Module 1: The Microbial World

1.1 What Is Microbiology?

Two closely related terms require careful distinction in this course.

Although the difference is subtle, it reflects a genuine biological distinction: bacteria, archaea, and microbial eukaryotes are all considered alive by standard biological criteria, whereas viruses occupy a conceptually distinct position as obligate intracellular entities that depend entirely on host cell machinery for reproduction.

1.2 Van Leeuwenhoek and the First Microscopes

The history of microbiology begins in earnest in the seventeenth century with two figures: Robert Hooke and Antonie van Leeuwenhoek. Hooke published Micrographia in 1665, demonstrating that optical instruments could reveal biological structures invisible to the naked eye, including the cell walls of cork. Van Leeuwenhoek made the more consequential leap: he was the first person to observe living bacteria.

Van Leeuwenhoek’s microscope was deceptively simple. It consisted of a single metal plate with a small hole drilled through it. Inside that hole was lodged a tiny spherical glass lens — the critical component. A sample was mounted on a fine metal point whose position could be adjusted by a series of screws, allowing the specimen to be moved toward or away from the lens until it came into focus. The entire device was held up to the eye and angled toward a bright light source, such as a candle or a window. Despite its apparent simplicity, van Leeuwenhoek’s proprietary lens-grinding technique allowed him to achieve magnifications of several hundred times — sufficient to observe bacteria for the first time and to describe them as “animalcules.”

The lens could be fabricated using a Pasteur pipette heated in a Bunsen burner flame: the glass curls and fuses into a tiny, highly curved sphere. That single sphere of glass, mounted between two layers of material with aligned holes, recapitulates the essential optics of van Leeuwenhoek’s original instrument. The magnification achievable depends on the curvature of the lens: more tightly curved spheres produce higher magnification.

1.3 Key Features of Microbial Life

Microorganisms share several key features that distinguish them from macroscopic organisms. They are small — most bacteria fall in the range of one to ten micrometres in length — which confers high surface-area-to-volume ratios and consequently rapid rates of nutrient uptake and metabolic activity. This also means that diffusion alone can supply metabolic needs without a circulatory system. Their small size, however, does not mean uniformity: microbial cells span an extraordinary range of shapes, sizes, and biochemical capabilities, as will be explored in subsequent modules.

1.4 Origins of Microbial Life

Life on Earth originated approximately 3.5–3.8 billion years ago, with the earliest fossils resembling modern prokaryotes. The cyanobacteria hold a position of special importance in this evolutionary narrative: they were the first organisms to perform oxygenic photosynthesis, using water as an electron donor and releasing molecular oxygen as a byproduct. Over hundreds of millions of years, the metabolic activity of cyanobacteria transformed Earth’s atmosphere from a reducing environment to the oxygen-rich one we inhabit today. The accumulation of atmospheric oxygen created the conditions for aerobic metabolism and, ultimately, for the evolution of complex multicellular life.

Endosymbiosis provides the framework for understanding the origin of eukaryotic organelles. According to this theory, the mitochondria of eukaryotic cells descended from an ancestral alphaproteobacterial endosymbiont that was engulfed but not digested by a host cell, while chloroplasts descended from an ancestral cyanobacterial endosymbiont. Evidence for endosymbiosis includes the double membranes of mitochondria and chloroplasts, the presence of their own circular genomes, their reproduction by binary fission independent of the host cell cycle, and the similarity of their ribosomes to those of bacteria.

1.5 Louis Pasteur and Germ Theory

Louis Pasteur made foundational contributions to microbiology in the nineteenth century. His elegant swan-neck flask experiments definitively refuted the doctrine of spontaneous generation, demonstrating that microbial contamination required the introduction of pre-existing microorganisms from the environment. Pasteur also developed the technique of pasteurization — controlled heating to reduce microbial loads in liquids — and contributed foundational work on fermentation and vaccination.

Koch’s postulates provide the logical framework for establishing that a specific microorganism causes a specific disease. The four postulates state: (1) the microorganism must be found in all cases of the disease; (2) it must be isolated from the diseased host and grown in pure culture; (3) the cultured microorganism must cause disease when introduced into a healthy host; and (4) the microorganism must be re-isolated from the experimentally infected host and shown to be identical to the original isolate. Koch’s postulates remain a touchstone in infectious disease microbiology, even as molecular methods have extended and in some cases complicated their application.

Module 2: Bacteria — Structure and Morphology

2.1 Cell Morphology

Bacterial cells display a limited but diagnostically useful set of morphologies. The five principal shapes are cocci (spheres), rods (also called bacilli), spirilla (rigid helices), spirochetes (flexible helices), and vibrios (comma-shaped). Cell shape is not merely descriptive: it can provide clues about a bacterium’s ecological niche and lifestyle. For example, elongated rod shapes increase surface area relative to volume, facilitating nutrient uptake in dilute environments, while helical shapes can help cells drill through viscous environments.

Two notable size extremes illustrate that “typical” bacterial dimensions are not universal. Epulopiscium fishelsoni, a symbiont of surgeon fish, reaches lengths visible to the naked eye. At the other extreme, ultramicrobacteria are extremely small cells that may pass through standard filtration membranes and that have reduced genomes suited to oligotrophic (nutrient-poor) environments. Being larger or smaller than the typical range confers specific adaptive advantages in the habitats these organisms occupy.

2.2 Internal Architecture of Bacterial Cells

The bacterial cell lacks the membrane-bound nucleus and complex endomembrane system characteristic of eukaryotes. Instead, genetic material is organized in the nucleoid — a region of the cytoplasm where the circular chromosome, associated proteins, and small RNA molecules are concentrated. The chromosome is not enclosed by a membrane but is extensively compacted by nucleoid-associated proteins and supercoiling.

In addition to the chromosome, many bacteria carry plasmids: small, circular, double-stranded DNA molecules that replicate independently of the chromosome. Plasmids frequently carry genes conferring selective advantages such as antibiotic resistance, virulence factors, or the ability to metabolize unusual substrates.

The ribosome is the universal machine of translation. Bacterial ribosomes are 70S particles, assembled from a 30S small subunit (containing 16S rRNA) and a 50S large subunit (containing 23S and 5S rRNA). This size difference from eukaryotic 80S ribosomes is clinically significant: many antibiotics, including streptomycin, tetracycline, and erythromycin, selectively inhibit bacterial ribosomes without affecting host cell ribosomes.

Some bacteria produce inclusions — intracellular storage granules of carbon, phosphate, sulfur, or other compounds — that serve as reserves during nutrient limitation. Gas vesicles are found in certain aquatic bacteria and archaea, providing buoyancy by enclosing gas within protein shells.

2.3 The Cell Membrane

The cytoplasmic membrane (also called the plasma membrane) is a phospholipid bilayer that serves as the primary permeability barrier of the cell. In bacteria, this membrane contains no sterols (with the notable exception of Mycoplasma and related organisms that incorporate cholesterol from their environment). Instead, bacterial membranes incorporate hopanoids — pentacyclic terpenoid lipids that fulfil a similar structural and regulatory role to sterols, stiffening the membrane and modulating its fluidity.

Transport across the membrane occurs by three principal mechanisms. Passive diffusion requires no energy and moves solutes down their concentration gradient. Facilitated diffusion employs specific membrane proteins (permeases or channels) to accelerate the passage of polar molecules without energy expenditure. Active transport uses energy — either ATP hydrolysis or the proton motive force — to move solutes against their concentration gradients. The phosphoenolpyruvate phosphotransferase system (PTS) is a specialized group translocation mechanism that chemically modifies sugars (primarily phosphorylation) as they cross the membrane, effectively trapping them inside the cell.

2.4 The Cell Wall and Gram Staining

The cell wall lies immediately external to the cytoplasmic membrane in most bacteria and provides mechanical strength to resist the osmotic pressure generated by the high solute concentration inside the cell. The principal structural polymer of bacterial cell walls is peptidoglycan (also called murein): a mesh-like macromolecule consisting of linear glycan strands cross-linked by short peptide bridges. The glycan strands are composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues. The peptide cross-links between adjacent NAM residues provide tensile strength.

The Gram stain, developed by Hans Christian Gram in 1884, is one of the most widely used differential stains in microbiology. It exploits the structural difference between the two major types of bacterial cell envelopes.

The procedure proceeds in four steps. First, a heat-fixed bacterial smear is flooded with crystal violet for approximately one minute; all cells stain purple. Second, Gram’s iodine (the mordant) is applied for one minute; iodine forms large crystal violet–iodine complexes within the cells and all cells remain purple. Third, the critical decolorization step — a brief rinse with alcohol or acetone — differentiates the two cell types. Gram-positive bacteria have a thick, multi-layered peptidoglycan wall that retains the crystal violet–iodine complex during decolorization and therefore remain purple. Gram-negative bacteria have a thin peptidoglycan layer sandwiched between two membranes; the alcohol disrupts the outer membrane and washes the dye complex out, leaving the cells colourless. Fourth, a counterstain — safranin — is applied for thirty to sixty seconds. Gram-positive cells remain purple, while gram-negative cells take up the safranin and appear pink.

The outer membrane of gram-negative bacteria is an asymmetric bilayer whose outer leaflet is composed largely of lipopolysaccharide (LPS). LPS consists of lipid A (the endotoxin component), a core polysaccharide, and the O-antigen (a repeating polysaccharide chain that varies in structure between strains and species). The outer membrane contains porins — barrel-shaped protein channels that permit the passive diffusion of small hydrophilic molecules.

Because the cell wall is essential and structurally unique to bacteria, it is an important antibiotic target. Penicillin and other beta-lactam antibiotics inhibit the transpeptidase enzymes (penicillin-binding proteins) responsible for cross-linking peptidoglycan strands. Without proper cross-linking, the cell wall weakens and the cell lyses under osmotic stress. Lysozyme, an enzyme present in tears, saliva, and mucus, cleaves the glycosidic bond between NAG and NAM residues, degrading the peptidoglycan backbone.

2.5 Cell Surface Structures

Flagella are the primary locomotory organelles of many bacteria. A bacterial flagellum has three structural components: the basal body, which is embedded in the cell membrane(s) and functions as a molecular motor; the hook, a short curved connector; and the filament, the long helical propeller. The basal body rotates using energy derived from a proton gradient across the cytoplasmic membrane — the proton motive force — rather than ATP hydrolysis directly. Rotation of the filament propels the cell through liquid environments. Depending on the number and arrangement of flagella, bacteria are classified as monotrichous (single flagellum), lophotrichous (a tuft at one or both poles), amphitrichous (single flagella at both poles), or peritrichous (flagella distributed around the entire cell surface).

Pili (singular: pilus) are thin, hair-like protein appendages shorter than flagella. They mediate adherence to surfaces and to other cells. Type IV pili can extend and retract, propelling the cell across surfaces in a process called twitching motility. Sex pili (or conjugation pili) are longer, specialized pili that form a bridge between two bacterial cells and facilitate the transfer of genetic material during conjugation.

Some bacteria produce an extracellular polysaccharide layer called a capsule that surrounds the cell wall. The capsule is loosely organized relative to the cell wall and can be as thick or thicker than the cell itself. It serves multiple functions: it prevents desiccation, facilitates adhesion to surfaces, promotes the formation of biofilms, and — critically in pathogenic bacteria — helps the organism evade phagocytosis by blocking complement deposition and preventing recognition by phagocyte receptors.

S-layers (surface layers) are crystalline arrays of identical protein or glycoprotein subunits that coat the outer surfaces of many bacteria and are particularly common in archaea. When visualized by electron microscopy, they resemble a regular tiling pattern. S-layers provide mechanical protection, act as a molecular sieve, and, like capsules, can help cells resist phagocytic attack.

Module 3: Archaea

3.1 Discovery and Domain Status

The archaea were long thought to be simply unusual bacteria, grouped among the prokaryotes by virtue of their lack of a membrane-bound nucleus. It was not until the 1970s that Carl Woese and George Fox, using comparative analysis of 16S ribosomal RNA sequences, demonstrated conclusively that archaea constitute a separate and fundamentally distinct domain of life — as different from bacteria as bacteria are from eukaryotes. This finding reshaped the tree of life from a two-domain (prokaryote/eukaryote) model to the three-domain model now universally accepted.

The reclassification arose because, although archaea look superficially similar to bacteria under the light microscope — they are generally small, lack a membrane-bound nucleus, and have a similar size range — molecular analyses revealed profound biochemical differences. The three domains of life are Bacteria, Archaea, and Eukarya.

3.2 Archaea Versus Bacteria: Similarities and Differences

Archaea and bacteria share several features of the prokaryotic cell plan: both lack a membrane-enclosed nucleus, both have circular chromosomes, both have 70S-type ribosomes, and both divide by binary fission. However, they differ in numerous important ways.

The most diagnostically significant difference is the cell membrane composition. Bacterial membranes are built from fatty acid chains esterified to glycerol. Archaeal membranes are built from isoprenoid side chains linked to glycerol by ether bonds rather than ester bonds. Furthermore, the glycerol backbone in archaeal membrane lipids has the opposite stereochemistry (sn-2,3-glycerol) compared to bacteria and eukaryotes. Many archaea also have monolayer membranes in which the isoprenoid chains from opposite sides are covalently linked end-to-end, forming a lipid monolayer rather than a bilayer — this adaptation greatly enhances membrane stability at high temperatures.

Archaea lack peptidoglycan in their cell walls. Instead, cell wall materials vary by group: many methanogens have pseudopeptidoglycan (which uses N-acetyltalosaminuronic acid instead of NAM and is insensitive to lysozyme), while others have S-layers as their only wall component, and some hyperthermophiles have unique proteinaceous walls.

Regarding gene expression, archaea combine bacterial and eukaryotic features in a mosaic fashion. Their RNA polymerase is more similar to the eukaryotic multi-subunit RNA polymerase than to the simpler bacterial version. Archaeal promoters resemble eukaryotic RNA polymerase II promoters. Yet their ribosomes are approximately 70S in size, and many of their metabolic pathways are more bacteria-like.

3.3 Archaea and Eukaryotes: Shared Features

Archaea share with eukaryotes several molecular features absent from bacteria. These include similar histone proteins that package DNA (though archaeal histones are structurally simpler), similar RNA polymerase subunit composition, use of TATA-box-binding protein and TFIIB-like transcription initiation factors, and intron-containing genes in some species. This mosaic of features supports the hypothesis — now widely favored — that eukaryotes arose from an archaeal ancestor, with the mitochondrion contributed by an alphaproteobacterial endosymbiont.

3.4 Extremophiles and Physiological Diversity

Archaea were initially discovered in extreme environments and were collectively branded extremophiles — organisms thriving in conditions lethal to most life. Research has subsequently revealed that many archaea inhabit completely “normal” environments including soils, oceans, and the human gut. Nevertheless, the archaeal extremophiles remain iconic and biotechnologically significant.

Halobacterium and relatives achieve osmotic balance with their hypersaline environment by accumulating high intracellular concentrations of potassium chloride. Their enzymes are adapted to function — and indeed require — high salt concentrations for structural stability. The purple membrane of Halobacterium salinarum contains bacteriorhodopsin, a light-driven proton pump that allows the cell to generate a proton motive force using light energy in a manner analogous to (but structurally unrelated to) the photosynthetic machinery of bacteria and chloroplasts.

Haloquadratum walsbyi is a remarkable halophile that forms nearly perfect flat, square cells — one of the most extraordinary cell shapes in the microbial world.

Methanogens are found wherever anaerobic organic matter decomposition occurs: in wetland sediments, marine sediments, landfills, the digestive tracts of ruminants and humans, and in the hindguts of termites. They play a critical role in the global carbon cycle, and the methane they produce is both a potent greenhouse gas and a potential renewable energy source.

Hyperthermophilic archaea such as members of Sulfolobus, Pyrococcus, and Methanopyrus thrive at temperatures exceeding 80°C, and some can grow above 100°C at high pressures. These organisms provided the heat-stable DNA polymerases (such as Pfu polymerase) that extended the utility of the polymerase chain reaction. Nanoarchaeum equitans has the smallest known archaeal genome and lives as an obligate symbiont on the surface of Ignicoccus, an unusual relationship described as “bizarre” because Nanoarchaeum appears to derive membrane lipids and other molecules directly from its host.

3.5 MreB and Cytoskeletal Homologues

The eukaryotic cytoskeletal protein actin has clear homologues in both bacteria and archaea. The bacterial homologue MreB organizes the cell shape of rod-shaped bacteria by forming a helical scaffold beneath the cytoplasmic membrane that guides cell wall synthesis. Without functional MreB, rod-shaped bacteria typically become spherical. Analysis of MreB protein sequences from bacteria and archaea, placed on a phylogenetic tree together with the eukaryotic actin sequence, reveals an informative pattern: some archaeal MreB sequences cluster with bacterial sequences, while others cluster closer to the eukaryotic actin sequence. This non-uniformity demonstrates that the evolutionary relationships between archaea, bacteria, and eukaryotes are not always as simple or clear-cut as the examples of histones or RNA polymerases might suggest. The tree reveals evolutionary mosaicism: different molecules within the same domain of life can have different evolutionary affinities to the other domains.

Module 4: Eukaryotic Microbes

4.1 Defining Features of Eukaryotic Cells

Eukaryotic cells are defined by the presence of a nucleus — a membrane-enclosed compartment housing the genome — and an extensive system of internal membranes. Additional distinguishing features include mitochondria (in virtually all eukaryotes), an endomembrane system comprising the endoplasmic reticulum and Golgi apparatus, a cytoskeleton built from actin filaments, microtubules, and intermediate filaments, and eukaryotic ribosomes of 80S (composed of 40S and 60S subunits).

In contrast to bacteria, eukaryotic chromosomes are linear and multiple, packaged into nucleosomes by histone proteins. Eukaryotes reproduce sexually via meiosis, which introduces genetic recombination, but many also reproduce asexually.

4.2 The Major Groups of Eukaryotic Microbes

The eukaryotic microbes encompass four broadly recognized groups: protists, algae, fungi, and slime molds. Understanding the distinguishing features of each is essential.

Protists are a heterogeneous, paraphyletic assemblage of unicellular eukaryotes that do not fit neatly into the animal, plant, or fungal kingdoms. They include heterotrophs such as the amoebae and flagellates, many of which are important parasites of humans and other animals.

Algae are photosynthetic eukaryotic microbes (and macrobes) that span a wide phylogenetic range. They carry out oxygenic photosynthesis using chloroplasts that, in different lineages, arose through primary, secondary, or even tertiary endosymbiosis. Marine phytoplankton, dominated by algal groups such as diatoms and dinoflagellates, are responsible for approximately half of global primary productivity.

Fungi are heterotrophic eukaryotes with cell walls composed primarily of chitin — a polymer of N-acetylglucosamine — rather than the cellulose found in plant cell walls or the peptidoglycan of bacteria. Fungi digest food externally by secreting hydrolytic enzymes and then absorbing the soluble products. They reproduce by spores and often have both sexual and asexual reproductive cycles.

Slime molds occupy a fascinating transitional position: they can exist as free-living unicellular amoebae that aggregate under nutrient stress into multicellular structures exhibiting coordinated behaviour and differentiation. They are studied as models of social behaviour and development.

4.3 Endosymbiosis and the Origin of Organelles

The endosymbiotic theory, championed by Lynn Margulis, proposes that the double-membrane organelles of eukaryotic cells — mitochondria and chloroplasts — were once free-living prokaryotes engulfed by a host cell in a permanent, mutually beneficial partnership. Evidence includes: (1) the double membrane of these organelles reflects the original inner (prokaryotic) membrane plus the phagosomal membrane; (2) their genomes are circular, resembling prokaryotic chromosomes; (3) their ribosomes (70S) are bacterial in size and sensitivity to antibiotics; and (4) phylogenetic analyses place mitochondrial genomes within the alphaproteobacteria and chloroplast genomes within the cyanobacteria.

Module 5: Viruses

5.1 Discovery of Viruses

Viruses were discovered at the end of the nineteenth century through experiments with tobacco mosaic disease. Dmitri Ivanowski showed in 1892 that extracts filtered through ceramic filters that removed bacteria could still cause disease. Martinus Beijerinck named the infectious agent a contagium vivum fluidum (living contagious fluid). Frederick Twort and Félix d’Hérelle independently discovered bacteriophages — viruses that infect bacteria — in the early twentieth century. Walter Reed demonstrated that yellow fever was caused by a filterable agent, providing early evidence for human viral disease.

5.2 Virus Structure

Viruses are not cells. They lack ribosomes, they do not carry out metabolism, and they cannot reproduce independently. A complete viral particle is called a virion. Its essential components are the genome (either DNA or RNA, single- or double-stranded) and the capsid — a protein shell assembled from repeating structural units called capsomeres. The capsid protects the genome and mediates attachment to host cells. Many animal viruses are additionally surrounded by a lipid envelope derived from host cell membranes, into which viral glycoproteins are inserted.

5.3 Viral Replication

All viruses follow a general replication scheme: attachment to a host cell receptor, entry, uncoating of the genome, replication of the genome and synthesis of viral proteins using host machinery, assembly of new virions, and release from the cell. The specific mechanisms differ substantially between enveloped and non-enveloped viruses and between animal and bacterial viruses.

The two major bacteriophage replication strategies are the lytic cycle and the lysogenic cycle. In the lytic cycle, the phage genome is immediately replicated, new phage particles are assembled, and the host cell is lysed to release progeny phage. In the lysogenic cycle, the phage genome integrates into the host chromosome as a prophage and is replicated passively with the host genome through many cell divisions. Under appropriate conditions (typically DNA damage or other stress), the prophage excises and enters the lytic cycle.

5.4 Viruses and the Environment

Despite their association with disease, viruses play broadly positive ecological roles. Approximately 20–40% of microbial biomass in the ocean is killed by viral lysis every day. This “viral shunt” returns cellular carbon, nitrogen, and phosphorus to the dissolved pool, preventing it from being sequestered in microbial biomass and supporting nutrient cycling. Viruses also drive horizontal gene transfer by carrying genes between bacterial hosts, accelerating the evolution and metabolic versatility of microbial communities.

5.5 Viroids and Prions

Beyond conventional viruses, two even more reduced infectious agents deserve mention. Viroids are small, circular, single-stranded RNA molecules that cause plant diseases. Unlike viruses, viroids have no protein coat; the RNA itself is the infectious agent. They are thought to disrupt host gene regulation. Prions are misfolded proteins that can induce normal versions of the same protein to adopt the misfolded conformation, propagating the abnormal state in a chain reaction. Prions cause fatal neurodegenerative diseases including Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE, or “mad cow disease”) in cattle. Prions represent perhaps the ultimate reduction of an infectious agent: they contain no nucleic acid at all.

5.6 CRISPR/Cas Systems

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and their associated Cas proteins constitute an adaptive immune system found in many bacteria and archaea. Arrays of short CRISPR spacer sequences, derived from previous viral infections, are stored in the chromosome. When foreign DNA matching a spacer sequence enters the cell, it is recognized and cleaved by the Cas nuclease complex.

The mechanism proceeds in three phases: adaptation (acquisition of new spacers from invading DNA), expression (transcription of the CRISPR array into precursor CRISPR RNA and processing into individual guide RNAs), and interference (the guide RNA directs the Cas effector nuclease to the matching target sequence, where it makes a double-stranded cut). A short sequence adjacent to the target, called the PAM (protospacer adjacent motif), distinguishes foreign DNA from the spacers stored in the CRISPR array itself, preventing self-cleavage.

The CRISPR/Cas9 system from Streptococcus pyogenes has been repurposed as a powerful genome-editing tool, enabling precise cuts to be made at any target sequence in any genome. A single guide RNA (sgRNA) is designed to match the target sequence; Cas9 creates a double-stranded break that can be repaired by error-prone non-homologous end joining (creating indels and gene knockouts) or by homology-directed repair using a provided template (enabling precise edits). This technology has revolutionized biomedical research and shows promise for therapeutic correction of genetic diseases.

Module 6A: Cultivating Microorganisms — Metabolism and Media

6.1 Metabolic Categories

Understanding what a microorganism needs to grow requires classifying its metabolic strategies. Two axes of classification are particularly important.

Energy source defines organisms as phototrophs (using light energy) or chemotrophs (using chemical energy from oxidation of organic or inorganic compounds).

Carbon source defines organisms as autotrophs (fixing inorganic CO₂ into organic carbon) or heterotrophs (requiring pre-formed organic carbon).

Combining these two axes gives four major nutritional categories. Photoautotrophs use light and fix CO₂ (e.g., cyanobacteria, algae). Photoheterotrophs use light but require organic carbon (e.g., some purple non-sulfur bacteria). Chemoautotrophs (also called chemolithotrophs) oxidize inorganic compounds to get energy and fix CO₂ (e.g., nitrifying bacteria, sulfur oxidizers). Chemoheterotrophs oxidize organic compounds for both energy and carbon (e.g., most animals and many bacteria).

6.2 Environmental Conditions for Growth

Microbial growth is sensitive to several environmental parameters.

Temperature determines which enzymes function efficiently. Bacteria are classified as psychrophiles (cold-adapted, optimum near 15°C), mesophiles (moderate, optimum 25–40°C, includes most pathogens), thermophiles (heat-adapted, optimum 45–80°C), or hyperthermophiles (optimum above 80°C).

pH is equally important. Most bacteria prefer near-neutral pH, but acidophiles thrive in low-pH environments (e.g., Acidithiobacillus ferrooxidans at pH < 2) and alkaliphiles prefer high pH.

Oxygen tolerance varies enormously. Obligate aerobes require oxygen for respiration. Obligate anaerobes are killed by oxygen. Facultative anaerobes can grow with or without oxygen, preferring aerobic conditions. Aerotolerant anaerobes tolerate oxygen but do not use it. Microaerophiles require oxygen at concentrations below atmospheric levels.

Water activity and osmolarity affect microbial growth. Osmophiles and halophiles are adapted to high solute concentrations.

6.3 Culture Media

Microbiologists manipulate and characterize microorganisms by growing them on defined media.

Module 6B: Microbial Growth and Control

6.4 Quantifying Microorganisms

Several methods exist for quantifying microbial populations. Direct counting under a microscope using a haemocytometer or Petroff-Hausser counting chamber counts total cells. Viable plate counts (also called colony counts or pour plates and spread plates) rely on the assumption that each colony originates from a single viable cell — a colony-forming unit (CFU). Serial dilutions are performed, and a known volume is plated; after incubation, colonies are counted and the original concentration back-calculated. Turbidimetry uses a spectrophotometer to measure optical density (OD) of the culture, which is proportional to cell concentration within a linear range. Most probable number (MPN) is a statistical method useful for quantifying organisms in environmental samples that cannot be easily cultured on plates.

6.5 The Bacterial Growth Curve

When a microbial culture is established in batch (closed) conditions, growth follows a characteristic pattern described by the bacterial growth curve, which has four phases.

In the lag phase, cells are metabolically active but not yet dividing — they are synthesizing enzymes and biosynthetic machinery needed for growth in the new medium. The length of the lag phase depends on the physiological state of the inoculum and the similarity between the inoculum environment and the new medium.

The exponential (log) phase follows: cells divide at the maximum rate possible under the given conditions, with the population doubling every generation time (g). During exponential growth, the number of cells follows a geometric progression (N = N₀ × 2ⁿ, where n is the number of generations). The specific growth rate (µ) and generation time are inversely related: g = ln2/µ.

The stationary phase is reached when nutrients become limiting or waste products accumulate. Cell division and cell death occur at equal rates, maintaining a constant total cell count.

In the death (decline) phase, cell death exceeds cell division and the viable population decreases, usually exponentially.

Continuous culture using a chemostat allows cells to be maintained indefinitely in exponential phase by continuously supplying fresh medium (and therefore limiting nutrient) while removing spent medium and cells at the same rate. This technique enables precise control of growth rate independent of nutrient concentration.

6.6 Controlling Microbial Growth

Microbial growth can be inhibited or eliminated by physical and chemical means. Sterilization refers to the complete elimination of all viable microorganisms including endospores. Disinfection refers to the elimination of most or all pathogens from inanimate surfaces. Antisepsis refers to the application of antimicrobial agents to living tissue.

Physical methods include sterilization by heat (autoclaving at 121°C and 15 psi for 15–20 minutes kills all life including endospores), dry heat ovens (160–170°C for 2 hours), ultraviolet radiation (which forms pyrimidine dimers in DNA), ionizing radiation (gamma rays for sterilizing medical devices), and filtration through membrane filters (typically 0.22 µm pore size, which removes bacteria but not viruses).

Chemical agents are classified by their mechanism of action. Bacteriostatic agents inhibit growth without killing. Bactericidal agents kill bacteria. Bacteriolytic agents cause lysis and cell death by targeting the cell wall.

Antibiotics are antimicrobial substances produced by microorganisms (or synthesized chemically) that inhibit or kill other microorganisms. They are classified as broad-spectrum (effective against many bacterial types) or narrow-spectrum (effective against a limited range). Key antibiotic classes and their mechanisms include: beta-lactams (inhibit cell wall synthesis via transpeptidase inhibition), aminoglycosides (inhibit protein synthesis at the 30S ribosome), tetracyclines (inhibit 30S ribosome), macrolides (inhibit 50S ribosome), fluoroquinolones (inhibit DNA gyrase and topoisomerase IV), and glycopeptides such as vancomycin (inhibit cell wall synthesis by binding the D-Ala-D-Ala terminus of peptidoglycan precursors).

Module 7A: Bacterial MVPs — Proteobacteria

7.1 Overview of Bacterial Diversity

The second half of the course shifts from individual mechanisms to a taxonomic and ecological survey of key bacteria. Module 7A and 7B introduce the “bacterial MVPs” — organisms that are particularly significant, well-studied, or representative of important metabolic categories. The phylum Proteobacteria is the most phylogenetically and metabolically diverse bacterial phylum and encompasses five classes: Alpha, Beta, Gamma, Delta, and Epsilon proteobacteria.

7.2 Taxonomic Framework: Bacterial Phyla

Before surveying specific MVP bacteria, it is useful to orient within the phylogenetic landscape of bacterial diversity. The 16S rRNA phylogenetic tree of bacteria reveals dozens of major lineages (phyla). The key phyla covered in this course are:

Proteobacteria — the most metabolically diverse phylum, including gram-negative rods and cocci with aerobic, anaerobic, and phototrophic members. Divided into five classes (Alpha through Epsilon), all gram-negative.

Firmicutes — gram-positive with low G+C genomes; includes endospore-formers (Bacillus, Clostridium), lactic acid bacteria, and Staphylococcus.

Actinobacteria — gram-positive with high G+C genomes; includes Streptomyces (antibiotic producers), Mycobacterium, and Corynebacterium.

Cyanobacteria — gram-negative oxygenic phototrophs; many are filamentous and some fix nitrogen.

Bacteroidetes — gram-negative; abundant in the mammalian gut and in marine environments; important degraders of complex polysaccharides.

Spirochaetes — gram-negative with distinctive helical morphology and internal flagella (axial filaments); includes Treponema pallidum (syphilis) and Borrelia burgdorferi (Lyme disease).

7.3 Alphaproteobacteria: Purple Non-Sulfur Bacteria and Nitrogen Fixers

Purple non-sulfur bacteria (family Rhodospirillaceae) are photoheterotrophs that can also grow as aerobic chemoheterotrophs in the absence of light. They are metabolically versatile and inhabit freshwater and marine sediments rich in organic matter.

Agrobacterium tumefaciens is an alphaproteobacterium of enormous agricultural significance. It causes crown gall disease in plants by transferring a segment of its Ti (tumour-inducing) plasmid into the host plant genome, where it directs the production of abnormal plant growth hormones causing tumour formation. The T-DNA transfer mechanism has been co-opted as a tool for plant genetic engineering.

Rhizobia are alphaproteobacteria that form symbiotic nitrogen-fixing nodules on the roots of leguminous plants. The interaction is highly specific: the plant releases flavonoids that activate nod genes in the rhizobium, triggering production of Nod factors (lipochitooligosaccharides) that stimulate cortical cell division in the root and nodule formation. Inside the nodule, bacteria differentiate into bacteroids that express nitrogenase, the enzyme complex that reduces atmospheric N₂ to NH₃. Because nitrogenase is irreversibly inactivated by oxygen, the nodule maintains a very low internal oxygen tension through the oxygen-binding protein leghemoglobin.

7.4 Gammaproteobacteria: Pseudomonads and Enteric Bacteria

Pseudomonas aeruginosa is the archetype of the pseudomonads: gram-negative, aerobic, rod-shaped bacteria with remarkable metabolic versatility. They can degrade an enormous variety of organic compounds, including xenobiotics, and are notable for their intrinsic antibiotic resistance and their ability to form biofilms. P. aeruginosa is an opportunistic pathogen particularly dangerous to immunocompromised individuals.

The enteric bacteria (family Enterobacteriaceae) are facultatively anaerobic gram-negative rods that colonize the intestines of animals. Escherichia coli is the central model organism of molecular biology and microbiology, owing to its rapid growth rate (~20 min generation time), ease of genetic manipulation, and thorough characterization. Not all E. coli strains are pathogenic; many are commensal members of the gut microbiome.

7.5 Betaproteobacteria: Nitrifiers and Methanotrophs

Nitrifying bacteria carry out two sequential steps of nitrification in the nitrogen cycle. Nitrosomonas oxidizes ammonium (NH₄⁺) to nitrite (NO₂⁻), and Nitrobacter oxidizes nitrite to nitrate (NO₃⁻). Both are chemolithotrophs, deriving energy from these inorganic oxidations and fixing CO₂.

Methanotrophs oxidize methane (CH₄) as their sole carbon and energy source. Methylotrophs grow on one-carbon compounds other than methane (e.g., methanol, methylamine). Both groups use specialized intracytoplasmic membranes to house the enzymes responsible for one-carbon compound oxidation.

7.6 Deltaproteobacteria: Predatory Bacteria

Myxobacteria are deltaproteobacteria with one of the most complex life cycles known among prokaryotes. When nutrients are limiting, myxobacterial cells aggregate and undergo coordinated development to form elaborate fruiting bodies containing resistant myxospores. This social behaviour requires intercellular communication and represents a degree of multicellularity remarkable for bacteria.

Bdellovibrio bacteriovorus is a tiny, fast-swimming predatory bacterium that attacks gram-negative bacteria. It penetrates the outer membrane of its prey, enters the periplasmic space, and digests the prey cell from within. Bdellovibrio has been studied as a potential “living antibiotic” against gram-negative pathogens.

Module 7B: Bacterial MVPs — Firmicutes, Actinobacteria, and Cyanobacteria

7.7 Firmicutes: Staphylococcus, Lactic Acid Bacteria, Bacillus, and Clostridium

The phylum Firmicutes contains primarily gram-positive bacteria with low G+C content in their genomes.

Staphylococcus aureus and Micrococcus are cocci ideally adapted to inhabit the skin and mucous membranes of mammals. The skin surface is dry, salty, slightly acidic, and often nutrient-poor — conditions that favour organisms with robust tolerance of osmotic stress and desiccation. S. aureus is a major human pathogen, causing infections ranging from superficial skin infections to life-threatening bacteremia and toxic shock syndrome. Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically important example of antibiotic resistance.

Lactic acid bacteria (LAB), including genera such as Lactobacillus, Streptococcus, and Leuconostoc, ferment sugars to lactic acid. This capacity underlies their roles in food production (cheese, yogurt, sauerkraut, kimchi) and in colonizing the human gut and vaginal microbiome. LAB are generally aerotolerant anaerobes.

Bacillus and Clostridium are both endospore-forming Firmicutes. Bacillus species are aerobic or facultatively anaerobic, while Clostridium species are obligate anaerobes. Bacillus subtilis is a principal model organism for studies of sporulation and gram-positive gene regulation. Clostridium botulinum produces the most lethal known toxin, while Clostridium difficile is a major cause of healthcare-associated diarrhoea.

7.8 Actinobacteria: Mycobacterium and Streptomyces

The phylum Actinobacteria comprises gram-positive bacteria with high G+C content. Many form branching filaments resembling fungal hyphae.

Mycobacterium tuberculosis, the causative agent of tuberculosis, has a distinctive cell envelope rich in mycolic acids — very long-chain branched fatty acids that render the cell wall extremely hydrophobic and impermeable. This gives mycobacteria their characteristic acid-fast staining property and contributes to their resistance to many antibiotics and to the host immune response.

Streptomyces species are soil actinomycetes famous for producing the majority of clinically useful antibiotics, including streptomycin, erythromycin, and vancomycin. They form complex multicellular structures and produce spores for dispersal.

7.9 Cyanobacteria

Cyanobacteria are gram-negative photoautotrophs that perform oxygenic photosynthesis using two photosystems (I and II) and generate molecular oxygen. They were responsible for the Great Oxidation Event ~2.4 billion years ago and remain major contributors to global primary productivity in aquatic environments. Some cyanobacteria, such as Anabaena, fix atmospheric nitrogen in specialized cells called heterocysts that are differentiated to maintain the low-oxygen environment required by nitrogenase.

Cyanobacteria are unusual among bacteria in their cell size and complexity. Filamentous forms can be several hundred micrometres in length and are visible under low magnification. They move by gliding motility — a mechanism that does not involve flagella but instead uses secreted slime and type IV pili-related machinery to propel cells along surfaces. Under the microscope, the gliding movement of large filamentous cyanobacteria is clearly visible and notably graceful.

Module 8: Regulation of Gene Expression

8.1 Transcription and Translation — A Refresher

Transcription is the synthesis of RNA from a DNA template, catalysed by RNA polymerase. In bacteria, the core RNA polymerase (consisting of subunits α₂ββ’ω) associates with a sigma factor (σ) to form the holoenzyme capable of recognizing promoters — specific DNA sequences that direct the polymerase to start transcription at the correct position and strand. The principal promoter elements in bacteria are the -10 and -35 regions, which are bound by the sigma factor.

Transcription terminates by two mechanisms. Intrinsic termination occurs when a stem-loop structure forms in the nascent RNA followed by a run of U residues, causing polymerase to stall and dissociate. Rho-dependent termination requires the RNA helicase Rho, which tracks along the RNA and promotes dissociation of the transcription complex at specific Rho utilization (rut) sites.

Translation converts the mRNA sequence into protein at the ribosome. The ribosome binding site (RBS) (or Shine-Dalgarno sequence) in bacteria base-pairs with the 3′ end of 16S rRNA in the 30S subunit to position the start codon (AUG) in the P-site.

8.2 Post-Translational Regulation

Before considering transcriptional control, bacteria can regulate protein activity after the protein has been synthesized. Allosteric regulation occurs when a small molecule binds to a site on a protein distinct from the active site, inducing a conformational change that alters activity. Covalent modifications include phosphorylation, methylation, and acetylation, which alter protein activity in a reversible manner. These post-translational mechanisms allow very rapid responses to environmental changes because they do not require new protein synthesis.

8.3 The Operon Model: Negative and Positive Control

Bacteria often organize co-regulated genes into operons — groups of contiguous genes transcribed from a single promoter into a polycistronic mRNA. The lac operon of E. coli is the canonical example.

The structural elements of a transcriptionally regulated operon include: the promoter (binding site for RNA polymerase), the operator (binding site for a repressor), the activator binding site (binding site for a transcriptional activator, often upstream of the promoter), and the structural genes encoding the enzymes or other proteins of the pathway.

Negative control of transcription occurs when a regulatory protein (a repressor) binds to the operator and physically blocks RNA polymerase from transcribing the structural genes. This blocking can be relieved (induction) by a small molecule called an inducer that binds the repressor and causes it to dissociate from the operator, allowing transcription to proceed. Alternatively, under repression, the repressor is only active when bound to a corepressor molecule.

Positive control of transcription occurs when a regulatory protein (an activator) binds to an activator binding site and directly stimulates RNA polymerase activity, either by stabilizing its binding to the promoter or by assisting in the transition to elongation. Activators are typically required when the promoter is inherently weak and cannot recruit RNA polymerase efficiently on its own.

8.4 The lac Operon in Detail

The lac operon encodes three structural genes: lacZ (β-galactosidase, which cleaves lactose into glucose and galactose), lacY (lactose permease, which transports lactose across the membrane), and lacA (thiogalactoside acetyltransferase).

Negative control by the Lac repressor (product of lacI): In the absence of lactose, the Lac repressor binds the operator and prevents transcription. When lactose (via its intracellular derivative allolactose) is present, it binds the repressor and induces a conformational change that reduces its affinity for the operator, freeing it and allowing transcription.

Positive control by catabolite activator protein (CAP): The lac operon is also subject to catabolite repression — it is not transcribed at high levels when glucose is plentiful. CAP (also called CRP) is an activator that binds the lac promoter only when associated with cyclic AMP (cAMP). cAMP levels are high when glucose is absent (because adenylate cyclase is active) and low when glucose is present (because glucose inhibits adenylate cyclase via the PTS). Therefore, maximum lac transcription requires both the absence of glucose (high cAMP, active CAP) and the presence of lactose (inducer inactivates the repressor).

8.5 Attenuation: The trp Operon

The tryptophan operon (trpLEDCBA) encodes enzymes for tryptophan biosynthesis and is regulated not only by a repressor (under negative control by tryptophan as corepressor) but also by a second mechanism called attenuation. The trp leader sequence (trpL) contains a short open reading frame encoding a peptide rich in tryptophan residues. When tryptophan is abundant, the ribosome translates the leader peptide efficiently and the mRNA folds into a terminator stem-loop structure that causes RNA polymerase to abort transcription before reaching the structural genes. When tryptophan is scarce, the ribosome stalls at the tryptophan codons and the mRNA folds into an antiterminator structure instead, allowing transcription to continue. This coupling of transcription and translation in bacteria allows a very rapid, fine-grained response to the intracellular tryptophan pool.

8.6 Quorum Sensing

Quorum sensing is a mechanism of intercellular communication in which bacteria produce and secrete small signalling molecules (autoinducers) that accumulate in proportion to population density. When the concentration of autoinducer reaches a threshold, it triggers co-ordinated changes in gene expression across the entire population.

The bioluminescent bacterium Aliivibrio fischeri (formerly Vibrio fischeri) is the model system for quorum sensing. It colonizes the light organ of the Hawaiian bobtail squid Euprymna scolopes, where it produces bioluminescence that the squid uses for counter-illumination camouflage. Bioluminescence genes (lux) are expressed only at high cell density, when the autoinducer N-(3-oxohexanoyl)-homoserine lactone (OHHL) reaches sufficient concentration to activate the transcriptional activator LuxR, which then drives lux operon expression.

8.7 Two-Component Regulatory Systems

Two-component regulatory systems are a widespread signal transduction strategy in bacteria (and some archaea) consisting of a sensor kinase (a membrane-spanning histidine kinase) and a response regulator (a cytoplasmic DNA-binding protein). Upon detecting an environmental signal, the sensor kinase autophosphorylates on a conserved histidine residue. It then transfers the phosphate group to a conserved aspartate residue on the response regulator, activating it to alter gene expression.

Agrobacterium tumefaciens uses a two-component system to detect plant wound compounds (acetosyringone and related phenolics), triggering expression of virulence (vir) genes required for T-DNA transfer. Chemotaxis (described below) represents an elaborate variant of a two-component system, with the sensor kinase CheA and multiple response regulators mediating both motor control and sensory adaptation.

8.8 Chemotaxis: Sensory Signal Transduction in Bacteria

Chemotaxis is the directed movement of a cell toward chemical attractants or away from chemical repellents. Bacteria cannot steer — their flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). CCW rotation causes the flagella to form a coordinated bundle that drives the cell forward in a run. CW rotation causes the bundle to fly apart and the cell to tumble, randomly reorienting it. By modulating the ratio of runs to tumbles based on the sensed chemical gradient, a bacterium achieves net movement up an attractant gradient or down a repellent gradient.

The molecular machinery of chemotaxis involves several proteins designated as Che (chemotaxis) proteins. Methyl-accepting chemotaxis proteins (MCPs) are transmembrane receptors that bind attractants or repellents and transmit a conformational signal to the cytoplasmic signalling complex.

CheA is the sensor kinase. In the absence of attractant (or presence of repellent), CheA autophosphorylates (using ATP, with the assistance of CheW, which couples CheA to the MCPs). Phosphorylated CheY (P-CheY) interacts with the flagellar motor switch protein FliM, causing CW rotation and a tumble. CheZ continuously dephosphorylates P-CheY, ensuring that tumbles are very brief — a single brief tumble is sufficient to reorient the cell.

Adaptation allows bacteria to detect changes in chemical concentrations rather than absolute concentrations — a form of sensory adaptation analogous to human visual adaptation. CheR methyltransferase continuously adds methyl groups to the MCPs. CheB, phosphorylated by CheA (CheA transfers a phosphate to CheB as well as to CheY), removes methyl groups from MCPs. The degree of MCP methylation modulates their sensitivity to attractants and repellents: higher methylation decreases sensitivity to attractants but increases sensitivity to repellents, and vice versa. This adjustment means that as a cell moves up an attractant gradient and the attractant concentration increases, the MCPs progressively become less sensitive to that attractant, allowing the cell to detect further increases rather than saturating.

To summarize the four scenarios: moving up an attractant gradient results in more runs and fewer tumbles; moving down an attractant gradient results in more tumbles; moving up a repellent gradient results in more tumbles; moving down a repellent gradient results in more runs. Good situations mean more runs; bad situations mean more tumbles.

8.9 Global Regulation: Sigma Factors and the SOS Response

A regulon is a set of operons or genes that share a common regulatory element and are co-regulated, even if they are not physically adjacent on the chromosome.

Sigma factors allow bacteria to redirect transcription globally in response to environmental conditions. By replacing the housekeeping sigma factor (σ⁷⁰ in E. coli) with an alternative sigma factor, RNA polymerase is redirected to a completely different set of promoters. For example, σ³² directs transcription of heat shock genes during thermal stress, and σ⁵⁴ is associated with nitrogen-regulated genes.

The SOS response is a global response to DNA damage. When DNA is damaged (e.g., by UV radiation), single-stranded DNA accumulates and activates the RecA protein. Activated RecA acts as a co-protease, stimulating the autocatalytic cleavage of the LexA repressor, which normally suppresses a large set of DNA repair genes. Cleavage of LexA allows expression of over forty SOS genes involved in DNA repair, including error-prone polymerases that can replicate past DNA lesions (translesion synthesis).

Module 8 Supplementary: Endospores

Endospores are dormant, highly resistant structures formed by certain gram-positive Firmicutes, most notably members of the genera Bacillus and Clostridium. They are not a reproductive structure but rather a survival strategy: when nutrient depletion or other stresses threaten cell viability, the cell undergoes a complex developmental programme to produce a spore that can persist indefinitely in the environment.

Endospores are extraordinarily resistant. They withstand exposure to strong acids, strong bases, desiccation, UV radiation, organic solvents, many disinfectants, and boiling water (100°C). This resistance stems from several structural features: the spore coat (layers of protective proteins), the cortex (a thick, modified peptidoglycan layer), low water content, and the presence of dipicolinic acid complexed with calcium ions, which stabilizes DNA and proteins against heat denaturation and UV damage. The spore core also contains small acid-soluble proteins (SASPs) that bind to and protect DNA.

Sporulation is initiated by the Spo0A transcription factor, which is activated by a phosphorelay system that integrates multiple nutrient and stress signals. The process involves asymmetric cell division to form a forespore within the mother cell, followed by engulfment and elaboration of the spore coat and cortex.

When environmental conditions become favourable again, the endospore germinates — it detects germinant molecules (often nutrients such as amino acids or sugars) through receptor proteins in the inner spore membrane, rapidly rehydrates, breaks down the cortex, and resumes vegetative growth. This germination process can be extremely rapid — within minutes of exposure to appropriate germinants.

The resistance of endospores to boiling water is the reason autoclaving (121°C, 15 psi for 15–20 minutes) rather than simple boiling is required to ensure sterilization in medical and laboratory settings.

Module 9: Microbial Genetics

9.1 Why Bacteria Are Ideal Genetic Tools

Bacteria offer several advantages as genetic research organisms. They have short generation times (20–30 minutes for fast-growing species), large population sizes (enabling detection of rare mutants), haploid genomes (so recessive mutations are immediately expressed phenotypically), and a rich toolkit of molecular techniques. These advantages mean that genetic experiments possible in bacteria would be impractical in diploid organisms with long generation times.

One third of all bacterial genes currently have unknown functions — a sobering fact that underscores how much remains to be discovered even in the best-studied microorganisms.

9.2 Genetic Terminology

Conventions for describing bacterial genotypes and phenotypes follow specific rules. Genotype designations use lowercase italicised three-letter gene symbols (e.g., metB). Phenotype designations use the same three letters, but capitalized and non-italicised (e.g., Met⁻ for inability to synthesize methionine, Met⁺ for prototrophy).

Selection of mutants uses conditions under which only the desired mutant grows (e.g., plating Met⁻ bacteria on medium lacking methionine selects against them, while plating on methionine-containing medium allows all to grow). Screening requires examining individual colonies for a phenotypic characteristic without directly selecting for it (e.g., testing for starch hydrolysis using iodine).

9.3 Using Auxotrophic Mutants

The logic of auxotrophic mutant analysis is best understood through a worked example. Consider a parental prototroph that is Met⁺ Pro⁺ — it can synthesize both methionine and proline. Two mutants are derived from it: Mutant 1 is Met⁻ Pro⁺ (cannot make methionine), and Mutant 2 is Met⁺ Pro⁻ (cannot make proline).

On complete medium (containing both methionine and proline), all three grow equally well. On minimal medium lacking both amino acids, only the prototroph grows; neither mutant can grow. On medium containing methionine but no proline, Mutant 1 grows (it can make its own proline) but Mutant 2 does not. On medium containing proline but no methionine, Mutant 2 grows but Mutant 1 does not. This logical use of defined media with specific supplements allows mutants to be identified, distinguished from one another, and characterized.

9.4 Restriction Enzymes and Cloning

Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA at specific recognition sequences, typically 4–8 base pairs in length. They are part of the restriction-modification system that protects bacteria against foreign DNA (such as bacteriophage DNA), while host DNA is protected from cleavage by methylation at the same recognition sequences.

Type II restriction enzymes (the kind used in molecular biology) cut at or near their recognition sequence. Many produce staggered cuts that leave short single-stranded sticky ends (also called cohesive ends), while others produce blunt ends.

Cloning is the insertion of a DNA fragment of interest into a self-replicating vector, which is then introduced into a host cell. The central steps are:

1. Both the vector and the source DNA are digested with the same restriction enzyme (in the example below, BamHI), producing compatible sticky ends.
2. The digested fragments are ligated together using DNA ligase, which forms phosphodiester bonds between the compatible ends.
3. The ligation mixture is transformed into E. coli (or another host).
4. Transformants are selected and screened to identify those carrying the desired insert.

A worked example uses the cloning of the Bacillus alpha-amylase gene into a plasmid vector that carries two selectable markers: ampicillin resistance and tetracycline resistance. The BamHI site in the vector lies within the tetracycline resistance gene. When BamHI cuts the vector, it disrupts the tet gene. Four outcomes are possible when the cut vector and cut Bacillus genomic DNA are ligated together and transformed into E. coli:

(1) The vector self-ligates with no insert: ampicillin resistant, tetracycline resistant — because the tet gene is reconstituted. Unwanted. (2) The linearized vector fails to recircularize: no plasmid, no transformant — the colony is lost. (3) A Bacillus DNA fragment (not the amylase gene) inserts into the vector: ampicillin resistant, tetracycline sensitive — because any insert disrupts tet. Partial interest. (4) The correct amylase gene inserts: ampicillin resistant, tetracycline sensitive, and the colonies show clearing around them on starch-iodine plates — because they express amylase. This is the desired outcome.

To distinguish outcomes (3) and (4), replica plating is used. The master plate grown on ampicillin is replica-plated onto both ampicillin and tetracycline plates. Colonies that grow on ampicillin but not tetracycline contain an insert. These candidate clones are then transferred to a starch-containing plate overlaid with iodine: clearing around a colony indicates amylase activity and confirms successful cloning of the functional gene.

9.5 Sanger DNA Sequencing

Sanger sequencing (also called dideoxy chain termination sequencing) was developed by Frederick Sanger and colleagues in 1977 and dominated DNA sequencing for three decades. The method exploits the chemistry of dideoxynucleoside triphosphates (ddNTPs) — nucleotides that lack the 3′-hydroxyl group needed for the next phosphodiester bond. Incorporation of a ddNTP terminates chain extension at that position.

The procedure begins by cloning the DNA of interest into a sequencing vector and rendering it single-stranded by heat denaturation. A short sequencing primer complementary to the vector sequence adjacent to the cloned insert is annealed. DNA polymerase extends from the 3′ end of the primer, copying the unknown insert sequence.

Four separate reactions are set up, each containing all four standard dNTPs and a small proportion of one of the four ddNTPs (ddGTP, ddATP, ddTTP, or ddCTP). Because only a small amount of ddNTP is present relative to the corresponding dNTP, chain extension proceeds normally most of the time but terminates at a low, stochastic frequency whenever a ddNTP is incorporated at the appropriate position. The result in each tube is a population of fragments all sharing the same 5′ end (the primer) but ending at every possible occurrence of one nucleotide type throughout the sequence.

To detect these fragments, a radioactive label (classically ³²P-dCTP) is incorporated. The four reactions are separated by polyacrylamide gel electrophoresis in four adjacent lanes (G, A, T, C). Smaller fragments (shortest extension products, closest to the primer) migrate farthest and appear at the bottom of the gel. After autoradiography, the sequence is read from bottom to top.

Modern Sanger sequencing replaces radioactivity with fluorophore-labelled ddNTPs (a different colour for each nucleotide), allows all four terminators to be combined in a single reaction, and uses capillary electrophoresis with laser detection for automated readout. The principle remains the same but the throughput and ease of use are dramatically greater.

Module 10: Microbial Genomics and Metagenomics

10.1 Genome Sequencing Methods

Sanger sequencing of whole genomes required a primer walking strategy or shotgun sequencing. In primer walking, the initial sequence from one primer is used to design the next primer, and so on, progressively reading along the chromosome. In shotgun sequencing (pioneered for microbial genomes by Craig Venter’s group), the genome is randomly fragmented into small pieces, each end is sequenced, and the resulting millions of short reads are assembled computationally using overlapping regions. The first complete bacterial genome (Haemophilus influenzae, 1995) was assembled this way.

454 pyrosequencing was a second-generation sequencing technology that detected light emitted during nucleotide incorporation rather than using chain termination. DNA fragments are clonally amplified on beads and the sequence is determined by flowing nucleotides one at a time and detecting pyrophosphate release via a luciferase reaction.

Illumina sequencing (sequencing by synthesis) is now the dominant next-generation sequencing platform. Libraries of DNA fragments are generated, ligated to adapters, and amplified into clusters on a flow cell surface. Fluorescently labeled nucleotides with reversible chain terminators are incorporated one at a time. After each cycle, the incorporated nucleotide is identified by its fluorescence before the terminator is removed and the next cycle begins. Illumina generates billions of reads per run at very low cost per base.

Bioinformatics — the application of computational tools to biological data — is indispensable for assembling, annotating, and interpreting genome sequences. De novo assembly of fragmented reads into a complete genome is computationally demanding. Gene calling algorithms identify open reading frames, and functional annotation uses database comparisons (e.g., BLAST) to assign predicted functions to gene products. Despite decades of bacterial genomics, approximately one third of genes in any given bacterium still have no known function.

10.2 Transcriptomics and Proteomics

Microarrays were the first high-throughput transcriptomics tool. A microarray is a glass slide onto which thousands of short DNA sequences (probes), each complementary to a different gene’s transcript, are spotted in a regular grid. mRNA extracted from cells is reverse-transcribed to cDNA, labelled with fluorescent dyes, and hybridized to the array. The intensity of fluorescence at each spot reflects the expression level of the corresponding gene. Comparing arrays from cells grown under different conditions reveals genes that are differentially expressed.

RNA-seq (RNA sequencing) has largely replaced microarrays for transcriptomics, combining the breadth of microarrays with the precision of sequencing and the ability to detect novel transcripts.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separates proteins first by isoelectric point (pI, the pH at which the protein carries no net charge) through isoelectric focusing, and then by molecular mass through standard SDS-PAGE. This two-dimensional separation can resolve thousands of individual protein spots from a complex cell lysate. Individual spots can be excised, digested with trypsin, and identified by mass spectrometry.

Mass spectrometry measures the mass-to-charge ratio (m/z) of ionized peptide fragments. Modern instruments such as tandem mass spectrometers (MS/MS) can sequence peptides de novo and identify proteins by matching fragmentation patterns against protein databases.

10.3 Metagenomics

Traditional microbiology faces a fundamental limitation: the vast majority of environmental microorganisms — estimates suggest over 99% — cannot be grown in pure culture under standard laboratory conditions. Metagenomics circumvents this limitation by extracting and sequencing all genomic DNA from a sample directly. This approach, pioneered in part by researchers at the University of Waterloo including Josh Neufeld and Josh Neufeld’s collaborators, has revealed the staggering diversity of uncultured microorganisms and their metabolic potential.

Metagenomic analyses have transformed our understanding of environments as diverse as ocean water (the Sargasso Sea and Global Ocean Sampling expeditions), the human gut microbiome, soil, hot springs, and aquifer sediments. They have led to the discovery of previously unknown metabolic pathways, the horizontal transfer of genes between distant lineages, and the existence of entire phyla of bacteria known only from environmental sequences.

Two broad strategies are used for metagenomic analysis. In sequence-based metagenomics, DNA is directly shotgun-sequenced and the reads assembled into larger contigs representing partial or complete genomes of individual organisms (metagenome-assembled genomes, MAGs). In function-based metagenomics, metagenomic DNA is cloned into expression vectors, transformed into a host like E. coli, and transformant colonies are screened for specific activities (e.g., cellulase activity, antibiotic resistance). This can reveal functional genes from uncultured organisms.

16S rRNA gene amplicon sequencing (sometimes called amplicon metagenomics or environmental genomics) is a complementary approach that amplifies and sequences the 16S ribosomal RNA gene from all bacteria and archaea in a sample. Because the 16S rRNA gene contains both conserved regions (useful for PCR primer design) and variable regions (useful for distinguishing species), it serves as a “barcode” for microbial communities. Comparisons with databases allow the diversity and relative abundance of taxa in an environmental sample to be determined without culturing a single organism.

10.4 The Winogradsky Column — Environmental Microbiology in Practice

The Winogradsky column is a classic enrichment culture device that replicates in miniature the chemical gradients found in aquatic sediments. It was developed by Sergei Winogradsky in the late nineteenth century during his studies of chemolithotrophy.

A Winogradsky column is constructed by filling a clear glass cylinder with mud and water supplemented with sources of sulfur (e.g., shredded newspaper as an electron donor, calcium sulfate as a sulfur source) and sealing or loosely capping the top. Over weeks to months, distinct zones develop from bottom to top corresponding to oxygen gradients, sulfide gradients, and light availability.

At the anaerobic base, sulfate-reducing bacteria reduce sulfate to hydrogen sulfide. Purple and green sulfur bacteria grow in the anoxic middle zones, using sulfide as an electron donor for anoxygenic photosynthesis. Cyanobacteria and other oxygenic phototrophs dominate the upper, oxygen-rich zone. Chemolithotrophs occupy gradient interfaces.

Observing a wet mount from the surface of an active Winogradsky column under the microscope reveals a remarkably diverse micro-ecosystem. Large, slow-moving cyanobacterial filaments advance by gliding motility. Rotifers with their crown of cilia are constantly active, sweeping bacteria into their digestive systems. Diatoms with their intricate silica shells are readily recognizable. Various protists and bacteria of many morphologies compete and interact in the small volume of a single microscope field. The column thus provides a tangible, visual demonstration of the principles covered throughout the course — metabolic diversity, trophic interactions, and environmental gradients — in a single vessel.

Module 9 Supplementary: Fungi — Mushrooms and Rhytisma

Fungi as Microbial Eukaryotes

Although many fungi form large macroscopic structures, their biology is fundamentally microbial. Fungal bodies are built from hyphae — long, thread-like filaments that grow by tip elongation. A mass of hyphae is called a mycelium. Hyphae penetrate substrates and secrete extracellular hydrolytic enzymes, absorbing the soluble breakdown products.

Mushrooms are the reproductive fruiting bodies produced by certain fungi (primarily Basidiomycetes), not the organism itself. The visible mushroom is a temporary structure for spore dispersal; the bulk of the fungal biomass is the mycelium ramifying invisibly through the substrate.

Dryad’s saddle (Polyporus squamosus) is a conspicuous bracket fungus found on living and dead deciduous trees in temperate forests, including those close to the University of Waterloo campus. It produces large, shelf-like fruiting bodies that can reach diameters of over 50 cm. The species name refers to the dryad — a tree nymph of classical mythology — suggesting the mushroom’s association with trees. Fruiting bodies emerge primarily in spring and sometimes again in autumn. They have a characteristic scaly, brown upper surface and a white underside covered in small pores (hence their classification as polypores). When a small piece is broken off, it releases a distinctive smell often described as mealy or reminiscent of watermelon rind. Dryad’s saddle fruiting bodies occur in multiple size classes on a single tree, from tiny primordia just beginning to form to fully mature specimens.

The mycelium of P. squamosus penetrates deeply into the heartwood of its host tree, causing white rot — degradation of lignin and cellulose. Because the mycelium is invisible from outside, a tree can appear relatively healthy while being heavily colonized. The emergence of fruiting bodies from a tree trunk or major branch reveals the extent of internal fungal colonization.

Rhytisma acerinum — Tar Spot of Maple

Rhytisma acerinum is an ascomycete fungus that causes tar spot disease on maple leaves (genus Acer). It is a biotrophic parasite — it grows only on living tissue and does not kill the leaf directly. The infection manifests as large, glossy, black spots on the upper surface of the leaf, hence the common name “tar spot.”

The disease cycle begins in spring when the overwintered dead leaves on the ground release ascospores from structures called apothecia embedded in the tar spots. The spores are dispersed by rain and wind onto newly emerged maple leaves. Germination and infection proceed through the growing season, with visible tar spots appearing by midsummer and expanding through autumn.

One particularly interesting observation: in autumn, the region of the leaf immediately surrounding the tar spot often retains green chlorophyll while the rest of the leaf has yellowed. This suggests that the fungus may manipulate host leaf senescence, possibly by producing cytokinins or other plant growth regulators that delay chlorophyll breakdown. Continued photosynthesis by the green halo surrounding the infection may provide carbohydrates that sustain the fungus as the rest of the leaf senesces. This is a striking example of parasitic manipulation of host physiology.

Although tar spot disease creates dramatic symptoms, it is largely cosmetic and does not cause severe long-term harm to the tree. The most effective management strategy is raking and removing fallen leaves in autumn, which disrupts the overwintering phase and reduces the spore load available to re-infect the tree in spring. Rhytisma is sensitive to air pollution, and its presence on maple trees is considered an indicator of relatively clean air quality — it is rarely found in heavily polluted urban environments.

Rhytisma is easy to find on maple leaves in the Waterloo region every summer and autumn and provides a vivid, accessible example of how fungal parasites interact with their hosts and how microbiology can be experienced directly in everyday natural environments.

Module 10 Supplementary: Microbial Ecology and Biogeochemistry

Microbial Community Analysis

Understanding microbial communities in nature requires tools that go beyond culturing individual organisms. The collection of genomes in an environmental sample constitutes the metagenome, the collection of all mRNA transcripts is the metatranscriptome, all proteins constitute the metaproteome, and all metabolites make up the metabolome. Together these omics approaches offer a comprehensive, multi-resolution view of who is present in a community, what genes they carry, which genes are being expressed, which proteins are active, and which metabolic reactions are occurring.

An important practical consideration in omics-based analysis is that the technology and bioinformatic tools are continually evolving. As more reference genomes are deposited in databases and as assembly algorithms improve, the same raw sequencing data can yield increasingly refined insights when re-analyzed. This means that datasets have a long life: raw sequence data deposited in repositories today will continue to be analyzed and re-analyzed as the field advances.

Life in Darwin’s Dust: Intercontinental Microbial Transport (Gorbushina et al., 2007)

The assigned micro-news reading “Life in Darwin’s dust: intercontinental transport and survival of microbes in the nineteenth century” (Environmental Microbiology, 2007) by Gorbushina, Kort, Schulte, Lazarus, Schnetger, Brumsack, Broughton, and Favet connects directly to themes of microbial ecology, endospore survival, and the global scale of microbial distribution.

Historical Context: Darwin, Ehrenberg, and Aeolian Dust

Charles Darwin, during the HMS Beagle voyage in January 1832, observed impalpably fine brown dust falling on the ship near the Cape de Verd archipelago. He collected samples and passed them to Christian Gottfried Ehrenberg — a pioneer of aerobiology — at the Royal Prussian Academy of Sciences in Berlin. Ehrenberg identified 67 different organic forms in Darwin’s five small packets. This collection was donated to the Prussian Academy before Ehrenberg’s death in 1876 and is now housed in the Museum für Naturkunde (Natural History Museum) of the Humboldt University in Berlin.

The Gorbushina et al. study accessed this irreplaceable collection in April 2006 to apply modern molecular-microbiological methods to nineteenth-century aeolian dust — samples gathered at the onset of the Industrial Revolution, before the globalization of industry, providing a relatively pristine baseline.

Geochemistry: Tracing the Dust to West Africa

Geochemical analyses (ICP-OES and ICP-MS elemental profiling) of samples collected over the Atlantic in 1838 showed enrichment patterns consistent with an origin in the Western Sahara — specifically the calcareous, carbonate-rich soils of that region. The Barbados samples from 1812 showed a distinct fractionation pattern reflecting the long-range transport across the Atlantic, with depletion of coarse-grained K-feldspar and heavy minerals. The largest single source of Saharan dust on the planet is the Bodélé Depression in northern Chad, where a gap between mountain ranges funnels winds onto a chalky diatomaceous desert, producing dust clouds stretching thousands of kilometres.

Microbial Results: What Survived for Nearly Two Centuries

From the historic dust samples, the researchers cultivated 48 bacterial isolates belonging to 17 distinct species, identified by 16S rRNA gene sequencing. All were spore-forming bacteria — specifically members of the Bacillaceae and related families. The species identified included:

• Bacillus megaterium, B. subtilis, B. pumilus, B. licheniformis, B. cereus, B. firmus, B. simplex, B. barbaricus, B. fusiformis, B. funiculus
• Brevibacillus brevis, Cohnella ginsengisoli, and multiple Paenibacillus species

Importantly, only two fungal species were cultivated from the historic dust: Aspergillus versicolor and Davidiella tassiana (a Cladosporium relative) — both slow-growing. In sharp contrast, contemporary museum air and dust harboured 17 fungal species and a much broader bacterial community including non-spore-forming Actinobacteria (Arthrobacter, Micrococcus, Curtobacterium). The stark microbiological differences between historic and museum samples confirm that the historic samples were not contaminated during their time in Berlin institutions.

The colony-forming unit counts from the oldest samples (Barbados, 1812) were approximately 10⁴ cfu g⁻¹ dust — remarkably high for nearly two-century-old specimens.

The Endospore Connection

The exclusively spore-forming bacterial community in the historic dust is consistent with the course’s material on endospores (Module 8 supplementary). The ability of Bacillus species to form endospores — metabolically dormant structures resistant to desiccation, UV radiation, and temperature extremes — explains their survival across centuries and during multi-week intercontinental transport.

To test this directly, the authors inoculated B. megaterium isolated from historic dust onto dry quartz sand. Within one week, all vegetative cells had converted to spores that clung to the sand grains. After 10 weeks at 20% relative humidity, the spores remained viable. As a non-spore-forming control, Rhizobium sp. NGR234 (a gram-negative rhizobial symbiont of legumes) was similarly inoculated onto dry sand. Its numbers declined steadily, but it still maintained almost 10⁴ viable cells g⁻¹ sand after 9 weeks — and those survivors retained full symbiotic competence when inoculated into Vigna unguiculata (cowpea) plants. This suggests that even non-spore-forming bacteria present in Saharan topsoils (Acacia-associated rhizobia are abundant in the Sahel) could potentially survive transport and seed new habitats.

Implications for Microbial Biogeography

The study provides direct empirical evidence for the long-suspected but difficult-to-prove concept that the diversity of microbial communities in geographically separated habitats is influenced by wind-driven dispersal. Saharan dust storms deposit enormous loads of minerals (providing nutrients, particularly phosphorus, to otherwise oligotrophic ecosystems like the Amazon rainforest and Caribbean coral reefs) along with viable microbial communities. Changes in land use and desertification in source areas may therefore alter the types of microorganisms being dispersed globally — with potential consequences for plant pathogens, coral reef microbiomes, and other distant ecosystems.

The paper’s concluding observation echoes Ehrenberg’s own prediction in 1851: “Probably even in 100 years research will find interest in carefully collected (dust) material be it on behalf of meteorology or of the study of organic life within.” The Ehrenberg collection — gathered at the onset of the Industrial Revolution — now provides a pre-industrial baseline against which the biological consequences of industrial land use and atmospheric change can be assessed.

Aquatic Metagenomics

Marine environments harbour extraordinary microbial diversity. The SAR11 clade (Candidatus Pelagibacter ubique) is one of the most abundant organisms on Earth, estimated to comprise one quarter of all marine bacteria. It was not described until 2002, despite the fact that it numerically dominates most ocean surface water samples — a direct consequence of the fact that it cannot be easily cultivated on standard media. Its discovery required metagenomic and cultivation efforts simultaneously.

Shotgun metagenomics of ocean water (the Global Ocean Sampling expedition led by Craig Venter) recovered millions of genes from uncultured organisms, including hundreds of previously unknown rhodopsin-like light-harvesting proteins — suggesting that light-driven proton pumping is far more widespread in marine bacteria than previously appreciated.

Soil Microbiology

Soil is arguably the most complex microbial habitat on Earth. A single gram of productive agricultural soil may contain one billion bacterial cells from thousands of distinct species, in addition to fungi, archaea, protozoa, nematodes, and viruses. Soil microbial communities drive the decomposition of plant litter, the mineralization of organic nitrogen and phosphorus, the formation of stable soil organic matter, and the maintenance of soil structure.

The Winogradsky column is a miniature model of this complexity. Its layered gradients of oxygen, sulfide, light, and organic matter create microenvironments that support diverse metabolic guilds simultaneously. The ability to observe cyanobacteria gliding, rotifers feeding, diatoms drifting, and protists hunting in a single wet mount from the column’s surface illustrates how much complexity can be packed into a few millilitres of mud — and by extension, into any handful of soil or sediment from the natural world.

The Microbiome

The human microbiome — the collective community of microorganisms inhabiting the human body — has attracted enormous research interest since the advent of 16S rRNA gene amplicon sequencing and metagenomics. The gut microbiome alone harbours approximately 10¹³ microbial cells and encodes a gene repertoire roughly 150 times larger than the human genome. These microorganisms carry out metabolic functions that the human genome cannot: fermenting dietary fibre to short-chain fatty acids (SCFAs) such as butyrate, propionate, and acetate; synthesizing vitamins B12 and K₂; training the immune system; and metabolizing xenobiotics and drugs.

Emerging research suggests that the gut microbiome may influence host behaviour and mental health through the gut-brain axis — bidirectional communication pathways involving neural, endocrine, and immune signals. Specific gut bacterial metabolites, including neurotransmitter precursors and direct neuroactive compounds, can influence the production of serotonin, dopamine, and gamma-aminobutyric acid (GABA). This is one of the most rapidly developing areas of microbiome research and represents a meeting point of microbiology, neuroscience, and psychiatry.

Course Conclusion

The course concludes with the observation that microbiology is not a discipline in which all answers have been found — far from it. One third of bacterial genes have unknown functions. Vast portions of the microbial world have never been cultivated. The biochemistry of archaea and their precise evolutionary relationship to eukaryotes continues to be refined as new genomic data accumulate. The human microbiome exerts influences on physiology and potentially even behaviour and mental health that are only beginning to be characterized.

BIOL 240 has introduced the foundational vocabulary, experimental methods, and conceptual frameworks needed to engage with contemporary microbiology: from van Leeuwenhoek’s metal-plate microscope to Illumina sequencers generating billions of reads; from Koch’s postulates to CRISPR-based genome editing; from the gram stain to metagenomics. The organisms discussed — bacteria, archaea, eukaryotic microbes, fungi, and viruses — are not isolated academic curiosities but essential participants in the planetary systems that sustain all life. Microbes cycle carbon, nitrogen, sulfur, and phosphorus through the biosphere. They underlie the food and pharmaceutical industries. They cause disease and they cure it. They have shaped Earth’s history and will shape its future.

The tools and concepts covered in this course — taxonomy and phylogeny, cell structure and function, metabolism and growth, genetics and gene regulation, genomics and community analysis — form the foundation for upper-level courses in microbial ecology, infectious disease, biotechnology, and environmental science. They also provide the conceptual vocabulary to engage as an informed citizen with the microbiological dimensions of climate change, pandemic preparedness, food security, and the human microbiome.

Two faculty members at the University of Waterloo contributed to the course materials and micro-news readings: Dr. Josh Neufeld and Dr. Josh Gilbert. Their co-authored article asking “what would a world without microbes be like?” was assigned as the first micro-news reading — an appropriate entry point for a course that ultimately argues the answer to that question is: a world without higher life as we know it. The Winogradsky column that appears in the Module 10 video footage was maintained in Dr. Neufeld’s office, and the mushroom and tar-spot observations were filmed in the woods and backyard close to the University of Waterloo campus — a reminder that microbiology is not confined to the laboratory but is woven into every square metre of the living landscape around us.

Appendix: Key Concepts Summary

Phylogenetic Foundations

The three-domain tree of life — Bacteria, Archaea, Eukarya — rests on comparative analyses of conserved molecular sequences, particularly 16S/18S ribosomal RNA. The 16S rRNA molecule is ideal for this purpose because it is present in all cellular life, functionally constrained (so it evolves slowly enough to preserve ancient relationships), and contains both conserved regions (useful as PCR primer targets) and variable regions (useful for distinguishing taxa). The phylogenetic tree built from 16S rRNA sequences was the key insight that allowed Carl Woese to recognize archaea as a separate domain, not merely exotic bacteria.

Importantly, no single gene tells the complete story of microbial evolution. Horizontal gene transfer (HGT) — the movement of genes between organisms other than through vertical inheritance from parent to offspring — is pervasive in the microbial world. Genes can move by transformation (uptake of naked DNA from the environment), transduction (phage-mediated transfer), and conjugation (direct cell-to-cell transfer through a pilus). HGT blurs phylogenetic boundaries, explains the rapid spread of antibiotic resistance, and contributes to the observation that different genes within the same organism can have very different evolutionary affinities — as seen clearly in the MreB/actin phylogenetic tree.

DNA Transfer Mechanisms

Transformation involves the uptake of DNA from the environment by naturally competent cells or cells made artificially competent by chemical or electrical treatment. In the laboratory, transformation is used to introduce plasmids into E. coli. In nature, transformation can transfer any genes present in environmental DNA.

Conjugation requires direct cell contact mediated by the sex pilus. The F (fertility) factor is the prototypical conjugative plasmid in E. coli. Cells carrying the F factor (F⁺ cells) can transfer it to F⁻ cells. When the F factor integrates into the bacterial chromosome, the cell becomes an Hfr (high-frequency recombination) strain. During conjugation between an Hfr cell and an F⁻ cell, chromosomal DNA is transferred beginning at the oriT site (origin of transfer), but the entire chromosome is rarely transferred because conjugation is usually interrupted before completion. The order in which chromosomal markers are transferred reflects their distance from oriT, enabling genetic mapping by interrupted mating experiments.

Transduction is the phage-mediated transfer of bacterial DNA. In generalized transduction, a phage accidentally packages a fragment of bacterial DNA (rather than phage DNA) during the lytic cycle; this DNA can be injected into a new host and recombined into the chromosome. In specialized transduction, an integrated prophage excises imprecisely, carrying adjacent bacterial genes along with phage DNA; upon infection of a new host, these bacterial genes can be expressed or integrated.

Transposons (mobile genetic elements) can move within and between genomes by a “cut-and-paste” (non-replicative) or “copy-and-paste” (replicative) mechanism. They carry genes for their own transposition (transposase) and often carry accessory genes including antibiotic resistance determinants.

Motility Beyond Flagella

The bacterial flagellum is the canonical motility organelle but it is not the only means by which bacteria move. Gliding motility occurs in the absence of flagella in several phylogenetically diverse groups: myxobacteria, cyanobacteria, Flavobacterium, and others. The molecular mechanisms of gliding differ between groups but often involve type IV pilus extension-retraction or the secretion of slime. Gliding motility is important in biofilm formation and in predation. Swarming motility is a flagella-dependent surface translocation involving co-ordinated multicellular movement facilitated by induction of additional flagella and production of biosurfactants. Spirochete motility is driven by the rotation of periplasmic flagella (axial filaments) that lie between the inner and outer membranes; their rotation causes the helical cell body to corkscrew through viscous environments — an important adaptation for spirochetes that infect tissues such as connective tissue and mucus.

Nitrogen Cycling

The global nitrogen cycle is almost entirely driven by microorganisms. Atmospheric nitrogen (N₂) is biologically inert and must be fixed (converted to NH₃) by nitrogen-fixing bacteria and archaea before it can be used by most living things. Nitrification converts NH₃ sequentially to NO₂⁻ and NO₃⁻ (by Nitrosomonas and Nitrobacter respectively). Plants take up nitrate and ammonium from soil. Denitrification returns fixed nitrogen to the atmosphere as N₂ or N₂O, completing the cycle. Ammonification releases NH₃ from the decomposition of organic nitrogen.

The nitrogen cycle illustrates a recurring theme of the course: major planetary biogeochemical processes are microbially catalysed and could not proceed without microbial activity. Removing microbes from Earth would collapse global elemental cycles within decades.

Horizontal Gene Transfer and Its Consequences

Horizontal gene transfer (HGT) is the movement of genetic information between organisms by means other than direct parent-to-offspring inheritance. It is pervasive among prokaryotes and has shaped their evolution far more profoundly than was appreciated before the genomic era. Analyses of fully sequenced genomes consistently show that a substantial fraction — often 15–20% — of any given bacterium’s genome was acquired from other organisms by HGT at some point in its evolutionary history.

The three mechanisms of HGT are transformation, conjugation, and transduction (all described in detail in Module 9). Their cumulative consequence is that beneficial genes — including those encoding antibiotic resistance, novel metabolic pathways, virulence factors, and degradative enzymes — can spread rapidly through bacterial populations and even jump between distantly related species. This is why antibiotic resistance genes found in hospital pathogens can appear shortly afterward in soil bacteria: the resistance plasmid has been transferred horizontally across phylogenetic boundaries.

HGT also complicates phylogenetic reconstruction. The standard assumption of phylogenetics — that all organisms inherit their genes from their ancestors — is violated by HGT. A “species” phylogenetic tree built from different genes can yield conflicting topologies, because different genes have different evolutionary histories. This is one reason why the boundaries between prokaryotic “species” are inherently fuzzy and why concepts such as the “core genome” (genes present in all strains of a species) and the “pan-genome” (all genes found in any strain of a species) have become central to microbial genomics.

Antibiotic Resistance

Antibiotic resistance arises through mutation and through acquisition of resistance genes by HGT. The major biochemical mechanisms of resistance include: (1) enzymatic inactivation of the antibiotic (e.g., beta-lactamases cleave the beta-lactam ring of penicillins and cephalosporins); (2) modification of the antibiotic target (e.g., altered penicillin-binding proteins in MRSA); (3) active efflux pumps that expel the antibiotic from the cell; and (4) reduced permeability through loss of porins. The rise of multi-drug resistant pathogens is one of the most pressing public health challenges of the twenty-first century and is driven by the selective pressure of antibiotic use in medicine, agriculture, and animal husbandry.

Microbial Evolution: Mutation and Natural Selection

Mutations are the ultimate raw material of evolution. In bacteria, mutations arise by replication errors, base modifications (oxidation, deamination), and DNA damage by UV or chemicals. The spontaneous mutation rate in bacteria is approximately 10⁻⁹ to 10⁻¹⁰ per base pair per replication. Although this rate per base is low, a culture of E. coli at exponential phase contains roughly 10⁹ cells per mL, and each cell replicates its entire 4.6 Mb chromosome every generation. The cumulative mutational output across a large bacterial population is therefore substantial.

Point mutations change a single base pair and can be silent (synonymous codon, no amino acid change), missense (non-synonymous codon, amino acid change), or nonsense (introduces a stop codon, truncating the protein). Insertion-deletion (indel) mutations alter the reading frame of a gene and are usually strongly disruptive to protein function downstream of the mutation.

Bacteria also undergo large-scale genomic changes including inversions, duplications, and deletions of genomic segments. Gene duplications are particularly important evolutionarily because one copy retains the original function while the other can accumulate mutations and potentially acquire a new function — a process called neofunctionalization. This is one mechanism by which the extraordinary metabolic diversity of bacteria has been generated over evolutionary time.

Biogeochemical Impact of Microorganisms

To appreciate the full scope of microbial influence, consider: cyanobacteria produced the oxygen atmosphere; methanogens are responsible for a significant fraction of annual methane emissions; sulfate-reducing bacteria in anoxic sediments couple the oxidation of organic matter to sulfate reduction, releasing hydrogen sulfide and driving the sulfur cycle; iron-oxidizing bacteria such as Acidithiobacillus ferrooxidans are used in bioleaching of metal ores; nitrogen-fixing symbionts in legume root nodules make leguminous crops possible without synthetic nitrogen fertilizer; and the decomposing fungi and bacteria in forest soils recycle the nutrients locked in dead plant biomass back into forms available to living plants.

Approximately half of all photosynthesis on Earth is performed by marine phytoplankton and cyanobacteria. Conversely, approximately 20–40% of marine microbial biomass is killed by viruses every day, releasing dissolved organic carbon and nutrients into the water column via the viral shunt. These numbers illustrate that microbial processes operate on planetary scales and are not peripheral to Earth’s biology — they are central to it.

Gram Stain Decision Table

The following logic summarizes how to interpret Gram stain results together with cell morphology — a fundamental diagnostic workflow in clinical and environmental microbiology.

A cell that retains crystal violet after decolorization (appears purple under the microscope) is Gram-positive, indicating a thick peptidoglycan wall and the absence of an outer membrane. A cell that is decolorized and then takes up the safranin counterstain (appears pink) is Gram-negative, indicating a thin peptidoglycan layer sandwiched between inner and outer membranes. Combining Gram reaction with cell morphology gives four basic categories immediately visible from a single stained slide: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods. Each category carries epidemiological and clinical implications: Gram-positive cocci include Staphylococcus and Streptococcus; Gram-negative rods include E. coli, Pseudomonas, and Salmonella; Gram-positive rods include Bacillus, Clostridium, and Lactobacillus.

The Gram stain is technically sensitive: over-decolorization can make Gram-positive cells appear Gram-negative (false negative), and under-decolorization can leave Gram-negative cells appearing Gram-positive (false positive). Organisms with unusually thin peptidoglycan, aged cultures, or cells damaged by antibiotics can give variable results. Some organisms, such as Mycobacterium, do not stain well by the Gram procedure because their thick, waxy mycolic acid layer is impermeable to the crystal violet–iodine complex, necessitating the alternative acid-fast stain (Ziehl-Neelsen) for diagnosis.

It is also worth noting that Gram staining a pure culture of unknown bacteria is one of the first steps in identification — together with colony morphology on a plate, presence or absence of motility (wet mount), and simple biochemical tests such as the catalase test (bubble production from H₂O₂ distinguishes catalase-positive Staphylococcus from catalase-negative Streptococcus). These classical approaches remain in routine clinical use today, complemented by and increasingly replaced by MALDI-TOF mass spectrometry for rapid species identification.

Notes compiled from lecture transcripts, module HTML content, and course materials for BIOL 240, Spring 2021, University of Waterloo. Textbook: Wessner, Dupont, Charles, Neufeld, Microbiology 3rd ed., Wiley 2020.