Microbial Genetics (II)
3 sub-topics · Pages 531–558
1. Regulation of Gene Expression
Gene expression is not constitutive — it is exquisitely regulated to match metabolic investment to environmental demand. The lac operon (induced by lactose, derepressed only when glucose is absent) and the trp operon (repressed when tryptophan is abundant) exemplify the two fundamental regulatory logics: inducible systems switch on catabolic genes when substrate arrives; repressible systems switch off biosynthetic genes when the product accumulates. These elegant molecular switches ensure that bacteria spend energy making enzymes only when they are needed.
✏️ Fill in the Blank
1. The transfer of genetic material between bacteria through direct cell-to-cell contact via a pilus is called _______.
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Conjugation2. The adaptive immune defence system found in bacteria that stores sequences from past phage infections and uses them to cleave matching foreign DNA is called _______.
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CRISPR-Cas3. The use of microorganisms to produce human proteins such as insulin is an example of _______ biotechnology.
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Recombinant DNA🔘 Multiple Choice
1. Transduction involves horizontal gene transfer mediated by:
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Correct: B) Bacteriophages2. Which mechanism of antibiotic resistance involves hydrolysis of the β-lactam ring?
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Correct: C) β-lactamase enzyme production3. In methicillin-resistant Staphylococcus aureus (MRSA), resistance to β-lactam antibiotics is conferred by:
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Correct: B) Acquisition of mecA gene (on SCCmec element) encoding an altered penicillin-binding protein (PBP2a) with low affinity for β-lactams4. Two-component regulatory systems in bacteria consist of:
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Correct: B) A membrane-bound sensor histidine kinase and a cytoplasmic response regulator5. The purpose of a selectable marker in a cloning vector is to:
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Correct: B) Allow identification of cells that have successfully taken up the vector💬 Open-Ended Questions
1. Compare transformation, transduction, and conjugation as mechanisms of horizontal gene transfer in bacteria. For each, describe the mechanism, the type of DNA transferred, and give an example of its ecological or medical significance.
Hint / Guidance
Transformation: naked DNA uptake (competent cells — natural or artificial); any DNA; Streptococcus pneumoniae naturally competent; spread of blaZ (β-lactamase), capsule genes. Transduction: generalised (random chromosomal fragments) or specialised (genes flanking integration site); Shiga toxin genes on Stx phage spread among E. coli. Conjugation: plasmid transfer via sex pilus (mating pair formation); large DNA possible; R-plasmids with multi-drug resistance spread globally; requires physical contact (blocked by DNase for transformation, requires living cells unlike transduction after phage purification).2. Describe how the lac operon is regulated by both negative (repressor) and positive (catabolite repression) mechanisms. Under what conditions is it maximally expressed?
Hint / Guidance
Negative control: LacI repressor binds palindromic operator (O1) between -10 and +1; blocks RNA pol progression. Inducer allolactose (formed from lactose by β-galactosidase) binds LacI → conformational change → dissociation from operator → derepression. Positive control: glucose catabolism → adenylyl cyclase inhibited → low cAMP; without cAMP, CAP (catabolite activator protein) cannot bind CAP site upstream of -35; transcription low even without repressor. High cAMP (glucose absent) → CAP-cAMP binds upstream → recruits RNA pol → 50-fold increase in transcription. Maximal expression: lactose present (allolactose derepresses) AND glucose absent (high cAMP activates CAP).3. Describe how integrons contribute to multidrug resistance in clinical pathogens. What is a gene cassette, and how do integrons capture and express them?
Hint / Guidance
Integron: platform element with integrase (IntI), attI recombination site, and Pc promoter. Gene cassettes: small mobile elements consisting of a single gene + attC recombination site; no promoter (rely on Pc of integron). Mechanism: IntI recognises attC in cassette and attI in integron → site-specific recombination → cassette inserted; transcription from Pc → cassette expressed. Class 1 integrons: most common in clinical resistance; usually carry multiple antibiotic resistance cassettes (aminoglycosides, sulfonamides, trimethoprim); often located on transposons on conjugative plasmids → triple mobility. Class 1 integrons also suppress host SOS response, increasing horizontal gene transfer. Associated with Gram-negative multi-drug resistant pathogens (Pseudomonas, Acinetobacter, Enterobacteriaceae).4. Discuss the environmental and clinical significance of integrative and conjugative elements (ICEs). How do they differ from plasmids and transposons?
Hint / Guidance
ICEs: self-transmissible elements that integrate into host chromosome (like prophage), excise when induced, form circular intermediate, transfer by conjugation, reintegrate in recipient. Differ from plasmids: no autonomous replication; reside stably as chromosomal insert. Differ from transposons: encode their own conjugation machinery; can transfer without plasmid co-residence. Clinical significance: ICESt3 and related elements carry erm (macrolide resistance) and tet genes in Streptococcus; SXT/R391 family carry multiple resistance genes and have spread globally in Vibrio cholerae (causing cholera strains to become multidrug resistant). Environmental: ICEs transfer metabolic genes (nitrogen fixation, hydrocarbon degradation) between soil bacteria; symbiosis islands (mesorhizobia) carry entire N₂ fixation gene clusters transferred by ICE conjugation. Estimated >20% of bacterial HGT may be ICE-mediated.5. Explain the concept of regulons and stimulons in bacterial gene regulation. How do global regulatory systems allow bacteria to rapidly adapt to stress?
Hint / Guidance
Stimulon: all genes whose expression changes in response to a particular environmental stimulus (may be controlled by multiple regulators). Regulon: all genes controlled by a single regulatory protein (subset within stimulon). Examples: SOS regulon (LexA; ~40 genes responding to DNA damage); heat shock (σ³²; ~30 chaperones/proteases); RpoS (σˢ stationary phase; >500 genes). Global regulators (pleiotropic): CRP (glucose signal, ~400 genes); Fur (iron homeostasis, ~90 genes); OmpR (osmoregulation). Rapid adaptation: pre-existing regulon = immediate coordinated response across metabolism, transport, repair; avoids need to evolve gene-by-gene. Hierarchy: master regulator activates/represses secondary regulators → cascading response.2. Transduction
Transduction is the bacteriophage-mediated transfer of host DNA between bacteria — a major route of horizontal gene transfer in natural environments. Generalised transduction (random chromosomal fragments mis-packaged into phage heads) can transfer any gene. Specialised transduction (imprecise prophage excision carrying flanking host genes) transfers specific genes. This mechanism has driven major evolutionary events: the Shiga toxin genes of enterohaemorragic E. coli O157:H7 were acquired by specialised transduction from a related phage.
✏️ Fill in the Blank
1. Mobile genetic elements that can move from one location to another within a genome are called _______.
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Transposons2. Efflux pumps, β-lactamase production, and target site modification are examples of mechanisms of antibiotic _______.
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Resistance3. The technique that uses specific primers and a thermostable DNA polymerase to amplify a target DNA sequence in vitro is called _______.
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PCR (Polymerase Chain Reaction)🔘 Multiple Choice
1. The CRISPR-Cas9 system in bacteria originally evolved as:
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Correct: B) An adaptive immune defence against invading bacteriophages and plasmids2. In generalised transduction, a bacteriophage:
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Correct: B) Accidentally packages a random fragment of host chromosomal DNA instead of phage DNA3. The operon model of gene regulation (Jacob and Monod) proposed that:
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Correct: B) Functionally related genes can be coordinately regulated as a unit by a single operator-repressor system4. In Southern blotting, which molecule is detected by the labelled probe?
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Correct: C) Denatured single-stranded DNA fragments on a membrane5. Which of the following techniques allows quantification of gene expression at the mRNA level across the entire genome simultaneously?
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Correct: B) DNA microarray or RNA-seq💬 Open-Ended Questions
1. Explain how mutations can lead to antibiotic resistance. Describe three different molecular mechanisms of resistance that can arise by mutation or horizontal gene transfer.
Hint / Guidance
(1) Target site modification: mutations in gyrA/gyrB → quinolone resistance; mutations in 16S rRNA → aminoglycoside resistance; acquisition of ermB (methylase) → macrolide resistance. (2) Efflux: mutations upregulate MexAB-OprM (Pseudomonas) → broad-spectrum resistance; acquired tet(A) efflux genes on transposons. (3) Enzymatic inactivation: TEM β-lactamase (plasmid-encoded) → penicillin hydrolysis; extended-spectrum β-lactamase (ESBL) variants → 3rd-gen cephalosporins; aminoglycoside acetyltransferases. Public health: combination resistance mechanisms (carbapenem-resistant Enterobacteriaceae) — OXA-48 + NDM-1 — leave few treatment options.2. What is a transposon and how does it contribute to bacterial genome evolution and antibiotic resistance spread?
Hint / Guidance
Transposon: mobile genetic element flanked by inverted terminal repeats; encodes transposase (and often additional genes). Types: IS elements (transposase only); composite transposons (two IS elements flanking resistance genes, e.g., Tn10 — tetracycline); complex transposons (Tn3 — β-lactamase). Mechanism: cut-and-paste (class II) or copy-and-paste (class I/retrotransposons). Contributions: (1) Transposons move resistance genes between plasmids and chromosomes; (2) Integrons — gene cassette capture systems; resistance genes accumulate; (3) New gene combinations; (4) Insertional mutagenesis creates new phenotypes; (5) Evolutionary engine — Tn916 conjugative transposon spreads tetM resistance across species boundaries.3. What are persister cells and how do they contribute to antibiotic treatment failure? Are persisters genetically resistant?
Hint / Guidance
Persisters: small subpopulation (~10⁻⁵–10⁻⁶) of phenotypically tolerant, metabolically dormant cells that survive bactericidal antibiotics without being genetically resistant. Formation: stochastic switching into dormant state (toxin-antitoxin modules: HipA/HipB — HipA kinase phosphorylates Ef-Tu, halting translation; RelE/RelB — RelE cleaves mRNA on ribosomes; MazF/MazE). Dormant state: antibiotic targets (cell wall synthesis, DNA replication, protein synthesis) not active → killing mechanisms don't apply. Not resistance: upon regrowth from persister state → progeny equally antibiotic-sensitive. Clinical relevance: explain why biofilm infections relapse after antibiotic treatment; chronic infections (P. aeruginosa in CF lungs, S. aureus in endocarditis); chronic Lyme disease debate. Treatment strategies: combination with drugs targeting dormant cells (metabolite addition to 'wake up' persisters); drugs targeting persister mechanisms.4. What is RNA-seq and how does it provide more information than DNA microarrays for analysing microbial gene expression?
Hint / Guidance
RNA-seq: extract total RNA → deplete rRNA → reverse transcribe to cDNA library → high-throughput sequencing → map reads to genome. Advantages over microarrays: (1) No prior sequence knowledge required (discovers new transcripts, sRNAs, antisense RNAs); (2) Greater dynamic range (count-based, no saturation); (3) Can detect single nucleotide variants and RNA editing; (4) Detects full-length transcript structure, alternative start sites, processing; (5) More sensitive for low-abundance transcripts; (6) Can study any organism including non-model. Microarray advantages: cheaper per sample; faster analysis. RNA-seq now standard for differential gene expression studies.5. How is recombinant human insulin produced using E. coli biotechnology? Describe the key molecular steps from gene to product.
Hint / Guidance
(1) Gene synthesis: insulin A and B chain genes codon-optimised for E. coli; (2) Cloned into expression vector (strong promoter, e.g., T7/trc; signal sequence for periplasmic secretion or fusion protein); (3) Transform E. coli; (4) Fed-batch fermentation in 10,000 L bioreactor; (5) Two separate chains expressed as Met-A and Met-B chain fusion proteins (with β-galactosidase or trpLE leader); (6) Cells disrupted; inclusion bodies solubilised in urea; (7) CNBr cleavage at Met removes leader; (8) A and B chains refolded in vitro + oxidative disulfide bond formation → proinsulin analogue or chain combination; modern process uses proinsulin expressed as single chain → trypsin/carboxypeptidase B to cleave C-peptide; (9) Purification by ion-exchange + HPLC; (10) Product: Humulin/Novolin.3. Plasmids
Plasmids are autonomously replicating extrachromosomal DNA elements that often carry genes conferring selective advantages under specific conditions: antibiotic resistance (R-plasmids), virulence factors, or specialised metabolic pathways. Conjugative plasmids encode their own transfer machinery (sex pilus, mating pair formation, rolling circle replication) and can spread between unrelated bacterial species — explaining the global dissemination of multidrug resistance genes across hospital pathogens. The clinical significance of plasmid-mediated resistance cannot be overstated.
✏️ Fill in the Blank
1. Large clusters of virulence genes (e.g., type III secretion system) that were acquired by HGT and are located contiguously on the chromosome are called _______ islands.
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Pathogenicity2. A recombinant plasmid carrying a gene of interest that is used to express that gene in a host cell is called an _______ vector.
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Expression🔘 Multiple Choice
1. Transformation in bacteria is defined as:
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Correct: C) Uptake and incorporation of free exogenous DNA from the environment2. In the lac operon, catabolite repression occurs when:
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Correct: B) Glucose is present, cAMP levels are low, and CAP cannot activate transcription3. Insertion sequences (IS elements) are the simplest type of transposable element. They encode:
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Correct: B) Only transposase flanked by inverted terminal repeats4. Conjugation in Gram-negative bacteria (F factor) involves which of the following?
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Correct: A) Rolling circle replication of the F plasmid; one strand transferred to recipient; complementary strand synthesised in both donor and recipient5. Site-directed mutagenesis is used to:
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Correct: B) Introduce specific, predetermined nucleotide changes at defined positions in a gene6. What is the role of a σ (sigma) factor in bacterial transcription?
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Correct: B) It recognises and binds to promoter sequences to direct RNAP to the correct transcription start site💬 Open-Ended Questions
1. A plasmid carrying antibiotic resistance genes is transferred from a hospital-acquired pathogen to a commensal gut bacterium during conjugation. Trace the molecular events of this transfer and discuss the public health consequences.
Hint / Guidance
Donor: tra genes activated → F pilus assembled → tip contacts recipient outer membrane; mating pair formation; OriT nicked; rolling circle replication → single strand transferred 5'→3'; complementary strand synthesised in recipient. Transconjugant: acquires plasmid → expresses resistance genes (transcription/translation immediate). Public health: gut commensal becomes reservoir; 'silent reservoir' in healthy carriers; re-transfer to incoming pathogen; selection by clinical antibiotic use amplifies resistant sub-populations. NDM-1 plasmid spread across species (Klebsiella, E. coli, Acinetobacter) this way.2. Explain how CRISPR-Cas systems work as an adaptive immune system in bacteria. How has this been adapted for use as a gene-editing tool?
Hint / Guidance
Natural function: CRISPR arrays store short sequences (spacers) from previous phage/plasmid infections. New infection: Cas proteins degrade foreign DNA; short sequences integrated as new spacers (adaptation). Defence: spacer-derived crRNA guides Cas-Cas protein complex to matching foreign DNA → double-strand break → phage/plasmid neutralised. Three stages: adaptation (spacer acquisition), expression (crRNA processing), interference (target cleavage). Biotechnology: Cas9 (Streptococcus pyogenes) guided by single guide RNA (sgRNA = crRNA + tracrRNA fusion); sgRNA directs Cas9 to any DNA with 20-nt match adjacent to PAM sequence (NGG); generates blunt-end DSB → NHEJ (insertions/deletions) or HDR (precise editing). Applications: disease gene therapy, crop engineering, diagnostics (SHERLOCK/DETECTR).3. What is the trp operon and how does it differ from the lac operon in terms of regulation logic?
Hint / Guidance
Trp operon: 5 genes (trpE-trpA) encoding tryptophan biosynthesis enzymes. Regulation: repressible system — opposite logic to lac operon. Trp repressor (TrpR) alone is inactive (aporepressor); tryptophan (corepressor) binds TrpR → conformational change → active repressor binds operator → blocks transcription. When Trp scarce: TrpR inactive → genes expressed → Trp synthesised. When Trp abundant: TrpR-Trp complex represses → prevents wasteful synthesis. Attenuation: second regulatory mechanism — leader peptide contains two Trp codons; if Trp-tRNATrp abundant, ribosome translates leader rapidly, RNA pol adopts terminator hairpin structure → early termination before structural genes. Contrast with lac: lac induced by substrate (lactose) — makes sense to express only when substrate present; trp repressed by product (Trp) — makes sense to stop synthesis when product abundant. Both represent efficient metabolic economy.4. Describe the construction of a genomic DNA library and an expression library. For what purposes would each be used?
Hint / Guidance
Genomic library: (1) Total genomic DNA partially digested with restriction enzyme → fragments; (2) Ligated into vector (lambda phage or cosmid for large inserts; plasmid for small); (3) Transformed/packaged into E. coli; each colony/plaque contains one genomic fragment. Use: clone specific genes by probe hybridisation, physical mapping, comparative genomics. Expression library: (1) Total mRNA extracted → reverse transcribed to cDNA (oligo-dT primer); (2) Second strand synthesised; (3) Ligated downstream of strong promoter in expression vector; (4) Screening by antibody (if protein expressed) or activity assay. Use: identify genes encoding specific proteins, produce recombinant proteins, antibody screening (phage display).5. What are riboswitches and small RNAs (sRNAs) and how do they regulate gene expression post-transcriptionally in bacteria?