Difficulty: Intermediate | Prerequisites: Exam 1 material (cell structure, metabolism, molecular information flow, microbial growth)
This second block builds on the molecular and metabolic foundations from Exam 1. You now learn how microbes regulate their gene expression in response to environmental signals, how viruses hijack cellular machinery to replicate, how bacteria exchange genetic material (horizontal gene transfer), and how humans exploit microbial genetics through biotechnology. The block closes with microbial evolution and systematics, which gives you the phylogenetic framework for classifying the enormous diversity of microbial life. If you missed the first exam material on transcription, translation, and operons, go back and review it before tackling this section, because regulation, viral replication, and genetic engineering all depend on that foundation.
Bacteria regulate gene expression at multiple levels (transcriptional, post-transcriptional, and post-translational) to adapt quickly to changing environments. Viruses are obligate intracellular parasites with diverse replication strategies dictated by their genome type. Bacteria share genes horizontally through transformation, transduction, and conjugation, fuelling rapid evolution. Biotechnology harnesses these genetic mechanisms for cloning, sequencing, and engineering organisms. Systematics uses molecular phylogenetics (primarily rRNA sequencing) to organise microbial diversity into a coherent tree of life.
Operon
A set of genes transcribed as a single mRNA under the control of one promoter and one or more regulatory elements. Allows coordinated expression of genes in a pathway.
Repressor
A regulatory protein that binds to the operator region of an operon and blocks RNA polymerase from transcribing the downstream genes. In simple terms, it is the "off switch."
Inducer
A small molecule that binds to a repressor and causes it to release from the operator, allowing transcription. For example, allolactose induces the lac operon. Think of it as the key that unlocks the off switch.
Lac operon
The classic inducible operon in E. coli for lactose metabolism. It contains lacZ (beta-galactosidase), lacY (permease), and lacA (transacetylase), regulated by the LacI repressor and subject to catabolite repression via the CAP-cAMP system.
Catabolite repression
A global regulatory mechanism ensuring that glucose is used preferentially over other carbon sources. When glucose is present, cAMP levels are low, so the CAP-cAMP activator complex does not form, and operons for alternative sugars (like the lac operon) remain poorly expressed even if the inducer is present.
Two-component regulatory system
A signal transduction mechanism consisting of a sensor kinase (in the membrane, detects an environmental stimulus) and a response regulator (in the cytoplasm, typically a transcription factor). The sensor kinase autophosphorylates and transfers the phosphate to the response regulator, activating it. Ubiquitous in bacteria and archaea.
Quorum sensing
Cell-density-dependent gene regulation. Bacteria produce and release small signalling molecules (autoinducers, such as acyl-homoserine lactones in Gram-negatives). When the population density is high enough, the autoinducer concentration reaches a threshold that triggers coordinated gene expression (e.g., bioluminescence in Vibrio fischeri, biofilm formation, virulence factor production).
Sigma factor
A dissociable subunit of bacterial RNA polymerase that directs the enzyme to specific promoter sequences. Alternative sigma factors allow cells to rapidly redirect transcription in response to stress (e.g., sigma-32 for heat shock, sigma-S for stationary phase).
Attenuation
A regulatory mechanism that controls transcription termination in the leader region of certain biosynthetic operons (e.g., the trp operon). Translation of a short leader peptide rich in tryptophan codons determines whether a terminator hairpin forms. When tryptophan is abundant, ribosomes translate the leader quickly, the terminator forms, and transcription stops early.
Riboswitch
A segment of the 5' untranslated region of an mRNA that can bind a small molecule (metabolite) directly, causing a conformational change that either terminates transcription or blocks translation. No protein regulator is needed. In simple terms, the mRNA itself acts as the sensor and the switch.
Virus (virion)
An acellular infectious agent consisting of a nucleic acid genome (DNA or RNA, single-stranded or double-stranded) enclosed in a protein coat (capsid), sometimes with a lipid envelope. Obligate intracellular parasites: they cannot replicate outside a host cell.
Capsid
The protein shell of a virus, assembled from repeating protein subunits (capsomeres). Common symmetries: icosahedral (20-faced, roughly spherical), helical (rod-shaped), and complex (e.g., bacteriophage T4 with head, tail, and baseplate).
Bacteriophage (phage)
A virus that infects bacteria. Phages are the most abundant biological entities on Earth. Key model phages include T4 (lytic), lambda (temperate), and M13 (filamentous).
Lytic cycle
A viral replication strategy in which the phage takes over the host cell, replicates its genome, assembles new virions, and lyses the cell to release progeny. Think of it as a smash-and-grab.
Lysogeny (temperate phage)
A viral replication strategy in which the phage genome integrates into the host chromosome (as a prophage) and replicates passively with the host. The prophage can later excise and enter the lytic cycle (induction), typically triggered by DNA damage (SOS response).
Lysogenic conversion
When a prophage confers new phenotypic properties on its bacterial host, such as toxin production. Classic examples: the cholera toxin gene is carried by phage CTXφ in Vibrio cholerae; the Shiga toxin gene is carried by a lambdoid phage in E. coli O157:H7.
Retrovirus
An RNA virus that uses reverse transcriptase to synthesise a DNA copy of its genome, which then integrates into the host chromosome. HIV is the most well-known example.
Transformation
The uptake of free DNA from the environment by a competent bacterial cell. Some species are naturally competent (e.g., Streptococcus pneumoniae, Bacillus subtilis); others can be made artificially competent (e.g., E. coli by CaCl₂ treatment or electroporation).
Transduction
Transfer of bacterial DNA from one cell to another via a bacteriophage. Generalised transduction: random fragments of host DNA are accidentally packaged into a phage head. Specialised transduction: genes adjacent to the prophage insertion site are excised along with the phage DNA.
Conjugation
Direct cell-to-cell transfer of DNA through a pilus (or direct contact). Requires a conjugative plasmid (e.g., the F plasmid in E. coli). The donor (F⁺) synthesises a pilus, makes contact with the recipient (F⁻), and transfers a single strand of the plasmid, which is then replicated in both cells.
F plasmid
The fertility plasmid of E. coli that encodes the genes for pilus formation and DNA transfer. When integrated into the chromosome, the cell is called Hfr (high-frequency recombination) and can transfer chromosomal genes to an F⁻ recipient.
Plasmid
A small, autonomously replicating, extrachromosomal DNA element. Common in bacteria. May carry genes for antibiotic resistance (R plasmids), virulence, metabolic functions, or conjugation.
Transposon (transposable element)
A mobile genetic element that can move from one location to another within or between DNA molecules. Insertion sequences (IS elements) carry only the transposase gene flanked by inverted repeats. Composite transposons carry additional genes (often antibiotic resistance) between two IS elements.
CRISPR-Cas
A prokaryotic adaptive immune system. Bacteria store short sequences (spacers) derived from past viral infections in CRISPR arrays. These are transcribed and processed into guide RNAs that direct Cas nucleases to cut matching foreign DNA. In simple terms, it is a bacterial "most wanted" database that lets the cell recognise and destroy returning invaders. Also the basis of the CRISPR-Cas9 genome editing tool.
Restriction enzyme (restriction endonuclease)
A bacterial enzyme that cuts DNA at specific short palindromic sequences. Part of the restriction-modification defence system against foreign DNA. Widely used in molecular cloning.
Polymerase chain reaction (PCR)
A laboratory technique that amplifies a specific segment of DNA through repeated cycles of denaturation, primer annealing, and extension by a thermostable DNA polymerase (e.g., Taq polymerase). In simple terms, it makes millions of copies of a target DNA sequence in a few hours.
Gel electrophoresis
A technique for separating DNA, RNA, or proteins by size using an electric field through an agarose or polyacrylamide gel. Smaller fragments migrate faster.
Recombinant DNA
DNA molecules formed by joining fragments from different sources, typically using restriction enzymes and DNA ligase. The foundation of genetic engineering.
Cloning vector
A DNA molecule (plasmid, phage, cosmid, BAC) used to carry a foreign DNA insert into a host cell for replication. Must contain an origin of replication, a selectable marker (e.g., antibiotic resistance gene), and a cloning site (multiple cloning site).
Gene expression system
A setup for producing large quantities of a protein from a cloned gene, typically using a strong, inducible promoter (e.g., the T7 promoter in the pET system) in a host like E. coli.
Metagenomics
The culture-independent study of genetic material recovered directly from environmental samples. Allows analysis of microbial communities without the need to isolate and culture individual species.
Phylogenetics
The study of evolutionary relationships among organisms, often inferred from molecular sequences. In microbiology, 16S rRNA (for bacteria and archaea) and 18S rRNA (for eukaryotes) are the gold-standard phylogenetic markers.
Molecular clock
The concept that mutations accumulate in conserved genes (like rRNA) at a roughly constant rate over time, allowing estimation of when lineages diverged.
Horizontal gene transfer (HGT)
The transfer of genes between organisms that are not parent and offspring. Includes transformation, transduction, and conjugation. A major driver of microbial evolution, it complicates the tree-of-life model by introducing "web-like" connections between lineages.
Systematics/taxonomy
The science of classifying and naming organisms. The hierarchy runs domain, phylum, class, order, family, genus, species. Bacterial species are defined operationally (e.g., >70% DNA-DNA hybridisation, or >95–97% 16S rRNA sequence identity for the same genus, >98.7% for the same species).
Levels of regulation:
Transcriptional: most common; controls whether and how much mRNA is made (repressors, activators, sigma factors, two-component systems)
Post-transcriptional: riboswitches, small regulatory RNAs (sRNAs), mRNA stability, attenuation
Post-translational: allosteric regulation of enzyme activity (feedback inhibition), covalent modification (phosphorylation, methylation), proteolytic degradation
Negative control (repression):
Repressor protein binds operator, blocking transcription
Inducible system (e.g., lac operon): repressor is active by default; inducer inactivates it
Repressible system (e.g., trp operon): repressor is inactive by default; corepressor (tryptophan) activates it
Positive control (activation):
Activator protein binds near the promoter and helps RNA polymerase bind or begin transcription
CAP-cAMP in the lac operon is a classic positive regulator
Catabolite repression and the lac operon in detail:
Full expression of the lac operon requires both the absence of glucose (so cAMP is high and CAP-cAMP activates) and the presence of lactose (so allolactose inactivates the LacI repressor)
This is an example of dual control: both positive and negative regulators must be in the correct state
Two-component systems: sensor kinase detects stimulus (e.g., osmolarity, nutrient availability), autophosphorylates a histidine residue, transfers the phosphate to an aspartate on the response regulator, which then activates or represses target genes
Quorum sensing: allows population-level coordination. Gram-negatives often use acyl-homoserine lactones (AHLs); Gram-positives often use processed oligopeptides. AI-2 is a "universal" signal used for interspecies communication
Global regulatory networks:
Regulons: sets of operons controlled by a single regulatory protein
Stimulons: all genes that respond to a specific environmental stimulus, regardless of which regulators are involved
Stringent response: under amino acid starvation, the alarmone ppGpp accumulates and redirects transcription away from growth-related genes toward survival genes
Chromosome replication is coordinated with cell division:
In fast-growing E. coli, new rounds of replication initiate before the previous round finishes (overlapping replication forks)
DnaA protein triggers initiation at oriC
Chromosome segregation: replicated origins move to opposite cell poles, aided by the Par system and condensins
Cell division machinery:
FtsZ forms a ring at midcell (the Z-ring), which recruits other division proteins (the divisome)
MinCDE system prevents Z-ring formation at the poles, ensuring division occurs at the centre
In some organisms, the nucleoid occlusion system also prevents division over unreplicated chromosomes
Viral structure:
Nucleic acid core: DNA or RNA, single-stranded (ss) or double-stranded (ds), linear or circular, segmented or non-segmented
Capsid: icosahedral, helical, or complex symmetry
Envelope: lipid bilayer derived from host membrane (present in some viruses, e.g., influenza, HIV)
Some phages have tails, baseplates, and tail fibres for host attachment
Baltimore classification: classifies viruses by genome type and replication strategy into seven groups (dsDNA, ssDNA, dsRNA, positive-sense ssRNA, negative-sense ssRNA, ssRNA-RT, dsDNA-RT)
Phage lytic cycle (e.g., T4):
Attachment (adsorption) to host surface receptor
Penetration (injection of DNA)
Biosynthesis: early genes (shut down host, degrade host DNA, modify RNA polymerase), late genes (structural proteins, lysis proteins)
Assembly (self-assembly of heads, tails, tail fibres)
Release (lysis via lysozyme and holin)
Lysogeny (e.g., phage lambda):
Phage DNA integrates into the host chromosome at attB (host) and attP (phage) sites via integrase
CI repressor maintains the lysogenic state by repressing lytic genes
Induction: DNA damage activates RecA, which stimulates self-cleavage of CI repressor, and the phage enters the lytic cycle
Lysogenic conversion: prophage genes expressed during lysogeny can alter host phenotype
Animal virus replication: similar stages (attachment, entry, uncoating, replication, assembly, release) but entry is usually by receptor-mediated endocytosis or membrane fusion (enveloped viruses); release by budding (enveloped) or cell lysis (non-enveloped)
Viruses are extraordinarily diverse in genome size (a few kb to over 1 Mb for giant viruses), structure, and host range
Viruses are the most abundant biological entities in the ocean and play critical roles in microbial population control and nutrient cycling (the "viral shunt" returns organic matter from lysed cells to the dissolved organic pool)
Viral metagenomics reveals vast uncharacterised diversity ("viral dark matter")
Systems biology aims to understand the cell as an integrated system rather than a collection of individual parts
Omics approaches:
Genomics: complete genome sequencing and annotation
Transcriptomics: global mRNA profiling (microarrays, RNA-seq)
Proteomics: global protein profiling (2D gel electrophoresis, mass spectrometry)
Metabolomics: global metabolite profiling
Integrating omics data reveals regulatory networks, metabolic flux, and emergent properties of microbial cells and communities
Mutations:
Point mutations: base-pair substitutions (transitions, transversions), insertions, deletions
Silent, missense, nonsense, frameshift mutations
Spontaneous mutations arise from replication errors, depurination, deamination
Induced mutations caused by chemical mutagens (base analogs, intercalating agents, alkylating agents) or radiation (UV causes pyrimidine dimers; ionising radiation causes strand breaks)
DNA repair:
Proofreading (3' to 5' exonuclease of DNA polymerase)
Mismatch repair (MutS/MutL system)
Excision repair: base excision repair (BER), nucleotide excision repair (NER)
SOS response: error-prone repair under extreme DNA damage; induces mutagenesis but allows survival
Horizontal gene transfer:
Transformation: uptake of naked DNA; requires competence
Transduction: phage-mediated transfer; generalised vs. specialised
Conjugation: requires cell-to-cell contact and a conjugative element; F plasmid, Hfr strains, F' plasmids
Transposable elements: IS elements, composite transposons, Tn3-family transposons. Can cause insertional inactivation, carry resistance genes, rearrange genomes
Recombination:
Homologous recombination (RecA-dependent): requires regions of sequence similarity
Site-specific recombination: integrase-mediated (e.g., phage lambda integration)
Molecular cloning workflow:
Cut target DNA and vector with the same restriction enzyme (or compatible enzymes)
Ligate insert into vector with DNA ligase
Transform into host (e.g., E. coli)
Screen or select for recombinants (blue-white screening with lacZ, antibiotic selection)
PCR: denaturation (94–98 °C), annealing (50–65 °C), extension (72 °C); repeated 25–35 cycles. Requires template, primers, dNTPs, thermostable polymerase
DNA sequencing: Sanger sequencing (dideoxy chain termination) for individual genes; next-generation sequencing (Illumina, PacBio, Oxford Nanopore) for whole genomes and metagenomes
Gene expression and protein production: expression vectors with strong promoters, ribosome binding sites, and sometimes fusion tags (His-tag, GST-tag) for purification
Applications of genetic engineering:
Production of recombinant proteins (insulin, growth hormone, industrial enzymes)
Genetically modified organisms (GMOs) in agriculture
Gene therapy
CRISPR-Cas9 genome editing: guide RNA directs Cas9 to a specific genomic location, Cas9 makes a double-strand break, and the cell's repair machinery introduces desired changes
Microbial biotechnology applications: biofuels (ethanol from cellulose), bioremediation (engineered organisms to degrade pollutants), biosynthesis of pharmaceuticals and fine chemicals, synthetic biology
Early Earth and the origin of life:
Prebiotic chemistry: Miller-Urey experiment demonstrated abiotic synthesis of amino acids
RNA world hypothesis: RNA may have preceded DNA and protein as the first self-replicating molecule
Last Universal Common Ancestor (LUCA): hypothetical ancestor of all extant life
Endosymbiotic theory: mitochondria and chloroplasts originated from endosymbiotic alpha-proteobacteria and cyanobacteria, respectively. Supported by double membranes, own DNA, 70S ribosomes, and phylogenetic evidence
Molecular phylogenetics:
16S/18S rRNA: slowly evolving, universally distributed, functionally conserved, large enough for meaningful comparison
Phylogenetic trees: constructed from sequence alignments using methods such as neighbour-joining, maximum likelihood, and Bayesian inference
Horizontal gene transfer complicates tree construction; whole-genome approaches and multi-locus sequence analysis help resolve ambiguities
Microbial taxonomy:
Domain → Phylum → Class → Order → Family → Genus → Species
Species concept in microbiology is operational; there is no universally agreed biological species concept for prokaryotes
Average nucleotide identity (ANI) >95–96% is increasingly used as a species boundary
Major bacterial phyla to know: Proteobacteria (alpha through epsilon classes), Firmicutes (Gram-positive, low G+C), Actinobacteria (Gram-positive, high G+C), Bacteroidetes, Cyanobacteria, Spirochaetes, Chlamydiae
Archaea: Euryarchaeota (methanogens, extreme halophiles, thermoacidophiles), Crenarchaeota (many thermophiles, Thaumarchaeota-like ammonia oxidisers), and newly discovered superphyla (Asgard archaea as closest relatives to eukaryotes)
Lac operon logic table:
Glucose | Lactose | cAMP level | CAP-cAMP bound? | LacI repressor bound? | Transcription |
|---|---|---|---|---|---|
Present | Absent | Low | No | Yes | Off |
Present | Present | Low | No | No | Low (basal) |
Absent | Absent | High | Yes | Yes | Off |
Absent | Present | High | Yes | No | High (full) |
PCR cycle: Denature (94 °C) → Anneal (50–65 °C) → Extend (72 °C) → Repeat
Antibiotic resistance genes spread rapidly through bacterial populations via conjugation, transduction, and transformation, which is why horizontal gene transfer is a central concern in public health
CRISPR-Cas9, originally discovered as a bacterial immune system, has been adapted into the most powerful genome-editing tool in modern biotechnology, with applications from treating genetic diseases to engineering crop resistance
Quorum sensing is a drug target: disrupting bacterial communication ("quorum quenching") is being explored as an alternative to conventional antibiotics, because it disarms pathogens without killing them, potentially reducing selection for resistance
Metagenomic sequencing of environmental samples (soil, ocean, human gut) has revealed that the vast majority of microbial diversity has never been cultured in a laboratory
Students often think the lac operon is simply "on when lactose is present." Full expression requires both the presence of lactose and the absence of glucose. Glucose repression via the CAP-cAMP system is the dominant control
"Lysogeny" does not mean the phage is dead or inactive. The prophage is replicated with the host chromosome and can be induced to enter the lytic cycle at any time. Lysogenic conversion can actively change the host's phenotype
Students confuse generalised and specialised transduction. Generalised transduction can transfer any host gene (random packaging). Specialised transduction only transfers genes adjacent to the prophage insertion site (imprecise excision)
PCR does not create new DNA from scratch. It amplifies existing target DNA. If the template is absent or degraded, PCR will not produce a product
⚠️ Be able to diagram the regulation of the lac operon under all four conditions (glucose present/absent × lactose present/absent) and explain the role of both the LacI repressor and the CAP-cAMP activator
⚠️ Know the difference between lytic and lysogenic cycles, including the molecular switch (CI repressor, RecA, SOS response) for phage lambda
⚠️ Compare and contrast transformation, generalised transduction, specialised transduction, and conjugation as mechanisms of horizontal gene transfer
⚠️ Be able to describe the steps of molecular cloning: cutting, ligating, transforming, screening/selecting
⚠️ Understand why 16S rRNA is the molecule of choice for phylogenetic analysis of prokaryotes
⚠️ Know how two-component regulatory systems work and be able to give an example
True or False: The lac operon is fully expressed when both glucose and lactose are present.
Fill in the blank: A temperate phage that has integrated into the host chromosome is called a ________.
True or False: In conjugation, the F⁻ recipient cell builds the pilus.
Fill in the blank: The enzyme used in PCR must be ________ because it must survive repeated heating to 94 °C.
True or False: Horizontal gene transfer only occurs between closely related species.
(Answers: 1. False, glucose repression via CAP-cAMP keeps expression low. 2. Prophage. 3. False, the F⁺ donor builds the pilus. 4. Thermostable (e.g., Taq polymerase). 5. False, HGT can occur between distantly related organisms, even across domains.)
Q: Explain why the lac operon is considered to be under both positive and negative control.
A: Negative control comes from the LacI repressor, which binds the operator and blocks transcription unless inactivated by the inducer allolactose. Positive control comes from the CAP-cAMP complex, which binds upstream of the promoter and enhances RNA polymerase binding. Full transcription requires both the removal of the repressor (lactose present) and the binding of the activator (glucose absent, cAMP high).
Q: A prophage in a lysogenic bacterium suddenly enters the lytic cycle. What molecular event most likely triggered this induction?
A: DNA damage (e.g., from UV light or a chemical mutagen) activates the SOS response. The RecA protein, stimulated by single-stranded DNA, promotes the self-cleavage (autoproteolysis) of the CI repressor that was maintaining lysogeny. Without CI repressor, the lytic genes are derepressed, and the phage enters the lytic cycle.
Q: How does generalised transduction differ from specialised transduction in terms of which host genes can be transferred?
A: In generalised transduction, random fragments of the host chromosome are accidentally packaged into phage heads during lytic replication, so virtually any host gene can be transferred. In specialised transduction, only host genes immediately adjacent to the prophage insertion site are transferred, because they are picked up when the prophage excises imprecisely.
Q: Describe how CRISPR-Cas functions as an adaptive immune system in bacteria.
A: When a bacterium survives a phage infection, short sequences (spacers) from the phage DNA are incorporated into the CRISPR array in the bacterial chromosome. These spacers are transcribed and processed into CRISPR RNAs (crRNAs) that guide Cas nuclease proteins to complementary sequences in incoming foreign DNA. The Cas nuclease cleaves the matching foreign DNA, preventing infection. This provides sequence-specific, heritable immunity.
Q: Why is 16S rRNA used as a molecular marker for bacterial phylogenetics rather than, say, a metabolic gene?
A: 16S rRNA is present in all bacteria and archaea (universally distributed), performs the same essential function in all of them (part of the ribosome, so functionally conserved), evolves slowly enough to retain phylogenetic signal across billions of years, yet contains enough variable regions to distinguish between species. Metabolic genes may be absent in some lineages, subject to horizontal transfer, or evolving at rates that make deep comparisons unreliable.
Regulation systems (two-component systems, quorum sensing) connect directly to the pathogenicity material in the final exam block (Ch. 25): many virulence factors are regulated by two-component systems and quorum sensing
Horizontal gene transfer and mobile genetic elements are the mechanism behind the antibiotic resistance problem covered in clinical microbiology (Ch. 28)
Viral ecology (the viral shunt) ties into the microbial ecosystems and nutrient cycles material (Ch. 20–21)
Biotechnology tools (PCR, cloning, sequencing) are the methods used in the clinical diagnostics and epidemiology chapters (Ch. 28–29)
gene regulation, lac operon, trp operon, catabolite repression, CAP-cAMP, inducer, repressor, activator, operon model, two-component system, sensor kinase, response regulator, quorum sensing, autoinducer, AHL, sigma factor, attenuation, riboswitch, stringent response, ppGpp, virus, virion, bacteriophage, phage, lytic cycle, lysogeny, prophage, lysogenic conversion, temperate phage, Baltimore classification, capsid, envelope, retrovirus, reverse transcriptase, transformation, transduction, generalised transduction, specialised transduction, conjugation, F plasmid, Hfr, pilus, plasmid, transposon, IS element, CRISPR, Cas9, restriction enzyme, PCR, gel electrophoresis, cloning vector, recombinant DNA, expression vector, metagenomics, phylogenetics, 16S rRNA, molecular clock, horizontal gene transfer, systematics, taxonomy, endosymbiosis, three domains, Proteobacteria, Firmicutes, Actinobacteria, Archaea, Brock Biology of Microorganisms Chapter 6 7 8 9 10 11 12 13 16 18 19, MCB 3020