energy: the energy required to break chemical bonds so a reaction can proceed.

Catalyst: a substance that lowers activation energy without being changed by the reaction.

Enzyme: a protein catalyst used by cells.

• Enzymes speed up reactions by reducing the energy barrier, making cellular processes efficient.

🔁 Redox Reactions: Electron Transfer

🔁 Oxidation and reduction basics

Oxidation-reduction (redox) reaction: electrons are passed from an electron donor to an electron acceptor.

• Oxidation: loss of electrons (the donor is oxidized).
• Reduction: gain of electrons (the acceptor is reduced).
• Mnemonic: OIL RIG = Oxidation Is Loss, Reduction Is Gain.
• Electrons represent energy, so a substance with many electrons to donate is energy-rich.

🔗 Conjugate redox pairs (half-reactions)

Conjugate redox pair (redox couple): the acceptor and donor forms of a substance in a half-reaction.

• Electrons do not exist freely; every redox reaction consists of two half-reactions.
• In one half-reaction, a reduced substance donates electrons and becomes oxidized.
• In another half-reaction, an oxidized substance accepts electrons and becomes reduced.
• Example: ½ O₂ / H₂O
  • H₂O can donate electrons → becomes O₂ (oxidized).
  • O₂ can accept electrons → becomes H₂O (reduced).
• A substance can be either donor or acceptor depending on its reaction partner.

⚖️ Standard reduction potential (E'0)

Standard reduction potential (E'0): a measurement (in volts or millivolts) of the tendency of the donor in a half-reaction to give up electrons.

• More negative E'0: stronger tendency to donate electrons in the reduced form → good electron donor.
• Less negative or positive E'0: weak tendency to donate electrons → better electron acceptor.
• Example: A substance with E'0 = -0.42 V (like 2H⁺/H₂) is a strong electron donor in its reduced form (H₂).

🗼 The Redox Tower and Energy Calculation

🗼 What is a redox tower?

Redox tower: a vertical list of redox couples ordered by their E'0 values.

• Top of the tower: most negative E'0 → best electron donors (reduced form on the right).
• Bottom of the tower: most positive E'0 → best electron acceptors (oxidized form on the left).
• Middle couples: can serve as either donor or acceptor depending on their partner.
• Example from the excerpt: glucose (top, best donor) → O₂ (bottom, best acceptor) produces CO₂ and H₂O.

📐 Calculating energy yield (ΔE'0 and ΔG°')

• ΔE'0 = acceptor E'0 minus donor E'0.
• Larger ΔE'0 → more potential energy for the cell (bigger "fall" down the tower).
• ΔG°' is proportional to ΔE'0, but also depends on the number of electrons transferred:
  • Formula: ΔG°' = -n F ΔE'0
    • n = number of electrons transferred.
    • F = Faraday constant (23,062 cal/mole-volt or 96,480 J/mole-volt).
• Don't confuse: ΔE'0 alone doesn't tell the full story; the number of electrons matters too.

🔄 Electron Carriers: The Middlemen

🔄 What are electron carriers?

Electron carriers: cellular intermediates that shuttle electrons from donor to acceptor, cycling between reduced (carrying electrons) and oxidized (after passing electrons on) forms without being consumed.

• Carriers allow chemically dissimilar donors and acceptors to interact indirectly.
• Carriers enable energy capture along the way.

🚫 Carriers are never the initial donor or final acceptor

• Key distinction: Carriers originate inside the cell and must be constantly recycled.
• The cell relies on external chemicals as the initial electron donor and final electron acceptor.
• Products (oxidized donor, reduced acceptor) are often waste and released to the environment.

🧬 Common electron carriers

Carrier	What it carries	Notes
NAD⁺ / NADH	2 electrons + 2 protons	Co-enzyme; closely related: NADP⁺ / NADPH (2e⁻ + 1H⁺)
FAD / FADH	2 electrons + 2 protons	Part of flavoproteins
FMN / FMNH	2 electrons + 2 protons	Flavin mononucleotide
Coenzyme Q (CoQ) / ubiquinone	2 electrons + 2 protons	Lipid-soluble carrier
Cytochromes	1 electron	Use iron in a heme group
Iron-sulfur (Fe-S) proteins (e.g., ferredoxin)	1 electron	Iron not in heme group

• Some carriers accept both electrons and protons; others accept electrons only—this becomes crucial for energy generation.

📉 Carriers must be arranged by E'0

• For the reaction to be energetically favorable, carriers must be ordered by standard reduction potential (going down the redox tower).
• Electrons pass from a carrier with more negative E'0 to one with less negative E'0.

⛓️ Electron Transport Chains

⛓️ How the chain works

Electron transport chain (ETC): a series of electron carriers embedded in a membrane, arranged in order of their E'0, through which electrons flow from an initial donor to a final acceptor.

• Start: external initial electron donor.
• Middle: electrons pass carrier to carrier, working down the electron tower.
• End: external final electron acceptor.
• Embedding carriers in a membrane (cell membrane in bacteria/archaea; mitochondrial membrane in eukaryotes) makes the process more efficient.

🔋 Energy capture along the chain

• As electrons "fall" down the tower (from more negative to less negative E'0), energy is released at each step.
• This energy is captured to do work (e.g., synthesize ATP).
• Example: Starting with glucose (top of tower) and ending with oxygen (bottom of tower) maximizes the energy yield because of the large ΔE'0.

🧩 Metabolism Overview

🧩 Definitions

Metabolism: the sum of all chemical reactions within a cell.

Catabolism: the breakdown of organic and inorganic molecules to release energy and derive molecules for other reactions.

Anabolism: the synthesis of complex molecules from simpler ones, requiring energy.

• Catabolism is exergonic (releases energy).
• Anabolism is endergonic (requires energy, often from ATP).

🔗 How catabolism and anabolism connect

• Energy released by catabolic (exergonic) reactions drives ATP synthesis.
• ATP then powers anabolic (endergonic) reactions.
• This coupling allows cells to build what they need while extracting energy from their environment.

🧭 Overview

🧠 One-sentence thesis

Chemoorganotrophy—the oxidation of organic chemicals for energy—can occur with oxygen (aerobic respiration, yielding up to ~32 ATP per glucose), without oxygen using alternative acceptors (anaerobic respiration, yielding less ATP), or via fermentation (yielding only 2 net ATP from glycolysis alone).

📌 Key points (3–5)

• What chemoorganotrophy is: oxidation of organic chemicals (e.g., glucose) to yield energy, with the organic compound serving as the initial electron donor.
• Aerobic respiration generates the most ATP: uses glycolysis + TCA cycle + oxidative phosphorylation (ETC + PMF + ATP synthase) with oxygen as the final electron acceptor; maximum ~32 ATP per glucose.
• Anaerobic respiration vs fermentation: both occur without oxygen, but anaerobic respiration uses an ETC with a non-oxygen final acceptor (e.g., nitrate, sulfate) and yields moderate ATP, while fermentation uses pyruvate as the final acceptor, lacks an ETC, and yields only 2 net ATP.
• Common confusion: fermentation ≠ anaerobic respiration; fermentation does not use an electron transport chain or oxidative phosphorylation, so it produces far less ATP.
• Why it matters: understanding these pathways explains microbial energy yields, metabolic flexibility in different environments, and industrial applications (fermented foods, biofuels).

🔋 Aerobic respiration: maximum energy harvest

🍬 Glycolysis: the universal starting pathway

Glycolysis: a nearly universal pathway for the catabolism of glucose to pyruvate.

• Occurs in two parts:
  • Part I: modifies the 6-carbon glucose; requires 2 ATP to phosphorylate/activate the sugar.
  • Part II: splits the 6-carbon compound into two 3-carbon molecules; generates 4 ATP by substrate-level phosphorylation.
• Net yield per glucose: 2 ATP, 2 NADH, 2 pyruvate.
• Substrate-level phosphorylation: a high-energy molecule directly transfers a phosphate (Pᵢ) to ADP to form ATP.
• Example: In aerobic respiration, the 2 NADH from glycolysis will later donate electrons to the electron transport chain to capture more energy.

🔄 TCA cycle: complete oxidation of pyruvate

Tricarboxylic acid (TCA) cycle: picks up at the end of glycolysis to fully oxidize each pyruvate molecule down to 3 CO₂.

• Connecting reaction: before entering the cycle proper, each pyruvate is converted to citrate, reducing 1 NAD⁺ to NADH.
• The cycle itself: citrate undergoes a series of oxidations, releasing electrons and producing many precursor metabolites for other pathways.
• Net yield per pyruvate: 3 NADH, 1 FADH₂, 1 GTP (ATP-equivalent) by substrate-level phosphorylation.
• Total from 2 pyruvate (from 1 glucose): 8 NADH, 2 FADH₂, 2 GTP.
• At this point, only 2 ATP (from glycolysis) + 2 GTP (from TCA) have been made by substrate-level phosphorylation; the bulk of ATP comes from the electron transport chain.

⚡ Oxidative phosphorylation: harnessing the electron transport chain

Oxidative phosphorylation: the synthesis of ATP from electron transport generated by oxidizing a chemical energy source.

• Electrons from NADH and FADH₂ are passed from carrier to carrier in order of standard reduction potential.
• Some carriers accept electrons and protons; others accept electrons only.
• Unaccepted protons migrate outward to line the outer part of the membrane (cell membrane in bacteria/archaea).

🔌 Proton motive force (PMF)

Proton motive force (PMF): the concentration gradient of protons that develops as positively charged protons accumulate outside the membrane, creating both a chemical and electrical potential difference.

• The cytoplasm becomes more alkaline and more negative.
• The PMF can do work for the cell: rotate the bacterial flagellum, uptake nutrients, or synthesize ATP.

🔧 ATP synthase

ATP synthase (ATPase): a large enzyme with two components—one spans the membrane, one sticks into the cytoplasm and synthesizes ATP.

• Protons are driven through the membrane-spanning component, generating torque that rotates the cytoplasmic portion.
• When the cytoplasmic component returns to its original configuration, it binds Pᵢ to ADP, generating ATP.

📊 Aerobic respiration summary

Source	Yield
Glycolysis (substrate-level)	2 net ATP
TCA cycle (substrate-level)	2 GTP (ATP-equivalents)
Glycolysis + TCA NADH (10 total)	10 × 2.5 = 25 ATP (via oxidative phosphorylation)
TCA FADH₂ (2 total)	2 × 1.5 = 3 ATP (via oxidative phosphorylation)
Grand total	~32 ATP per glucose

• The process is not completely efficient; some "leakage" occurs.
• Current estimates: 2.5 ATP per NADH, 1.5 ATP per FADH₂.
• Why so much ATP? Large distance between the initial electron donor (glucose) and the final electron acceptor (oxygen), plus glucose has many electrons to donate.

🌫️ Anaerobic chemoorganotrophy: energy without oxygen

🧪 Anaerobic respiration: using alternative electron acceptors

Anaerobic respiration: starts with glycolysis and can use the TCA cycle, just like aerobic respiration, but the final electron acceptor is not oxygen.

• Still uses oxidative phosphorylation (ETC + ATP synthase).
• Common alternative electron acceptors: nitrate (NO₃⁻), ferric iron (Fe³⁺), sulfate (SO₄²⁻), carbonate (CO₃²⁻), or certain organic compounds like fumarate.
• Best electron acceptor: the one lowest on the electron tower in oxidized form (left-hand side of the redox couple).
• ATP yield: less than aerobic respiration, because the alternative acceptor is higher on the electron tower than oxygen, shortening the distance between donor and acceptor and reducing ATP production.
• Example: A microbe using nitrate as the final acceptor will generate less ATP than one using oxygen, but more than one using fermentation.

🍺 Fermentation: no ETC, only substrate-level phosphorylation

Fermentation: catabolism of glucose in the absence of oxygen, using pyruvate (an organic compound) as the final electron acceptor; does not use an electron transport chain.

• Key difference from anaerobic respiration: fermentation lacks an ETC (or represses ETC synthesis when oxygen is absent), so it does not use the TCA cycle at all.
• Process: starts with glycolysis, yielding 2 net ATP and 2 NADH.
• Why not stop after glycolysis? Eventually all NAD⁺ would become reduced (to NADH); the cell must re-oxidize NADH to continue glycolysis.
• Solution: use pyruvate as the final electron acceptor, re-oxidizing NADH and producing fermentation products (e.g., ethanol, CO₂, various acids like lactate).
• Net ATP yield: only 2 ATP per glucose (from glycolysis alone).
• Don't confuse: fermentation ≠ anaerobic respiration; fermentation does not use oxidative phosphorylation, so it produces far less ATP.

🧀 Fermentation products and applications

• Fermentation products are waste for the cell but vitally important for humans.
• Applications: fermented foods (beer, wine, bread, cheese, tofu), industrial processes.
• Example: Lactate fermentation produces lactic acid; ethanol fermentation produces ethanol and CO₂.

🔍 Comparing the three pathways

Pathway	Electron donor	Final electron acceptor	Uses ETC?	Uses TCA cycle?	ATP yield per glucose
Aerobic respiration	Organic (e.g., glucose)	Oxygen (O₂)	Yes	Yes	~32 ATP
Anaerobic respiration	Organic (e.g., glucose)	Non-oxygen inorganic (e.g., NO₃⁻, SO₄²⁻, Fe³⁺)	Yes	Yes	Moderate (less than aerobic)
Fermentation	Organic (e.g., glucose)	Organic (pyruvate)	No	No	2 net ATP

• Why the difference in ATP yield?
  • Aerobic respiration: oxygen is the best electron acceptor (lowest on the electron tower), maximizing the distance electrons travel and the PMF generated.
  • Anaerobic respiration: alternative acceptors are higher on the electron tower, shortening the distance and reducing ATP yield.
  • Fermentation: no ETC or oxidative phosphorylation, so only substrate-level phosphorylation from glycolysis contributes ATP.

🧩 Key mechanisms and concepts

🔄 Why NADH must be re-oxidized

• Glycolysis reduces NAD⁺ to NADH.
• If NADH is not re-oxidized back to NAD⁺, glycolysis cannot continue (the cell runs out of NAD⁺).
• In aerobic/anaerobic respiration: NADH donates electrons to the ETC, re-oxidizing to NAD⁺.
• In fermentation: NADH donates electrons to pyruvate, re-oxidizing to NAD⁺ and producing fermentation products.

⚙️ Substrate-level vs oxidative phosphorylation

Type	Mechanism	Where it occurs
Substrate-level phosphorylation	A high-energy molecule directly transfers Pᵢ to ADP	Glycolysis, TCA cycle
Oxidative phosphorylation	ATP synthase uses the PMF (from the ETC) to bind Pᵢ to ADP	Electron transport chain + ATP synthase

• Don't confuse: substrate-level phosphorylation is direct; oxidative phosphorylation requires the ETC and PMF.

🌍 Metabolic flexibility and environment

• Microbes can perform chemoorganotrophy in the presence or absence of oxygen, depending on what is available and whether they have enzymes to deal with toxic oxygen by-products.
• Aerobic respiration: requires oxygen; generates the most ATP.
• Anaerobic respiration: uses alternative acceptors; allows microbes to live in oxygen-poor environments (e.g., deep soil, sediments).
• Fermentation: no external electron acceptor needed; allows survival in strictly anaerobic conditions but yields minimal ATP.

🧭 Overview

🧠 One-sentence thesis

Chemolithotrophy enables microbes to generate energy by oxidizing inorganic chemicals instead of organic compounds, and these organisms play essential roles in the nitrogen cycle by converting nitrogen between different chemical forms that other life depends on.

📌 Key points (3–5)

• What chemolithotrophy is: oxidation of inorganic chemicals (not organic) to generate energy through oxidative phosphorylation.
• Variety of electron donors: hydrogen gas, sulfur compounds, nitrogen compounds, and ferrous iron can all serve as electron donors.
• Energy yield varies widely: ATP production depends on the distance between electron donor and acceptor; always less than aerobic respiration using glucose.
• Common confusion—autotrophs vs heterotrophs: chemolithoautotrophs fix CO₂ for carbon (often requiring reverse electron flow), while chemolithoheterotrophs use inorganic chemicals for energy but rely on organic chemicals for carbon.
• Nitrogen cycle roles: microbes perform nitrogen fixation (N₂ to NH₃), nitrification (NH₃ to NO₃⁻), denitrification (NO₃⁻ to N₂), assimilation (inorganic to organic nitrogen), and anammox (anaerobic ammonia oxidation).

⚡ Core mechanism of chemolithotrophy

⚡ What chemolithotrophy is

Chemolithotrophy: the oxidation of inorganic chemicals for the generation of energy.

• Uses oxidative phosphorylation just like aerobic and anaerobic respiration.
• The key difference: the substance being oxidized (electron donor) is inorganic, not organic.
• Electrons pass through the electron transport chain (ETC), generating a proton motive force (PMF) that drives ATP synthase to produce ATP.

🔋 Electron donors used

Chemolithotrophs use a variety of inorganic compounds as electron donors:

Electron donor	Organism type	Key enzyme/process
Hydrogen gas (H₂)	Hydrogen oxidizers	Hydrogenase enzyme; aerobic types reduce O₂ to water
Sulfur compounds (H₂S, S⁰, S₂O₃²⁻, SO₃²⁻)	Sulfur oxidizers	Sulfite oxidase; stepwise oxidation often produces sulfate (SO₄²⁻)
Nitrogen compounds (NH₃, NO₂⁻)	Nitrogen oxidizers	Two-step nitrification: NH₃ → NO₂⁻ → NO₃⁻
Ferrous iron (Fe²⁺)	Iron oxidizers	Oxidize Fe²⁺ to Fe³⁺; challenging because Fe²⁺ spontaneously oxidizes in O₂

• Example: Hydrogen oxidizers use hydrogenase to oxidize H₂; aerobic versions eventually reduce oxygen to water.
• Example: Sulfur oxidizers can oxidize hydrogen sulfide (H₂S) stepwise to sulfate (SO₄²⁻) with sulfite oxidase.

🎯 Electron acceptors

• Chemolithotrophy can occur aerobically or anaerobically.
• Oxygen is the best electron acceptor (creates the biggest distance between donor and acceptor).
• Non-oxygen acceptors allow greater diversity and wider environmental range, but sacrifice energy production.

📉 Energy yield

• ATP generated varies widely depending on the specific donor and acceptor used.
• Always produces less ATP than aerobic respiration (which uses glucose as donor and oxygen as acceptor, yielding up to 32 ATP).
• The smaller the distance between donor and acceptor on the electron tower, the less ATP formed.
• Example: An organism using Fe²⁺ as donor faces unfavorable bioenergetics even with oxygen as acceptor, because Fe²⁺ has a very positive standard reduction potential.

🌱 Carbon metabolism strategies

🌱 Chemolithoautotrophs

Chemolithoautotrophs: chemolithotrophs that are autotrophs, fixing atmospheric carbon dioxide to assemble organic compounds.

• Most chemolithotrophs are autotrophs.
• Require both ATP and reducing power (NADH/NADPH) to convert oxidized CO₂ into reduced organic compounds like glucose.
• Reverse electron flow: if the electron donor has a higher redox potential than NAD⁺/NADP⁺, the organism must push electrons back up the electron tower.
  • This is energetically unfavorable, consuming energy from the PMF to drive electrons in reverse through the ETC.
• Example: An organism using an electron donor with higher potential than NAD⁺ must spend extra energy to generate the reducing power needed for CO₂ fixation.

🔄 Chemolithoheterotrophs (mixotrophs)

Chemolithoheterotrophs (also called mixotrophs): organisms using an inorganic chemical for energy and electrons, but relying on organic chemicals for carbon needs.

• Use inorganic chemicals for energy, but do not fix CO₂.
• Require both inorganic and organic compounds for growth and reproduction.
• Don't confuse: chemolithoautotrophs fix CO₂; chemolithoheterotrophs obtain carbon from organic sources.

🔁 Nitrogen cycle overview

🔁 Why the nitrogen cycle matters

• Nitrogen is an essential element for life (component of amino acids and nucleotides).
• The nitrogen cycle depicts how nitrogen is used and converted by organisms into different chemical forms.
• Most chemical conversions are performed by microbes as part of their metabolism, providing valuable services to other organisms.

🧬 Key nitrogen transformations

The excerpt describes six major nitrogen metabolism processes:

Process	Conversion	Organism type	Purpose
Nitrogen fixation	N₂ → NH₃	Diazotrophs	Make nitrogen usable for life
Assimilation	Inorganic N → organic N	Various microbes	Cellular growth and reproduction
Nitrification	NH₃ → NO₂⁻ → NO₃⁻	Chemolithotrophs	Energy generation
Denitrification	NO₃⁻ → N₂	Denitrifying microbes	Anaerobic respiration
Anammox	NH₃ + NO₂⁻ → N₂	Marine bacteria	Energy generation

🌾 Nitrogen fixation

🌾 What nitrogen fixation is

Nitrogen fixation: the conversion of relatively inert dinitrogen gas (N₂) into ammonia (NH₃), a much more useable form of nitrogen for most life forms.

• Performed by diazotrophs, a limited number of bacteria and archaea.
• Essential for Earth's organisms because nitrogen is required for amino acids and nucleotides.
• No eukaryote is known that can fix nitrogen; plants, animals, and other organisms rely on bacteria and archaea.

⚙️ Requirements and challenges

• Extremely energy and electron intensive to break the triple bond in N₂ and reduce it to NH₃.
• Requires the enzyme nitrogenase, which is inactivated by O₂.
• Nitrogen fixation must occur in an anaerobic environment.
• Aerobic nitrogen-fixing organisms must devise special conditions to protect nitrogenase.

🤝 Symbiotic nitrogen-fixing organisms

• Partner with a plant host to obtain an appropriate environment for nitrogenase.
• Bacteria live in plant tissue, often in root nodules, fixing nitrogen and sharing the results.
• The plant provides the location and additional nutrients to support the energy-taxing process.
• Bacteria and host exchange chemical recognition signals to facilitate the relationship.
• Example: Rhizobium partners with plants of the legume family (clover, soybeans, alfalfa).

🆓 Free-living nitrogen-fixing organisms

• Fix nitrogen for their own use; nitrogen is shared when the organism dies or is ingested.
• Anaerobic free-living organisms do not need special adaptations for nitrogenase.
• Aerobic organisms must make adaptations:
  • Example: Cyanobacteria (multicellular bacterium) make specialized cells called heterocysts where nitrogen fixation occurs.
  • Since Cyanobacteria produce oxygen during photosynthesis, an anoxygenic version occurs within the heterocyst, keeping nitrogenase active.
  • Heterocysts share fixed nitrogen with surrounding cells; surrounding cells provide nutrients to heterocysts.

🔬 Other nitrogen metabolism processes

🔬 Assimilation

Assimilation: a reductive process by which an inorganic form of nitrogen is reduced to organic nitrogen compounds such as amino acids and nucleotides, allowing for cellular growth and reproduction.

• Only the amount needed by the cell is reduced.
• Ammonia assimilation: ammonia (NH₃) or ammonium ion (NH₄⁺) formed during nitrogen fixation is incorporated into cellular nitrogen.
• Assimilative nitrate reduction: multi-step process where nitrate is reduced to nitrite, then ammonia, and finally into organic nitrogen.

♻️ Nitrification (energy generation)

• A 2-step process performed by chemolithotrophs using reduced or partially reduced nitrogen as an electron donor to obtain energy.
• One group performs ammonia oxidation (NH₃ → NO₂⁻).
• A different group performs nitrite oxidation (NO₂⁻ → NO₃⁻).
• A non-nitrogen compound serves as the electron acceptor.
• ATP is gained through oxidative phosphorylation (ETC, PMF, ATP synthase).
• Don't confuse: this is the same nitrification mentioned earlier under nitrogen oxidizers; it's an energy-generating process, not assimilation.

💨 Denitrification

Denitrification: the reduction of NO₃⁻ to gaseous nitrogen compounds, such as N₂.

• Denitrifying microbes perform anaerobic respiration, using NO₃⁻ as an alternate final electron acceptor to O₂.
• This is a type of dissimilatory nitrate reduction: nitrate is reduced during energy conservation, not for making organic compounds.
• Produces large amounts of excess byproducts, resulting in loss of nitrogen from the local environment to the atmosphere.

🌊 Anammox

Anammox (anaerobic ammonia oxidation): performed by marine bacteria that utilize nitrogen compounds as both electron acceptor and electron donor to generate energy.

• Relatively recently discovered.
• In this chemolithotrophic reaction:
  • Ammonia is oxidized anaerobically (electron donor).
  • Nitrite is utilized as the electron acceptor.
  • Dinitrogen gas (N₂) is produced as a byproduct.
• Reactions occur within the anammoxosome, a specialized cytoplasmic structure constituting 50–70% of total cell volume.
• Like denitrification, anammox removes fixed nitrogen from the local environment, releasing it to the atmosphere.

🧭 Overview

🧠 One-sentence thesis

Phototrophy encompasses diverse microbial strategies for converting sunlight into ATP, ranging from simple single-photosystem anoxygenic processes in purple and green bacteria to the two-photosystem oxygenic Z pathway in cyanobacteria that produces oxygen and enabled aerobic life on Earth.

📌 Key points (3–5)

• Phototrophy vs photosynthesis: phototrophy is sunlight-to-ATP conversion; photosynthesis adds CO₂ fixation (photoautotrophs do both; photoheterotrophs only make ATP and use pre-made organics).
• Anoxygenic vs oxygenic: purple and green bacteria use one photosystem without producing oxygen; cyanobacteria use two photosystems (PSII and PSI) and split water, releasing oxygen.
• Cyclic vs non-cyclic photophosphorylation: cyclic returns electrons to the original pigment and repeats indefinitely; non-cyclic diverts electrons elsewhere (e.g., to NADP⁺) and requires an external electron donor.
• Common confusion—reverse electron flow: purple bacteria need it because their excited reaction center (P870) is still not negative enough to reduce NAD(P)⁺ directly; green bacteria avoid it because their initial carrier (ferredoxin) is already more negative than NAD(P)⁺.
• Why it matters: oxygenic photosynthesis by cyanobacteria oxygenated Earth, enabling aerobic respiration; different pigments and wavelengths let multiple phototrophs coexist without competing.

🌈 Pigments and light capture

🌈 Types of pigments

Pigment	Color / wavelengths absorbed	Found in
Chlorophylls	Green (absorb red ≈675 nm and blue ≈430 nm)	Plants, algae, cyanobacteria
Bacteriochlorophylls	Absorb higher wavelengths (≈870 nm)	Purple and green bacteria
Carotenoids	Yellow/orange/red (absorb blue 400–550 nm)	Accessory pigments in many phototrophs
Phycobiliproteins	Phycoerythrin (red) and phycocyanin (blue)	Accessory pigments in some phototrophs

• Why different wavelengths matter: bacteriochlorophylls absorb higher (longer) wavelengths than chlorophylls, so different phototrophs can occupy the same environment without competing for the same light.
• Accessory pigments serve two roles:
  1. Expand the range of wavelengths absorbed → better light utilization.
  2. Act as antioxidants → protect the organism.

📡 Antennae and reaction centers

Antennae: light-harvesting pigments that funnel energy to reaction centers.
Reaction centers: the molecules that actually convert light energy into ATP.

• In bacteria and archaea, pigments are housed in membrane invaginations or in a chlorosome.
• The antennae absorb photons and channel the energy to a special pair of chlorophyll/bacteriochlorophyll molecules in the reaction center.
• Example: many antenna pigments collect light and pass it to one reaction center, amplifying efficiency.

⚡ General photophosphorylation mechanism

⚡ How light becomes ATP

1. Photon absorption: antennae absorb light and funnel energy to the reaction center's special chlorophyll/bacteriochlorophyll pair.
2. Excitation: the molecules become excited → their reduction potential becomes more negative (they "jump up the electron tower").
3. Electron transport chain: excited electrons pass through carriers (e.g., ferredoxin, cytochromes) → proton motive force develops.
4. ATP synthesis: protons flow back through ATPase → ATP is generated.

Photophosphorylation: ATP synthesis driven by sunlight (as opposed to chemical energy).

🔄 Cyclic vs non-cyclic photophosphorylation

Type	Electron fate	External electron donor needed?	Purpose
Cyclic	Electrons return to the original chlorophyll/bacteriochlorophyll pair	No	Repeatable ATP generation
Non-cyclic	Electrons diverted elsewhere (e.g., to NAD(P)⁺)	Yes	Generate reducing power (NADPH) for CO₂ fixation

• Don't confuse: cyclic is self-sustaining for ATP; non-cyclic requires an external electron source to replenish the system.
• Example: a photoautotroph needs both ATP and NADPH to fix CO₂, so it must use non-cyclic at least part of the time.

🟣 Anoxygenic phototrophy (purple and green bacteria)

🟣 Purple phototrophic bacteria

Anoxygenic phototrophy: phototrophy that does not generate oxygen.

• One photosystem with bacteriochlorophyll.
• Reaction center P870: E₀′ = +0.5 V when not excited; after absorbing a photon, E₀′ = −1.0 V.
• Problem for photoautotrophs: −1.0 V is still not negative enough to reduce NAD(P)⁺ (E₀′ = −0.32 V).

🔁 Reverse electron flow in purple bacteria

• What it is: using energy from the proton motive force to push electrons "up" the electron tower to a more negative potential.
• Why needed: to generate NAD(P)H for CO₂ fixation.
• Cost: energetically unfavorable.
• External electron donor required: typically H₂S or elemental sulfur → various sulfur byproducts produced.

🌿 Photoheterotrophy in purple bacteria

• When organic compounds are available, purple bacteria often switch to photoheterotrophy:
  • Use cyclic photophosphorylation → ATP.
  • Get organic compounds from the environment.
  • Advantage: no need for reverse electron flow or external electron donors.

🟢 Green phototrophic bacteria

• Also anoxygenic, with one photosystem and bacteriochlorophyll.
• Use cyclic photophosphorylation for ATP.
• Periodically draw off electrons to NAD(P)⁺ for reducing power (non-cyclic).

✅ No reverse electron flow needed

• Why: the initial carrier, ferredoxin (Fd), has a more negative E₀′ than NAD(P)⁺.
• Electrons can flow "downhill" from ferredoxin to NAD(P)⁺ without extra energy input.
• External electron donor still required: typically H₂S or thiosulfate.

🔀 Alternating strategy

• Green bacteria operate as photoautotrophs by alternating their photosystem use:
  • Sometimes for ATP (cyclic).
  • Sometimes for NADPH (non-cyclic).

🆚 Purple vs green bacteria comparison

Feature	Purple bacteria	Green bacteria
Photosystems	One (P870)	One
Reverse electron flow	Yes (for NAD(P)H)	No (ferredoxin E₀′ is negative enough)
External electron donor	H₂S, S⁰	H₂S, thiosulfate
Photoautotroph strategy	Cyclic + reverse flow + external donor	Alternate cyclic and non-cyclic
Photoheterotroph option	Yes (cyclic only, use environmental organics)	Not emphasized in excerpt

🌊 Oxygenic phototrophy (cyanobacteria and the Z pathway)

🌊 Two-photosystem design

Oxygenic phototrophy: phototrophy that produces oxygen as a byproduct.

• Used by cyanobacteria (and plants) with chlorophyll a.
• Two distinct photosystems: photosystem II (PSII) and photosystem I (PSI), each with separate reaction centers.
• Allows simultaneous generation of ATP and reducing power in one process → facilitates photoautotrophic growth (true photosynthesis).

⚡ The Z pathway step-by-step

1. PSII (P680): light decreases the reduction potential of P680 chlorophyll a molecules.
2. Electron transport chain 1: electrons pass through carriers → proton motive force → ATP via ATPase.
3. Electrons move to PSI: passed from PSII to PSI.
4. PSI (P700): another photon excites electrons again, decreasing their reduction potential even more.
5. Electron transport chain 2: electrons pass through a different chain → eventually reduce NADP⁺ → NADPH.

• Non-cyclic photophosphorylation: electrons do not return to the original photosystem.
• External electron donor required: water (H₂O).

💧 Water splitting and oxygen evolution

• Challenge: water (O₂/H₂O redox couple) is normally a poor electron donor due to its extremely positive reduction potential.
• Solution: P680 chlorophyll a, when not excited, has an even more positive reduction potential → can accept electrons from water.
• Hydrolysis of water: H₂O → electrons + protons + O₂ (oxygen gas released).
• Historical impact: cyanobacteria are thought to have oxygenated Earth ≈3.5 billion years ago, enabling aerobic respiration.

🔬 Anoxygenic mode in heterocysts

• Heterocysts: specialized cyanobacterial cells for nitrogen fixation.
• Problem: oxygen inactivates nitrogenase enzymes.
• Solution: heterocysts degrade PSII → only PSI remains → anoxygenic phototrophy (no oxygen byproduct) while still producing ATP.
• Example: cyanobacteria can switch modes depending on metabolic needs.

🔴 Rhodopsin-based phototrophy (archaea)

🔴 Unique mechanism without chlorophyll

Bacteriorhodopsin (archaearhodopsin): a retinal molecule (related to vertebrate eye pigments) used by some archaea for phototrophy.

• How it works:
  1. Rhodopsin absorbs light.
  2. Undergoes a conformational change.
  3. Pumps a proton across the cell membrane.
  4. Proton motive force develops → ATP synthesis.
• No electron transport chain involved: direct proton pumping.
• Simplicity: bypasses the need for complex pigment systems and electron carriers.

🆚 Comparison with other phototrophy

Feature	Chlorophyll/bacteriochlorophyll-based	Rhodopsin-based
Pigment	Chlorophyll or bacteriochlorophyll	Bacteriorhodopsin/archaearhodopsin
Mechanism	Electron excitation → electron transport chain → PMF	Direct proton pumping via conformational change
Organisms	Bacteria (purple, green), cyanobacteria, plants	Archaea
Complexity	Requires antennae, reaction centers, carriers	Simpler: one molecule does it all

• Don't confuse: rhodopsin-based phototrophy is not photosynthesis (no CO₂ fixation mentioned); it is purely ATP generation.

🌍 Evolutionary and ecological context

🌍 Metabolic diversity timeline (from excerpt)

• Early Earth: hot, anoxic, reduced inorganic chemicals abundant.
• Primitive cells: likely had simple electron transfer systems → proton motive force → energy conservation.
• Chemolithoautotrophs: proliferated → organic material accumulated.
• Chemoorganotrophs: evolved to oxidize organics → longer electron transport chains → faster growth.
• ≈3.5 billion years ago: phototrophic pigments evolved → anoxygenic phototrophy (using sulfur products as electron donors).
• Later: oxygenic photosynthesis (cyanobacteria) → oxygen accumulation → aerobic respiration became possible.

🌱 Ecological coexistence

• Different pigments absorb different wavelengths: chlorophylls (≈675 nm, 430 nm) vs bacteriochlorophylls (≈870 nm).
• Result: multiple phototrophs can occupy the same environment without direct competition for light.
• Example: green bacteria and purple bacteria can coexist in the same water column by using different parts of the light spectrum.

🌏 Cyanobacteria's legacy

• Oxygenation of Earth: oxygenic photosynthesis by cyanobacteria released O₂ as a byproduct.
• Enabled aerobic respiration: oxygen became available as a terminal electron acceptor → much more ATP per glucose.
• Evolutionary milestone: paved the way for complex multicellular life.

🧭 Overview

🧠 One-sentence thesis

Microbial evolution from Earth's earliest cells to modern diversity explains how metabolic pathways, photosynthesis, and eukaryotic organelles arose, and molecular phylogeny now allows us to classify organisms based on their evolutionary relationships.

📌 Key points (3–5)

• Timeline: Earth is 4.6 billion years old; first cells appeared ~3.8 billion years ago under hot, anoxic conditions with reduced inorganic chemicals.
• Major evolutionary milestones: RNA world → metabolic diversity → anoxygenic phototrophy (~3.5 bya) → oxygenic photosynthesis (~2.5–3.3 bya, Great Oxidation Event) → ozone shield (~2 bya) → endosymbiosis (eukaryotic organelles).
• Endosymbiosis: chloroplasts and mitochondria originated from ingested free-living bacteria that became mutually dependent organelles.
• Molecular phylogeny: uses ribosomal RNA (rRNA) sequences as a "molecular chronometer" because they change slowly and resist horizontal gene transfer, enabling accurate evolutionary comparisons.
• Common confusion: taxonomy vs phylogeny—taxonomy is the organization and naming of organisms; phylogeny is the evolutionary relationships among them; modern classification uses a polyphasic approach combining phenotype, genotype, and evolutionary data.

🌍 Early Earth and the origin of life

🔥 Conditions on early Earth

• Earth formed 4.6 billion years ago; first cells appeared ~3.8 billion years ago.
• Early conditions: extremely hot, anoxic (no oxygen), abundant reduced inorganic chemicals.
• Initial cells were likely adapted to these harsh conditions.

🧬 RNA world hypothesis

RNA world: a hypothesized stage in which self-replicating RNA both stored genetic information and catalyzed protein synthesis.

• RNA plays crucial roles in modern cells; the hypothesis suggests it was even more central in primitive cells.
• Over time, proteins took over catalytic functions and DNA became the primary information storage molecule.
• Example: early cells might have relied on RNA for both "instructions" and "tools," before specialization into DNA (storage) and proteins (catalysis).

🔄 Evolution of metabolism and energy systems

⚡ Early metabolic systems

• Initial cells probably had a primitive electron transport system—perhaps just one carrier—that still generated a proton motive force to conserve energy.
• As chemolithoautotrophs (organisms using inorganic chemicals for energy and CO₂ for carbon) proliferated, organic material accumulated in the environment.

🍃 Development of chemoorganotrophy

• Accumulation of organic material enabled chemoorganotrophic organisms (those oxidizing organic compounds).
• Organic compounds have more negative redox potentials and more electrons, likely lengthening electron transport chains.
• Result: faster growth and accelerated diversification.

☀️ Phototrophy and the Great Oxidation Event

🌅 Anoxygenic phototrophy (~3.5 billion years ago)

• Some cells evolved phototrophic pigments, converting light energy into chemical energy.
• Initially used anoxygenic phototrophy: sulfur products served as electron donors during CO₂ fixation (no oxygen produced).

🪨 Stromatolites as evidence

Stromatolites: layered rocks formed when minerals are incorporated into thick microbial mats growing on water surfaces.

• Ancient stromatolites contain fossilized microbial mats of cyanobacteria-like cells, indicating their presence early in Earth's history.

💨 Oxygenic photosynthesis and the Great Oxidation Event (~2.5–3.3 billion years ago)

• Cyanobacterial ancestors developed oxygenic photosynthesis by acquiring two photosystems and chlorophyll a.
• Water became the electron donor, producing oxygen as a byproduct.
• Great Oxidation Event: oxygen accumulated in Earth's atmosphere, fundamentally changing possible metabolisms and allowing oxygen to be used as a final electron acceptor.

🛡️ Ozone shield formation (~2 billion years ago)

Ozone shield: a layer of ozone (O₃) around Earth that blocks much ultraviolet (UV) radiation from the sun.

• UV radiation damages DNA; the ozone layer made surface habitats viable.
• Mechanism: accumulated O₂ was converted to O₃ when exposed to UV light.
• Result: organisms could inhabit Earth's surface instead of only ocean depths or soil layers.

🔬 Endosymbiosis and the origin of eukaryotes

🤝 The endosymbiotic theory

Endosymbiosis: the process by which a eukaryotic ancestor arose when a cell ingested a free-living bacterium but did not digest it, and the two became mutually dependent.

• The ingested cell (endosymbiont) had capabilities the proto-eukaryotic cell lacked:
  • Phototrophy → became chloroplasts (derived from cyanobacterial ancestors).
  • Oxidative phosphorylation → became mitochondria (derived from gram-negative bacillus ancestors).
• Over time, the endosymbiont became an organelle.

🧪 Evidence for endosymbiosis

Mitochondria and chloroplasts share these bacterial traits:

Feature	Description
Chromosome	Single, circular (like bacteria)
Reproduction	Binary fission, separate from the eukaryotic cell
Ribosomes	70S sized (bacterial type)
Membrane	Lipid bilayer with 2:1 protein-to-lipid ratio
rRNA sequences	Phylogenetically group with bacteria (most important)

• Don't confuse: organelles are not independent bacteria today; they are mutually dependent components of eukaryotic cells, but their ancestry is bacterial.

🌳 Phylogeny and molecular techniques

🧬 What is phylogeny?

Phylogeny: the evolutionary development of an organism.

• Molecular phylogeny: uses genomes or ribosomal RNA (rRNA) nucleotide sequences to assess evolutionary relationships, generally believed to provide the most accurate information about microbial relatedness.

🔗 DNA-DNA hybridization

Nucleic acid hybridization (DNA-DNA hybridization): a technique comparing genome similarities by melting (heating) DNA strands, then cooling to allow complementary strands to re-anneal.

• Strands with complementary sequences re-anneal; non-complementary strands remain unpaired.
• Typically one DNA source is labeled (e.g., with radioactivity) to identify each source.
• Used to measure genetic similarity between organisms.

📜 Nucleic acid sequencing

• Direct comparison of sequences, typically using rRNA from small ribosomal subunits.
• Why rRNA is ideal:
  • Genes encoding it change very slowly over time.
  • Not strongly influenced by horizontal gene transfer.
  • Serves as a molecular chronometer: tracks genetic changes over long periods, even between closely related organisms.

🌲 Phylogenetic trees

Phylogenetic tree: a pictorial representation of how organisms are believed to be related evolutionarily.

Component	Definition
Root	The last common ancestor for the organisms being compared (LUCA = Last Universal Common Ancestor for all living cells)
Internal node (branchpoint)	Represents a divergence event based on a genetic change
Branch	Connects nodes; length can indicate the amount of molecular changes over time (if scaled)
External node	Represents specific taxa, organisms, or genes
Clade	A group of organisms sharing a particular common ancestor

• Example: if two organisms share a recent internal node, they diverged more recently and are more closely related.

📚 Taxonomy and classification systems

🏷️ What is taxonomy?

Taxonomy: the organization of organisms based on their relatedness, involving classification schemes, identification of isolates, and nomenclature (naming).

• Taxonomy is the system for organizing and naming; phylogeny is the evolutionary relationships that inform taxonomy.

🗂️ Classification systems

System	Basis	What it uses
Phenetic	Phenotypes or physical appearances	Observable traits
Phylogenetic	Evolutionary relationships	Ancestry and divergence
Genotypic	Genes or genomes	DNA/RNA sequences
Polyphasic (most popular)	Combination of all three	Phenotype + genotype + evolutionary data

• Don't confuse: phenetic looks at appearance, phylogenetic at ancestry, genotypic at genes; polyphasic integrates all three for a comprehensive view.

🦠 Microbial species definition

• No widely accepted single definition exists for microbial species.

• Most common approach: polyphasic, using both genetic and phenotypic information.

• Threshold criteria for two organisms to belong to the same species:

  • ≥70% DNA-DNA hybridization.
  • ≥97% 16S rRNA sequence identity.

• Example: two bacterial isolates with 75% DNA-DNA hybridization and 98% 16S rRNA identity would be considered the same species; isolates with 65% and 95% would not.

🧭 Overview

🧠 One-sentence thesis

Bacteria and archaea acquire genetic variability and new genes through horizontal gene transfer mechanisms—conjugation, transformation, and transduction—which allow them to survive environmental challenges without sexual reproduction.

📌 Key points (3–5)

• Why HGT matters: Bacteria lack sexual reproduction, so horizontal gene transfer (HGT) is their primary means of acquiring new genes for survival (e.g., breaking down nutrients or resisting antibiotics).
• Three HGT mechanisms: conjugation (plasmid transfer via pilus), transformation (uptake of naked DNA from environment), and transduction (virus-mediated gene transfer).
• Common confusion: Transformation vs conjugation—transformation does not require cell-to-cell contact; DNA comes directly from the environment, whereas conjugation requires physical contact via a pilus.
• Recombination is essential: For transformation and transduction, acquired DNA must be recombined into the recipient's chromosome (via RecA or site-specific recombination) to be expressed.
• Transposable elements: "Jumping genes" can activate or inactivate genes by moving within a DNA molecule, adding another layer of genetic variability.

🔄 Horizontal Gene Transfer mechanisms

🔗 Conjugation

Conjugation: the process by which a donor bacterium transfers a copy of a plasmid to a recipient bacterium through a pilus.

• Requires cell-to-cell contact.
• Key players:
  • Donor cell (F+): has a conjugative plasmid coding for pilus proteins.
  • Recipient cell (F-): lacks the conjugative plasmid.
  • Pilus: threadlike filament that binds the recipient and brings it close to the donor.
• How it works:
  1. Pilus binds the F- cell.
  2. A channel opens between the two cells.
  3. A single-stranded DNA copy of the plasmid enters the recipient.
  4. Both cells synthesize the complementary strand, resulting in two F+ cells.
• Why it matters: The plasmid carries its own promoters, so genes can be expressed immediately without recombination into the chromosome.

🧬 Transformation

Transformation: the process by which a bacterial cell acquires new genes directly from the environment, without cell-to-cell contact.

• Does not require cell-to-cell contact.
• Key components:
  • Naked DNA: DNA released into the environment, typically from a lysed donor cell.
  • Competent cell: a recipient cell capable of taking up DNA from the environment.
• Natural competence:
  • Determined genetically.
  • Typically occurs at the end of exponential phase or beginning of stationary phase, under high cell density and limited nutrients.
  • Specific proteins are made: DNA-binding proteins (DNA translocase), endonucleases, transmembrane channel proteins, and (in Gram-negative cells) cell wall autolysin.
• How it works:
  1. Random DNA pieces bind to receptors on the cell surface.
  2. DNA translocase transports DNA through the transmembrane channel.
  3. Endonuclease may degrade one strand of dsDNA (if only ssDNA can enter) or cleave DNA into smaller fragments.
  4. Inside the cell, DNA must be incorporated into the chromosome by RecA for gene expression.
• Don't confuse: Transformation brings in "naked" environmental DNA; conjugation transfers plasmid DNA through direct contact.

🦠 Transduction

Transduction: the use of a virus (bacteriophage) to shuttle bacterial genes from one cell to another, eliminating the need for cell-to-cell contact.

• Two types: generalized transduction and specialized transduction.

🔀 Generalized transduction

• Can occur with: virulent or temperate bacteriophage in the lytic cycle.
• How it works:
  1. Bacteriophage infects a host cell (absorption, penetration, synthesis).
  2. During assembly, random pieces of bacterial DNA are mistakenly packaged into a phage head, creating a transducing particle.
  3. The transducing particle binds to a new host cell and injects the bacterial DNA.
  4. If the DNA is incorporated into the recipient's chromosome, the genes can be expressed.
• Key point: The transducing particle is not a functional virus; it carries bacterial DNA instead of viral DNA.

🎯 Specialized transduction

• Can occur only with: temperate bacteriophage (involves lysogenic cycle).
• How it works:
  1. Viral DNA integrates into the host chromosome, forming a prophage.
  2. During induction, the prophage is excised from the chromosome.
  3. Excision error: bacterial genes immediately adjacent to the viral genes are excised along with the viral DNA.
  4. All resulting virions contain a hybrid of viral and bacterial DNA.
  5. These virions infect new host cells, injecting the hybrid DNA, which can be incorporated if a prophage forms.
• End result: The second host cell can contain its own DNA, DNA from the first host, and viral DNA.
• Don't confuse: Generalized transduction packages random bacterial DNA; specialized transduction packages specific bacterial genes adjacent to the prophage insertion site.

🧩 Molecular recombination

🔄 Why recombination is needed

• In conjugation, genes are on a plasmid with their own promoters, so they can be expressed immediately.
• In transformation and transduction, naked DNA enters the cell and could be degraded unless it is recombined with the recipient's chromosome.

🧬 Homologous recombination

Homologous recombination: the most common mechanism of molecular recombination, involving the RecA protein.

• How it works:
  1. DNA from two sources are paired based on similar nucleotide sequences.
  2. An endonuclease nicks one strand.
  3. RecA pairs bases from different strands (strand invasion).
  4. The cross-over is resolved by resolvase, which cuts and rejoins the DNA into two separate dsDNA molecules.
• Key requirement: Similar sequences in the donor and recipient DNA.

🎯 Site-specific recombination

• Used by: viruses to insert their genome into the host chromosome; also used by transposable elements.
• Does not require extensive sequence similarity (unlike homologous recombination).

🧬 Transposable elements

🧬 What are transposable elements?

Transposable elements (or "jumping genes"): DNA sequences that can move from one location to another within a DNA molecule by a process known as transposition.

• Role: Can activate or inactivate genes within an organism.
• Structure:
  • Code for transposase (the enzyme that allows transposition).
  • Have short inverted repeats (IRs) at each end.

🔀 Types of transposable elements

Type	Structure	Transposition mechanism
Insertion sequence (IS)	Contains transposase and IRs of varying lengths	Simplest transposable element
Transposon	Contains transposase, IRs, and additional genes (type varies)	Can use conservative or replicative transposition

🔄 Transposition mechanisms

• Conservative transposition (cut-and-paste model):
  • The transposon is removed from one location and relocated to another.
• Replicative transposition:
  • The transposon is copied; the copy is inserted at a second site while the original remains.

🧪 Why transposable elements matter

• They can activate or inactivate genes depending on where they insert.
• Example: If a transposon inserts into the middle of a gene, it may disrupt that gene's function; if it inserts near a promoter, it may activate a nearby gene.
• Historical note: Barbara McClintock won the Nobel Prize in 1983 for demonstrating this in corn.

🧭 Overview

🧠 One-sentence thesis

Genetic engineering deliberately manipulates DNA in the laboratory to alter genes in organisms, often using microbial substances and techniques adapted for more complex organisms, with applications ranging from DNA replication to protein production.

📌 Key points (3–5)

• Core process: Gene cloning involves isolating target DNA, cutting it with restriction enzymes, combining it with vector DNA using ligase, and introducing the recombinant DNA into host cells.
• Key distinction: Restriction endonucleases can make blunt cuts or staggered cuts; staggered cuts produce "sticky ends" that are invaluable for cloning because unpaired bases recombine with complementary sequences.
• Multiple delivery methods: Introducing recombinant DNA into cells varies by organism—bacteria use transformation or electroporation; plants and animals may use Ti plasmids, gene guns, viral vectors, or CRISPR-Cas9.
• Common confusion: A genomic library contains many recombinant DNA combinations (not just the desired one); shotgun cloning with random fragments can yield thousands of clones requiring screening.
• Practical applications: GMOs can serve as sources of DNA, RNA, or proteins of value (e.g., insulin, human growth hormone, antisense RNA for disease prevention).

🧬 The gene cloning workflow

🎯 Isolate target DNA

• First, identify the gene(s) of interest—the target DNA.
• Example: To make E. coli glow in the dark, find an organism with that trait (e.g., jellyfish with green fluorescent protein, GFP) and identify the responsible gene.

Cloning vector: typically a plasmid or virus capable of independent replication that stably carries target DNA from one location to another.

• Plasmid vectors are available from bacteria and yeast.
• The vector acts as a vehicle to replicate and transport the target DNA.

✂️ Cut DNA with restriction endonucleases

Restriction endonucleases: enzymes that recognize short DNA sequences (4–8 bp long) that are palindromic inverted repeats (read the same on each strand in the 5' to 3' direction).

• Both target DNA and vector DNA are cut with the same restriction enzyme.
• Widespread in bacteria and archaea; each enzyme recognizes a specific sequence.

Two types of cuts:

Cut type	Description	Cloning value
Blunt cut	Straight across the DNA	Less useful for recombination
Staggered cut	Produces short single-stranded regions on each strand	Creates "sticky ends"

Sticky ends: very short single-stranded DNA regions produced by staggered cuts; unpaired bases will recombine with any DNA having the complementary base sequence.

• Sticky ends are invaluable in molecular cloning because they allow precise pairing with complementary sequences.

🔗 Combine target and vector DNA

• After both DNAs are cleaved by the same restriction enzyme, mix them together.
• Add DNA ligase, an enzyme that repairs the covalent bonds on the sugar-phosphate backbone.

Recombinant DNA (also called chimeras): DNA molecules that contain DNA from two or more sources.

• The result is stable combination of target DNA inserted into vector DNA.

🚪 Introduce recombinant DNA into host cells

• The recombinant DNA must enter a host cell so genes can be replicated or expressed.
• Method depends on the complexity of the host organism.

For bacteria:

• Transformation: use competent cells to pick up recombinant DNA molecules (often the easiest method).
• Electroporation: expose cells to a brief pulse of high-voltage electricity, making the plasma membrane temporarily permeable to DNA.

Challenge: mixed outcomes

• Some cells acquire the desired configuration (target + vector).
• Others get alternate combinations (plasmid + plasmid, or target + target).

Genomic library: the mixture of cells carrying various recombinant DNA combinations; must be screened to select the appropriate clone.

Shotgun cloning: when random DNA fragments are used instead of isolated target genes; can yield thousands or tens of thousands of clones to screen.

• Don't confuse: a genomic library is not a pure collection of the desired clone—it requires screening to find the right one.

🌱 Delivery methods for non-bacterial cells

🦠 Agrobacterium tumefaciens and the Ti plasmid

• Agrobacterium tumefaciens is a plant pathogen causing crown gall disease (tumor formation).

Ti (tumor inducing) plasmid: a plasmid that inserts bacterial DNA into the host plant genome.

• Scientists exploit this natural process for plant genetic engineering:
  • Insert foreign DNA into the Ti plasmid.
  • Remove genes necessary for disease.
  • Result: production of transgenic plants without causing disease.

🔫 Gene gun

Gene gun: uses very small metal particles (microprojectiles) coated with recombinant DNA, blasted at plant or animal tissue at high velocity.

• If the DNA is taken up by the cell's DNA, the genes are expressed.
• Physical method suitable for both plant and animal tissue.

🦠 Viral vectors

Viral vector: virulence genes are removed from a virus and foreign DNA is inserted, allowing the virus capsid to shuttle genetic material into plant or animal cells.

• Marker genes are typically added to identify cells that took up the genes.
• Exploits the virus's natural ability to enter cells.

✂️ CRISPR-Cas9 genome editing

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats): a family of DNA sequences commonly found in bacterial and archaeal genomes, originally from lysogenic bacteriophage.

• Cells use these DNA fragments combined with Cas9 (CRISPR-associated protein 9) enzyme to identify and destroy similar DNA, preventing subsequent bacteriophage infections.
• This natural gene editing process can directly edit genomes in eukaryotic organisms.
• Typical uses: deleting a gene or inserting a gene.

🧪 DNA manipulation techniques

🧫 Gel electrophoresis

Gel electrophoresis: a technique to separate nucleic acid fragments based on size.

How it works:

• Prepare a porous agarose gel (concentration adjusted based on expected fragment size).
• Deposit nucleic acid samples into wells in the gel.
• Apply electrical current.
• Nucleic acid (negatively charged) moves toward the positive electrode (placed at the bottom).
• Smallest pieces encounter least resistance and move fastest.

Uses:

• Identify particular fragments.
• Verify that a technique was successful.
• Compare fragment length to a DNA ladder (fragments of known size).

🔁 Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR): a method to copy or amplify DNA in vitro; can yield a billionfold copies of a single gene within a short period.

Required components:

• Template DNA: the original DNA to be copied.
• Primers: small oligonucleotides that flank the gene(s) of interest by recognizing sequences on either side.
• Nucleotides: the building blocks of DNA.
• DNA polymerase: the enzyme that synthesizes new DNA strands.

Three-step cycle (repeated 20–30 times):

Step	Temperature	What happens
Denature	High heat	Separate the DNA strands
Anneal	Lower temperature	Primers bind to template DNA
Extend	Moderate heat	DNA polymerase extends primers using original DNA as template

• The cycle exponentially increases the amount of target DNA in a few hours.

🎯 Applications of genetically modified organisms

🧬 Source of DNA

• GMOs can rapidly replicate a piece of DNA, providing a large source.
• Example: A breast cancer gene spliced into E. coli allows rapid production of the gene for sequencing, study, and manipulation without repeated human tissue donations.

📜 Source of RNA

Antisense RNA: single-stranded RNA complementary to the mRNA that codes for a protein; made by cells as a way to control target genes.

• Increasing interest in using antisense RNA to prevent diseases caused by production of a particular protein.
• The antisense RNA binds to the target mRNA, blocking protein production.

🧪 Source of protein

• Microbes replicate rapidly, making them advantageous for manufacturing proteins of interest.
• With the right promoters, bacteria will express genes for proteins not naturally found in bacteria (e.g., cytokine).

Examples of proteins produced by genetically engineered cells:

• Insulin
• Human growth hormone
• Wide variety of other proteins of use to humans

Genetically modified organism (GMO) or transgenic organism: a genetically modified organism that contains a gene from a different organism.

• Typically created so the GMO will provide needed information or a product of value to society.

🧭 Overview

🧠 One-sentence thesis

Genomics studies entire genomes through sequencing and computational analysis to understand not only what genes an organism has but also how those genes are expressed and function in context.

📌 Key points (3–5)

• What genomics encompasses: sequencing, analyzing, and comparing the complete DNA collection of organisms, with microbes being especially well-studied due to their smaller genome size.
• Sequencing evolution: methods have progressed from shotgun sequencing (random fragments aligned by computer) to second-generation (massively parallel), third-generation (single molecules), and fourth-generation (non-optical detection).
• Bioinformatics role: computers analyze sequences to locate genes (genome annotation), identify open reading frames (ORFs), and compare against databases like GenBank using tools like BLAST.
• Common confusion: comparative genomics (what genes exist and how genomes differ) vs functional genomics (what genes actually do when expressed—transcriptome and proteome).
• Why it matters: genomics reveals not just gene sequences but also gene expression patterns, protein functions, metabolic pathways, and even entire environmental communities without culturing organisms.

🧬 Sequencing methods

🧬 Shotgun sequencing

Shotgun sequencing: a method that breaks the genome into randomly sized fragments, inserts them into vectors to create a genomic library of clones, sequences the fragments, and uses computer analysis to find overlapping regions and assemble the complete genome.

• The genome is fragmented randomly, not systematically.
• Overlapping sequences are aligned by computer to form longer stretches.
• Why coverage matters: many clones contain identical or near-identical sequences, reducing errors through redundancy.
• Example: if Fragment A ends with "ATCG" and Fragment B starts with "ATCG," the computer infers they overlap and joins them into a longer sequence.

🔬 Second-generation sequencing

Second-generation DNA sequencing: uses massively parallel methods where multiple samples are sequenced side-by-side.

• DNA fragments (a few hundred bases each) are amplified by PCR.
• Each fragment is attached to a small bead, so each bead carries several copies of the same DNA section.
• Beads are placed into a plate with more than a million wells (one bead per well), and all fragments are sequenced simultaneously.
• Key advantage: parallelism—many sequences are read at once, greatly increasing throughput.

🧪 Third- and fourth-generation sequencing

Generation	Key feature
Third	Sequences single molecules of DNA (no amplification needed)
Fourth	"Post-light sequencing"—uses methods other than optical detection

• Third-generation removes the need for PCR amplification, working directly with individual DNA molecules.
• Fourth-generation explores alternative detection technologies beyond light-based systems.

🖥️ Bioinformatics and gene identification

🖥️ What bioinformatics does

Bioinformatics: a field combining biology, computer science, and statistics to use computers to analyze information in genomic sequences.

• After sequencing, raw data must be interpreted.
• Genome annotation: locating specific genes within a genome.
• Computers handle the vast scale—the excerpt notes that the biggest bottleneck is not lack of data but lack of computing power to process it.

🧩 Open reading frames (ORFs)

Open reading frame (ORF): a possible protein-coding gene, typically with at least 100 codons before a stop codon and 3' terminator sequences.

• For double-stranded DNA, there are six reading frames (two strands × three possible starting positions per strand).
• DNA is read in sets of three bases (codons) at a time.
• Functional ORF: an ORF that the organism actually uses to encode a protein (not all ORFs are functional).
• Computers search DNA sequences for ORFs; bioinformaticists then analyze which ones likely encode proteins.

🔍 GenBank and BLAST

• GenBank: a database of over 200 billion base pairs of sequences that scientists can access.
• BLAST (basic local alignment search tool): a database search tool with programs for comparing both nucleotide and amino acid sequences.
• BLAST ranks results in order of decreasing similarity, helping identify matches to sequences of interest.
• Example: a scientist finds an unknown ORF and uses BLAST to compare it against GenBank; if a high-similarity match is found, the ORF likely codes for a similar protein.

🧬 Comparative genomics

🧬 What comparative genomics reveals

Comparative genomics: assessing genomes for information about size, organization, and gene content by comparing multiple organisms or strains.

• Helps understand what genes organisms share and what makes them different.
• Provides insight into genes acquired through horizontal gene transfer.

🧩 Core genome vs pan genome

Term	Definition	What it tells us
Core genome	Genes coding for essential cellular functions shared by all strains in a group	What is universal and necessary
Pan genome	All genes found in all members of a species	The full diversity of the group

• Most "extra" genes (beyond the core) are probably picked up by horizontal gene transfer.
• Example: multiple bacterial strains of the same species all share core genes for basic metabolism, but individual strains may have additional genes for antibiotic resistance or toxin production.

🧬 Paralogs vs orthologs

• Paralogs: genes within a single organism that likely arose from gene duplication.
  • Often one copy is altered to take on a new function.
• Orthologs: genes in different organisms that arose from gene duplication in a common ancestor.
• Don't confuse: paralogs are within one organism (duplication within a lineage); orthologs are across organisms (inherited from a shared ancestor).

🧪 Functional genomics

🧪 What functional genomics studies

Functional genomics: placing genomic information in context by understanding what the cell does with its genes—i.e., what happens when genes are expressed.

• Sequence and gene location are only part of the picture.
• Functional genomics examines the transcriptome (all RNA a cell can make) and proteome (all proteins encoded by the genome).
• Goal: understand gene expression in real cellular conditions.

🧬 Microarrays (gene chips)

Microarrays or gene chips: solid supports with multiple spots of DNA arranged in a grid, where each spot represents a single gene or ORF.

• Known fragments of nucleic acid are labeled and used as probes.
• A signal is produced if binding occurs (indicating the gene is present or expressed).
• Application: determine which genes are turned on or off under particular conditions.
• Example: compare a bacterial pathogen's gene expression inside the host versus outside—microarrays reveal which genes are active in each environment.

🧬 Proteomics

Proteomics: the study of an organism's proteome (all its proteins).

• Functional proteomics: examines protein functions and how proteins interact with one another.
• Two-dimensional gel electrophoresis: a common technique that first separates proteins by isoelectric point (using a pH gradient based on amino acid content), then by size (using a polyacrylamide gel).
• Structural proteomics: focuses on three-dimensional protein structure, often determined by computer modeling that predicts folding based on amino acid sequences and known patterns.

🧪 Metabolomics

Metabolomics: identifying the complete set of metabolic intermediates produced by an organism.

• Extremely complicated because many metabolites are used in multiple pathways.
• Provides a snapshot of cellular metabolism beyond just genes and proteins.

🌍 Metagenomics

🌍 What metagenomics does

Metagenomics or environmental genomics: extracting pooled DNA directly from a specific environment without first isolating and identifying individual organisms.

• Allows study of all organisms in an environment, including those difficult or impossible to culture in the lab.
• Scientists can consider the entire microbial community at once.

🧬 Metagenome and phylotype

• Metagenome: the collective genetic material from an environmental sample.
• Phylotype: a taxon identified using nucleic acid sequences alone, in the absence of organism isolation.
• Example: soil DNA is extracted and sequenced; computer analysis reveals sequences from dozens of bacterial species, some of which have never been cultured, each identified as a phylotype.

🧭 Overview

🧠 One-sentence thesis

Symbiotic relationships between microbes and other organisms range from mutually beneficial partnerships to harmful infections, with profound impacts on human health, industrial systems, and microbial survival strategies.

📌 Key points (3–5)

• What symbiosis means: an intimate relationship between two organisms that can be beneficial, neutral, or harmful to either partner.
• The human microbiome: trillions of microbes (10^14) living in and on humans play essential roles in digestion, nutrition, and potentially influence obesity and disease.
• Biofilms as microbial communities: complex aggregations of cells encased in a matrix that provide protection, nutrient access, and communication advantages, making them highly resistant to antibiotics.
• Common confusion: symbiosis vs mutualism—symbiosis is the general term for any intimate relationship; mutualism specifically means both partners benefit.
• Quorum sensing mechanism: bacteria communicate using chemical signals (autoinducers) to coordinate gene expression only when population density reaches a threshold, avoiding wasted energy at low densities.

🤝 Types of Symbiotic Relationships

🤝 Defining symbiosis

Symbiosis: an intimate relationship between two organisms.

• The term itself does not specify whether the relationship is good, bad, or neutral for either partner.
• Many people mistakenly use "symbiosis" to mean only mutually beneficial relationships, but the excerpt clarifies it encompasses all types of intimate partnerships.

🌟 Three relationship categories

Relationship Type	Definition	Benefit Distribution
Mutualistic	Both partners benefit	Win-win
Commensalistic	One partner benefits, the other is unaffected	Win-neutral
Pathogenic	One partner benefits at the expense of the other	Win-lose

• Don't confuse: All three are forms of symbiosis; the term "symbiosis" alone doesn't tell you who benefits.

🦠 The Human Microbiome

🦠 What the microbiome includes

Human microbiome: the genes associated with all the microbes that live in and on a human.

• Total population: 10^14 microbes (trillions).
• Types: mostly bacteria, but also archaea, fungi, and eukaryotic microbes.
• Locations: skin, upper respiratory tract, stomach, intestines, and urogenital tracts.
• Colonization begins soon after birth through contact with people, surfaces, and objects.

🍽️ Gut microbes and metabolism

• Most human-associated microbes live in the gut, with populations dramatically increasing 1-4 hours after eating.
• Estimated 500-1000 bacterial species in the gastrointestinal tract (often described as 5-8 pounds of bacteria).

Essential digestive functions:

• Break down carbohydrates that humans cannot digest on their own.
• Liberate short-chain fatty acids from indigestible dietary fibers.
• Produce vitamins such as biotin and vitamin K.

Example: Without gut microbes, humans would be unable to extract nutrients from certain plant fibers, losing access to important energy sources and vitamins.

⚖️ Gut microbes and obesity

• Research shows obese mice have different gut microbial communities compared to non-obese mice.
• Obese mice have more Firmicutes bacteria and methanogenic Archaea.
• Hypothesis: these microbes may be more efficient at absorbing nutrients.
• Important: The excerpt states this is "currently hypothetical"—the role is suggested but not proven.

🏥 Microbiome and disease

Dysbiosis: microbiota changes associated with diseased states.

• Preliminary research links microbiota to:
  • Rheumatoid arthritis
  • Colorectal cancer
  • Diabetes
  • Obesity

Research context:

• The Human Microbiome Project (HMP): international research program (U.S.-based) with ~200 researchers.
• Used advanced DNA-sequencing to determine which microbes are present and their population levels.
• Current research focuses on the microbiome's role in both health and disease.
• The excerpt emphasizes knowledge is still growing.

🧱 Biofilms: Microbial Communities

🧱 What biofilms are

Biofilms: a complex aggregation of cells encased within an extracellular matrix and attached to a surface.

• Can form on almost any surface.
• Common locations: rocks, caves, pipes, boat hulls, cooking vessels, medical implants.
• Ancient: fossil record shows evidence going back 3.4 billion years.
• Usually contain many different bacterial species, each influencing others' gene expression and growth.

🔄 Four stages of biofilm development

🔄 Stage 1: Cell deposition and attachment

Planktonic cells: free-floating cells.

• Planktonic cells must collide with a suitable surface.
• Surfaces are typically preconditioned with environmental proteins and other molecules.

🔄 Stage 2: Colonization

• Cell-to-cell signaling occurs, leading to expression of biofilm-specific genes.
• These genes are associated with communal production of extracellular polymeric substances (EPS).
• DNA released by some cells can be taken up by others, stimulating new gene expression.

🔄 Stage 3: Maturation

• The EPS matrix fully encases all cells.
• Biofilm continues to thicken and grow, forming a complex, dynamic community.
• Water channels form throughout the structure.

🔄 Stage 4: Detachment and sloughing

• Individual cells or pieces of the biofilm are released to the environment as active dispersal.
• Can be triggered by environmental factors such as nutrient or oxygen concentration.

🛡️ Why cells form biofilms

Protection advantages:

• Increased protection from harmful conditions: UV light, physical agitation, antimicrobial agents, and phagocytosis.
• Bacteria within biofilms are up to 1,000 times more resistant to antibiotics than free-floating cells.

Nutrient access:

• Allows cell populations to "put down roots" and stay near nutrient-rich areas.
• Example: A biofilm on a dairy plant pipe has continual access to fresh food instead of being swept away with the final product.

Community benefits:

• Easy cell-to-cell communication and genetic exchange within microbial populations.

⚠️ Biofilm impacts on humans

Medical problems:

• Medical implants (catheters, artificial joints) are particularly susceptible to biofilm formation.
• Responsible for many chronic infections due to increased resistance to antimicrobial compounds and antibiotics.
• Dental plaque is a biofilm that affects almost everyone and can lead to cavity formation.

Industrial problems:

• Affect industries relying on pipes to convey water, food, oil, or other liquids.
• Their resistance makes biofilms particularly difficult to completely eliminate.

📡 Quorum Sensing: Bacterial Communication

📡 What quorum sensing is

Quorum sensing: the ability of some bacteria to communicate in a density-dependent fashion, allowing them to delay activation of specific genes until it is most advantageous for the population.

• The term "quorum" refers to having a minimum number of members needed for an organization to conduct business.
• Bacteria use this to coordinate behavior based on population density.

🔬 How the mechanism works

The autoinducer system:

Autoinducers: small diffusible substances used for cell-to-cell communication.

Step-by-step process:

1. A cell produces an autoinducer molecule.
2. The autoinducer diffuses across the plasma membrane and is released into the environment.
3. As the cell population increases, the concentration of autoinducer increases.
4. Once a threshold concentration is reached, the molecule binds to specific cellular receptors.
5. The autoinducer diffuses into the cell, often binding to a specific transcription factor.
6. This produces a conformational change allowing the transcription factor to bind to the cell's DNA.
7. Binding triggers expression of specific genes.

Why it matters: Cells avoid wasting energy on activities that only work at high population densities.

💡 Example: Bioluminescent bacteria and the bobtail squid

The mutualistic relationship:

• Aliivibrio fischeri (bioluminescent bacterium) lives in the bobtail squid's light organ.
• The squid evolved a light organ specifically to house the bacterium.
• The squid relies on bacterial luminescence to provide camouflage against predators.

How quorum sensing helps:

• At low cell density, luminescence would not provide the desired camouflage effect.
• Producing light at low density would waste energy for the bacterial population.
• The lux gene (codes for luciferase enzyme necessary for luminescence) is only activated when bacterial population reaches sufficient density.
• This ensures energy is only spent on light production when it benefits both the bacteria and the squid.

Don't confuse: This is a mutualistic relationship (both benefit), not just the bacteria benefiting from the squid's resources.

🧭 Overview

🧠 One-sentence thesis

Bacterial pathogenicity depends on a dynamic interplay between the pathogen's virulence factors (adherence, invasion, and toxins) and the host's defenses, with transmission modes and host conditions determining whether exposure leads to disease.

📌 Key points (3–5)

• Pathogenicity vs virulence: pathogenicity is the ability to cause disease; virulence is the measurement of that ability—highly virulent pathogens are more likely to cause disease.
• Host-pathogen interaction is dynamic: exposure does not guarantee disease; virulence, number of microbes, entry location, host health, and host defenses all matter.
• Virulence factors: physical structures (capsules, pili) and chemical substances (enzymes, toxins) that enable bacteria to adhere, colonize, invade, and damage the host.
• Common confusion—exotoxins vs endotoxins: exotoxins are heat-sensitive proteins released by living bacteria and cause specific diseases; endotoxins are heat-stable lipid A from gram-negative cell walls, released only when bacteria lyse, and cause the same general symptoms regardless of species.
• Transmission modes: direct contact, droplet, indirect (fomites), airborne, fecal-oral, and vectorborne—each involves different intermediaries and distances.

🦠 Core definitions and the host-pathogen relationship

🦠 Key terms

Pathogen: a microbe capable of causing disease.

Host: the organism being infected.

Pathogenicity: the ability to cause disease.

Virulence: the measurement of pathogenicity; highly virulent pathogens are more likely to cause disease.

Opportunistic pathogen: a microbe that typically infects a host compromised by a weakened immune system or breach to natural defenses (e.g., a wound).

⚖️ The dynamic interaction

• Host-pathogen interaction is constantly changing and involves many variables:
  • Virulence of the pathogen
  • Number of microbes that entered
  • Location of entry
  • Overall health of the host
  • State of the host's defenses
• Key insight: exposure to a pathogen does not ensure disease will occur—the host might fight off the infection before signs or symptoms develop.

🚪 Pathogen transmission

🏠 Reservoirs and carriers

Reservoir: the natural site or home for a pathogen; can be animate (human or animal) or inanimate (water, soil, food).

Carrier: a person who carries the pathogen but shows no obvious symptoms of disease; carriers play an important role in spreading disease.

Zoonosis: a disease that primarily occurs in animal populations but can spread to humans.

Nosocomial infection: a hospital-acquired infection.

🚦 Modes of transmission

Mode of transmission: the mechanism by which a pathogen is picked up by a host.

Mode	Description	Key features
Direct contact	Host-to-host contact (kissing, sexual intercourse, skin or body fluid contact)	Includes vertical transmission: mother to infant across placenta, during birth, or while breastfeeding
Droplet transmission	Respiratory droplets expelled by coughing or sneezing, inhaled by nearby host	Often considered a form of direct contact; droplets do not travel long distances and do not remain infectious long
Indirect contact	Transfer through an intermediary	Fomite: contaminated inanimate object (toy, doorknob, keyboard); or healthcare worker transmitting between patients
Airborne transmission	Small particles or droplets in the environment	Remain infectious over time and distance (e.g., fungal spores inhaled during a dust storm)
Fecal-oral transmission	Pathogen shed in feces contaminates food or water consumed by next host	Common for gastrointestinal pathogens
Vectorborne transmission	Arthropod vector (mosquitoes, flies, ticks) carries pathogen	Vector picks up agent when biting infected host, spreads to next host when biting again

Don't confuse: droplet vs airborne—droplets travel short distances and lose infectivity quickly; airborne particles remain infectious over long distances and time.

🧬 Virulence factors and pathogenicity islands

🧬 What are virulence factors?

Virulence factors: physical structures or chemical substances that allow a bacterium to infect a host and contribute to virulence.

• These can be:
  • Physical structures (capsules, pili, flagella)
  • Chemical substances (enzymes, toxins)

🏝️ Pathogenicity islands

Pathogenicity islands: clusters of genes coding for virulence factors, found on the pathogen's chromosome or plasmid DNA.

• Distinguished by:
  • G+C content that differs from the rest of the genome
  • Presence of insertion-like sequences flanking the gene cluster
• Why they matter: pathogenicity islands facilitate horizontal gene transfer, allowing virulence factors to be shared between bacteria and leading to the development of new pathogens over time.

🎛️ Quorum sensing control

• Genes for virulence factors are often controlled by quorum sensing.
• Why: ensures gene activation only when the pathogen population is at optimal density.
• Triggering genes too soon could alert the host's immune system and cut short the bacterial infection.

🔗 Adherence, colonization, and invasion

🔗 Adherence

Adherence: the ability of bacterial pathogens to grab onto host cells or tissue and resist removal by physical (sneezing) or mechanical (ciliated cell movement) means.

• Mechanisms:
  • Polysaccharide layers (capsule or slime layer): provide adhesion and resistance to phagocytosis
  • Physical structures (pilus, flagellum)

🌱 Colonization

Colonization: once cells adhere to a surface, they increase in number by utilizing available resources at the site.

• Important for:
  • Pathogen survival
  • Invasion to other sites (yielding more nutrients and space)

⚔️ Invasion

Invasion: the ability of the pathogen to spread to other locations in the host by invading host cells or tissue.

• Typically when disease or obvious signs/symptoms occur.
• Mechanisms:
  • Physical structures still play a role
  • Enzymes are key:
    • Collagenase: breaks down collagen in connective tissue, allowing spread
    • Leukocidins: destroy white blood cells, decreasing host resistance
    • Hemolysins: lyse red blood cells, releasing iron (a growth-limiting factor for bacteria)

🩸 Bacteremia and septicemia

Bacteremia: bacteria in the bloodstream.

Septicemia: a massive, systemic infection that can result from bacteremia; can cause septic shock and death as the host becomes overwhelmed by the bacterial pathogen and its products.

💉 Toxins: exotoxins and endotoxins

💉 General toxin concepts

Toxins: substances produced by some bacterial pathogens that are poisonous to the host.

Toxigenicity: an organism's ability to make toxins.

• Two categories for bacteria: exotoxins and endotoxins.

🧪 Exotoxins

Exotoxins: heat-sensitive soluble proteins released into the surrounding environment by a living organism.

• Characteristics:
  • Incredibly potent
  • Can spread throughout the host's body, causing damage distant from the original infection site
  • Associated with specific diseases (botulism, tetanus, diphtheria)
  • Toxin genes often carried on plasmids or by prophages

🧪 Type I: Cell surface-active

• Bind to cell receptors and stimulate cell responses.
• Example: superantigen stimulates host T cells (immune system component).
  • Stimulated T cells produce excessive cytokine (signaling molecule).
  • Result: massive inflammation and tissue damage.

🧪 Type II: Membrane-damaging

• Exert effect on host cell membrane, often by forming pores.
• Result: cell lysis as cytoplasmic contents rush out and water rushes in, disrupting osmotic balance.

🧪 Type III: Intracellular

• Gain access to a particular host cell and stimulate a reaction within the target cell.
• Example: AB-toxin:
  • Composed of two subunits: A portion and B portion
  • B subunit: binding portion; recognizes and binds to correct cell type
  • A subunit: enzymatic activity portion; once delivered into correct cell by B subunit, enacts mechanism leading to decreased function or cell death
  • Example: tetanus toxin produced by Clostridium tetani:
    • Delivered to a neuron
    • A subunit cleaves cellular synaptobreven
    • Result: decreased neurotransmitter release → spastic paralysis
  • Each AB-toxin is associated with a different disease.

🧱 Endotoxins

Endotoxins: made by gram-negative bacteria as a component of the outer membrane of their cell wall.

• The outer membrane contains lipopolysaccharide (LPS).
• The toxic component is the lipid part known as lipid A.

Feature	Exotoxins	Endotoxins
Heat sensitivity	Heat-sensitive	Heat-stable
Release	Released by living bacteria	Released only when bacterial cell is lysed
Specificity	Associated with specific diseases	Effect on host is the same regardless of what bacterium made it
Effects	Vary by toxin type	Fever, diarrhea, weakness, blood coagulation
Severe outcome	Depends on toxin	Massive release can cause endotoxin shock (deadly)

Don't confuse: exotoxins are proteins released by living cells and cause disease-specific effects; endotoxins are lipid A from the cell wall, released when bacteria die, and cause general systemic symptoms.

🧭 Overview

🧠 One-sentence thesis

The Baltimore Scheme classifies viruses into seven classes based on how their genome type relates to mRNA production, determining the replication strategies and enzymes each virus must use or provide.

📌 Key points (3–5)

• Why viruses need a special classification: viruses lack ribosomes (and rRNA), so they cannot fit into the Three Domain Classification used for cellular organisms.
• What the Baltimore Scheme does: it focuses on the relationship between a viral genome and how it produces mRNA, recognizing seven classes.
• Core challenge for all viruses: each genome type (dsDNA, ssDNA, dsRNA, +ssRNA, -ssRNA, retroviruses, reverse-transcribing DNA) requires different enzymes and strategies to make mRNA and replicate.
• Common confusion—plus vs. minus strand: plus-strand RNA can be used directly as mRNA; minus-strand RNA is complementary to mRNA and cannot be used directly.
• Beyond viruses: viroids (infectious ssRNA without protein) and prions (infectious protein without nucleic acid) represent even simpler infectious agents.

🧬 DNA viruses (Classes I, II, VII)

🧬 Class I: dsDNA viruses

Class I viruses have a double-stranded DNA genome, identical in structure to the host cell's DNA.

• Why they are simpler: many host enzymes can be used for replication and protein production.
• Information flow: dsDNA → mRNA → protein (conventional pathway).
  • DNA-dependent RNA-polymerase produces mRNA.
  • DNA-dependent DNA-polymerase replicates the genome (from virus or host).
• Gene expression control: viruses like bacteriophage T4 ensure orderly protein production by modifying the host RNA polymerase at different stages (early, middle, late genes).
• Concatemers: several viral genomes linked together due to short single-stranded regions with terminal repeats; a viral endonuclease cuts them to appropriate length during packaging.

Example: T4 modifies host RNA polymerase so it no longer recognizes host promoters, ensuring only viral genes are transcribed.

🧬 Productive vs. latent infections (herpesviruses)

Infection type	What happens	Location
Productive infection	Explosive viral replication, cell death, disease signs	Neurons infected
Latent infection	Virus remains undetected in neurons; genome stays dormant	Neurons

• If reactivated, latent infection becomes productive again.
• Don't confuse: latent does not mean the virus is gone; it can reactivate.

🧬 Class II: ssDNA viruses

Class II viruses have a single-stranded DNA genome.

• Two possibilities:
  • Plus-strand DNA: same base sequence as mRNA.
  • Minus-strand DNA: complementary to mRNA.
• Replicative form (RF): a double-stranded intermediate formed by synthesizing a complementary strand; used for both protein production and genome replication.
• Rolling-circle replication: one strand is nicked, replication enzymes extend the free 3' end, and the 5' end is peeled off as a complementary strand is synthesized around the circular DNA, producing a long displaced strand.

Example: For plus-strand DNA viruses, a complementary strand must be made first to form the RF before mRNA and genomes can be produced.

🧬 Class VII: DNA viruses using reverse transcriptase

Class VII viruses (hepadnaviruses) contain a partially double-stranded DNA genome with a single-stranded region.

• Steps:

  1. Host enzymes fill in the gap to form a closed dsDNA loop in the nucleus.
  2. Transcription yields a plus-strand RNA called the pregenome and the enzyme reverse transcriptase (an RNA-dependent DNA-polymerase).
  3. Reverse transcriptase uses the pregenome as a template to produce minus-strand DNA genomes.
  4. A small piece of pregenome serves as a primer to produce the double-stranded region.

• Don't confuse: these are DNA viruses that use reverse transcriptase, not retroviruses (which are RNA viruses).

🧪 RNA viruses (Classes III, IV, V, VI)

🧪 Class III: dsRNA viruses

Class III viruses have a double-stranded RNA genome.

• Problem: cells do not use dsRNA and have systems to destroy it; cells also lack RNA-dependent RNA-polymerases.
• Solution: the virus must protect its dsRNA genome and provide its own RNA-dependent RNA-polymerase.
  • Transcriptase: transcribes mRNA from the minus-strand.
  • Replicase: replicates the RNA genome.

Example (rotavirus): the viral nucleocapsid remains intact in the cytoplasm; replication occurs inside, keeping dsRNA protected. New nucleocapsids form around RNA replicase and plus-strand RNA, and minus-strand RNA is synthesized within the nucleocapsid.

🧪 Class IV: +ssRNA viruses

Class IV viruses have plus-strand RNA that can be used directly as mRNA.

• Information flow: genome enters cell → host ribosome translates it immediately → one viral protein is an RNA-dependent RNA-polymerase (RNA replicase).
• Replication: RNA replicase creates minus-strand RNA from the plus-strand genome; minus-strand serves as template for more plus-strand RNA (used as mRNA or new genomes).

🧪 Strategies to generate multiple proteins from unsegmented +ssRNA

Class IV viruses face a challenge: how to make multiple proteins from a single RNA strand?

Strategy	How it works
Polyprotein	Large protein with protease activity cleaves itself into smaller proteins (e.g., poliovirus)
Subgenomic mRNA	Portions of viral RNA are skipped during translation, yielding different proteins
Ribosomal frame-shifting	Ribosome reads mRNA in different open reading frames (ORF #1, #2, #3), producing entirely different amino acid sequences
Readthrough mechanism	Ribosome ignores some stop codons and continues, making longer proteins; combined with frame-shifting for even more variety

Example: Poliovirus produces a polyprotein that cleaves itself into three smaller proteins, which are further cleaved to yield all capsid proteins and an RNA-dependent RNA-polymerase.

🧪 Class V: -ssRNA viruses

Class V viruses have minus-strand RNA genomes that cannot be used directly as mRNA.

• Key requirement: the virus must carry an RNA-dependent RNA-polymerase in its capsid.
• Steps:
  1. Upon entry, the polymerase generates plus-strand RNAs.
  2. Plus-strand RNAs are used as mRNA for protein production.
  3. Plus-strand RNAs also serve as templates to make minus-strand RNA genomes.

Example: Influenza virus, rabies virus, and Ebola virus are all Class V viruses.

🧪 Class VI: +ssRNA retroviruses

Class VI viruses (retroviruses) have plus-strand RNA genomes, but the RNA is not used as mRNA.

• Steps:

  1. Reverse transcriptase synthesizes ssDNA complementary to the viral genome.
  2. Reverse transcriptase's ribonuclease activity degrades the RNA strand of the RNA-DNA hybrid.
  3. Reverse transcriptase acts as a DNA polymerase to make a complementary copy, yielding dsDNA.
  4. The dsDNA is inserted into the host chromosome, forming a provirus.

• Provirus vs. prophage: a provirus can remain latent indefinitely or express viral genes without excision; a prophage must excise for gene expression.

Example: A provirus integrated into the host chromosome can produce new viruses without ever leaving the chromosome.

🦠 Other infectious agents

🦠 Viroids

Viroids are small, circular ssRNA molecules that lack protein and cause plant diseases.

• Challenge: ssRNA is highly susceptible to enzymatic degradation.
• Solution: extensive complementary base pairing causes the viroid to take on a hairpin configuration that resists enzymes.
• Replication: viroids rely on a plant RNA polymerase with RNA replicase activity.

🦠 Prions

Prions are infectious agents made entirely of protein, completely lacking nucleic acid.

• Diseases: bovine spongiform encephalopathy (BSE/"mad cow disease"), Creutzfeldt-Jakob disease (humans), scrapie (sheep), and a prion in yeast.
• Mechanism:
  • PrP^C (Prion Protein Cellular): normal prion protein in healthy neurons with a particular secondary structure.
  • PrP^SC (Prion Protein Scrapie): pathogenic form with a different secondary structure; capable of converting PrP^C into the pathogenic form.
• Pathology: accumulation of PrP^SC destroys brain and nervous tissue, causing memory loss, lack of coordination, and death.

Example: An organism with healthy PrP^C encounters PrP^SC; the pathogenic form converts the normal protein, leading to accumulation and disease.

Don't confuse: prions replicate by converting existing host protein, not by encoding new protein from nucleic acid.

🔑 Summary table: Baltimore Scheme classes

Class	Genome type	Can genome be used as mRNA?	Key enzyme(s) needed	Replication strategy
I	dsDNA	No	DNA-dependent RNA-polymerase, DNA-dependent DNA-polymerase	Conventional: DNA → mRNA → protein
II	ssDNA	No	DNA polymerase	Form replicative form (RF); rolling-circle replication
VII	Partially dsDNA	No	Reverse transcriptase (RNA-dependent DNA-polymerase)	Pregenome (RNA) → DNA → integrate or replicate
III	dsRNA	No (must be protected)	RNA-dependent RNA-polymerase (transcriptase + replicase)	Transcribe mRNA from minus-strand; replicate inside capsid
IV	+ssRNA	Yes	RNA-dependent RNA-polymerase (replicase)	Genome used as mRNA; replicase makes minus-strand template
V	-ssRNA	No	RNA-dependent RNA-polymerase (carried in capsid)	Make plus-strand RNA for mRNA and as template for genomes
VI	+ssRNA (retrovirus)	No	Reverse transcriptase, ribonuclease	RNA → ssDNA → dsDNA → integrate as provirus