Microbial genetics

In short

Microbial genetics is the study of how bacteria, archaea, fungi and viruses store, express, mutate and exchange genetic information. Because bacteria have a single small haploid genome, divide in minutes and take up foreign DNA, they are both the model system in which the gene was dissected and the workhorse of modern biotechnology. The topic carries the heaviest weight in B.Sc Microbiology general-microbiology papers, in CSIR-UGC NET / GATE Biotechnology (BT) and Life Sciences (XL), and in ICMR/DBT-JRF screening. Employers test it in interviews at Serum Institute, Biocon, Bharat Biotech, Syngene, Dr Reddy's, Panacea and in molecular-diagnostic labs under NABL ISO 15189, because plasmid handling, strain stability, antibiotic-resistance markers and PCR all rest on it.

Explained

Organisation of the microbial genome

• Bacterial chromosome: usually one circular, double-stranded, covalently closed DNA molecule in the nucleoid; *E. coli* K-12 is about 4.6 Mbp with roughly 4,300 genes. No nuclear membrane, no histones; DNA is condensed by nucleoid-associated proteins (HU, H-NS, Fis, IHF) and negative supercoiling from DNA gyrase.
• Haploid and gene-dense: little non-coding DNA, no introns in most bacteria, genes of a pathway lie together as an operon transcribed into one polycistronic mRNA.
• Plasmids: small, self-replicating extrachromosomal circular DNA, 1-200 kbp, dispensable but often advantageous. Types: F (fertility), R (resistance), Col (colicin), virulence (e.g. Ti plasmid of *Agrobacterium*, pXO1/pXO2 of *Bacillus anthracis*), degradative/catabolic (TOL plasmid for toluene).
• Plasmid properties: copy number (relaxed, e.g. pUC ~500; stringent, e.g. F ~1-2), incompatibility group (two plasmids of the same Inc group cannot be stably maintained), episome (a plasmid able to integrate into the chromosome, e.g. F), curing (loss on treatment with acridine orange, ethidium bromide or elevated temperature).
• Transposable elements: IS elements (e.g. IS1, IS10) carry only transposase flanked by inverted repeats; composite transposons (Tn5, Tn10) carry extra genes such as kanamycin or tetracycline resistance between two IS elements; Tn3 uses replicative transposition via a cointegrate and resolvase; conjugative transposons (Tn916) move between cells. Transposition is non-homologous and generates direct repeats at the target site.
• Integrons capture resistance gene cassettes by site-specific recombination (IntI integrase, attI site) - the main reason multidrug resistance accumulates so fast.
• Genome in viruses: DNA or RNA, single- or double-stranded, linear or circular; ϕX174 single-stranded circular DNA, λ linear dsDNA with cos ends, M13 filamentous ssDNA used as a cloning/phage-display vector.

Mutation: types, causes and detection

• Mutation is a stable heritable change in the DNA base sequence; the altered organism is a mutant and the normal one the wild type.
• By size: point mutation (single base) versus gross changes (deletion, insertion, duplication, inversion, translocation).
• Point mutations: transition (purine↔purine or pyrimidine↔pyrimidine), transversion (purine↔pyrimidine). Effects on protein: silent (same amino acid), missense (different amino acid), nonsense (stop codon), frameshift (insertion/deletion not a multiple of three - usually the most damaging).
• By origin: spontaneous (replication error, tautomeric shift, depurination, deamination of cytosine to uracil, oxidative damage, transposition) - rate about 10⁻⁶ to 10⁻¹⁰ per base per generation; induced by mutagens.
• Chemical mutagens: base analogues (5-bromouracil, 2-aminopurine → transitions); deaminating agents (nitrous acid); alkylating agents (EMS, MNNG, nitrogen mustard); intercalating agents (acridine orange, ethidium bromide, proflavine → frameshifts); hydroxylamine (specific C→T).
• Physical mutagens: UV at 254 nm forms cyclobutane thymine dimers (hence germicidal lamps); ionising radiation (X-rays, gamma) causes strand breaks.
• Phenotypic classes of mutants: auxotroph (lost a biosynthetic ability, e.g. His⁻, needs histidine), prototroph (wild type), conditional/temperature-sensitive, resistant mutants, regulatory mutants.
• Selection and screening: direct selection on selective plates for resistant mutants; replica plating (Lederberg) to detect auxotrophs, which cannot be directly selected; penicillin enrichment for auxotrophs.
• Reversion and suppression: back mutation restores the original sequence; suppressor mutation restores phenotype by a second change elsewhere (intragenic or intergenic, e.g. an altered tRNA reading a nonsense codon).
• Ames test: histidine-requiring *Salmonella* Typhimurium strains (TA98, TA100) with defective excision repair and an LPS defect for permeability; reversion to His⁺ on minimal medium measures mutagenicity, with rat-liver S9 microsomal fraction to mimic mammalian metabolic activation. Used in Indian safety dossiers under the Drugs and Cosmetics Rules (Schedule Y framework) and CDSCO/NDCT genotoxicity requirements, following OECD Test Guideline 471.
• Luria-Delbrück fluctuation test (1943) and the Newcombe spreading and Lederberg replica-plating experiments showed mutations arise spontaneously before exposure, not as an adaptive response - the foundation of bacterial genetics.

DNA replication and repair in bacteria

• Replication: bidirectional and semi-conservative from a single oriC, theta (θ) mode, terminating at ter; DNA Pol III holoenzyme extends, Pol I removes RNA primers, primase (DnaG) makes primers, helicase (DnaB), SSB, gyrase relieves supercoils, ligase seals nicks. Rolling-circle mode in plasmid transfer and ϕX174; Pol III proofreads by 3'→5' exonuclease.
• Mismatch repair: MutS-MutL-MutH recognises mismatches using hemimethylated Dam sites to identify the new strand; loss of mutS/mutL gives mutator strains.
• Excision repair: nucleotide excision repair (UvrABC) removes bulky lesions including thymine dimers; base excision repair uses specific glycosylases (e.g. uracil-DNA glycosylase) plus AP endonuclease.
• Direct repair: photoreactivation by photolyase (needs visible light) splits thymine dimers; O6-methylguanine methyltransferase (Ada) removes alkyl groups.
• Recombinational (post-replication) repair: RecA-mediated strand exchange; SOS response - DNA damage activates RecA, which promotes cleavage of the LexA repressor, inducing uvr, recA, sulA and error-prone Pol IV/Pol V (translesion synthesis, hence UV mutagenesis).
• Homologous recombination requires extensive sequence identity, RecBCD (exonuclease V) to generate single strands at chi sites, RecA for invasion, and RuvABC to resolve Holliday junctions.

Gene transfer: transformation, conjugation, transduction

• Transformation: uptake of free naked DNA from the environment. Natural competence in *Streptococcus pneumoniae*, *Bacillus subtilis*, *Haemophilus influenzae*, *Neisseria*; proven by Griffith (1928) and Avery-MacLeod-McCarty (1944). Artificial: CaCl₂ + heat shock in chemically competent *E. coli*; electroporation; protoplast/PEG method in Gram-positives and yeast.
• Conjugation: cell-to-cell transfer through a sex pilus requiring physical contact and the tra genes of the F plasmid. F⁺ × F⁻ transfers only the plasmid by rolling-circle replication, converting the recipient to F⁺; Hfr × F⁻ transfers chromosomal genes from oriT onwards, the recipient usually remains F⁻ because the F genes transfer last; F′ carries a piece of the chromosome (formed by imprecise excision) and gives partial diploids (merodiploids) used for complementation tests.
• Interrupted mating (Wollman and Jacob) - time of entry of each marker maps the chromosome in minutes (*E. coli* total map = 100 min).
• Transduction: gene transfer by a bacteriophage. Generalised (lytic phage such as P22 in *Salmonella*, P1 in *E. coli*) packages any chromosomal fragment by a packaging error; specialised (temperate λ) transfers only genes adjacent to the attachment site (gal, bio) after faulty excision of the prophage; abortive transduction - the DNA never integrates or replicates.
• Lysogeny and λ control: integration at attB/attP by Int and IHF; the cI repressor maintains lysogeny, cro drives the lytic cycle; induction by UV via the SOS response. Transposition and phage-mediated transfer together with conjugative plasmids drive horizontal gene transfer (HGT) and the spread of resistance.
• Protoplast fusion in fungi and streptomycetes, and gene mapping by co-transduction frequency, complete the toolkit.

Regulation of gene expression

• Constitutive genes are always expressed; inducible and repressible genes respond to signals - economical for a cell with limited energy.
• Operon model (Jacob and Monod, 1961): promoter + operator + structural genes under one control; the regulatory gene may lie elsewhere.
• lac operon (inducible, catabolic): lacZ (β-galactosidase), lacY (permease), lacA (transacetylase); lacI makes the repressor; allolactose (natural) or IPTG (non-metabolisable) is the inducer. Negative control: inducer binds repressor, which leaves the operator. Positive control: low glucose → high cAMP → cAMP-CAP complex binds the promoter and raises transcription (catabolite repression explains diauxic growth). Mutant classes: lacI⁻ constitutive, lacI^s super-repressed, O^c operator-constitutive and *cis*-dominant, lacZ⁻ no enzyme. X-gal/blue-white screening comes from lacZ.
• trp operon (repressible, anabolic): five structural genes; tryptophan acts as a co-repressor on the aporepressor. Additionally regulated by attenuation - a leader sequence with two Trp codons; when charged tRNA^Trp is plentiful the ribosome allows a terminator hairpin to form.
• Other mechanisms: ara operon (AraC acts as both repressor and activator), two-component systems (sensor kinase + response regulator, e.g. EnvZ/OmpR), sigma-factor switching (σ³² heat shock, σ⁵⁴ nitrogen, σ²⁸ flagella), quorum sensing (LuxI/LuxR acyl-HSL in Gram-negatives, AIP in *S. aureus*), riboswitches, small regulatory RNAs, stringent response (ppGpp on amino-acid starvation).
• Eukaryotic microbes: *Saccharomyces* GAL regulon (Gal4 activator, Gal80 inhibitor), mating-type switching, and *Neurospora* as the original one gene-one enzyme system (Beadle and Tatum).

Recombinant DNA and applied microbial genetics

• Restriction endonucleases: Type II cut at palindromic sites (EcoRI G^AATTC sticky ends, SmaI blunt); host DNA protected by methylation. Ligase joins; alkaline phosphatase prevents self-ligation; polylinker/MCS allows choice of site.
• Vectors: plasmid (pBR322 with amp^R and tet^R; pUC19 with lacZα and ampicillin), phage λ replacement vectors, cosmids (40 kb), BAC, shuttle and expression vectors (pET with T7 promoter), Ti plasmid for plants.
• Selection and screening: antibiotic selection, insertional inactivation, blue-white screening on X-gal/IPTG, colony hybridisation, PCR screening, sequencing.
• PCR: denaturation 94-95 °C, annealing 50-65 °C, extension 72 °C with *Taq* polymerase; variants RT-PCR, qPCR (TaqMan/SYBR), multiplex, nested. Core of RT-PCR diagnostics in NABL ISO 15189 labs and of ICMR-approved kits.
• Other tools: Sanger and next-generation sequencing, Southern/Northern/Western blotting, site-directed mutagenesis, gene knockout by homologous recombination or λ-Red, transposon mutagenesis libraries, CRISPR-Cas9 (originally a bacterial adaptive immune system with CRISPR arrays, spacers and Cas proteins).
• Indian regulation: rDNA work follows the Rules for the Manufacture, Use, Import, Export and Storage of Hazardous Micro-organisms/GMOs or Cells, 1989 under the Environment (Protection) Act, 1986, administered through RCGM (DBT), IBSC at institute level, GEAC (MoEFCC) for environmental release, and the DBT Regulatory Guidelines for Recombinant DNA Research and Biocontainment, 2017; risk-group classification and BSL-1 to BSL-4 containment apply. Biosafety cabinets and containment follow national guidance; clinical labs follow NABL ISO 15189 and biomedical waste rules (Biomedical Waste Management Rules, 2016 as amended). Access to Indian microbial resources is governed by the Biological Diversity Act, 2002 (amended 2023) and the NBA.

Key points

• Bacterial genome - single circular haploid dsDNA chromosome in a nucleoid; *E. coli* K-12 ≈ 4.6 Mbp, no histones, supercoiled by gyrase.
• Plasmid - independently replicating extrachromosomal DNA; F, R, Col, virulence and degradative types; defined by copy number and Inc group.
• Episome - plasmid that can also integrate into the chromosome (F is the classic example).
• Transposon - mobile element moving by non-homologous recombination; IS elements, composite (Tn5, Tn10) and replicative (Tn3).
• Transition vs transversion - purine↔purine/pyrimidine↔pyrimidine versus purine↔pyrimidine substitution.
• Frameshift - insertion or deletion not a multiple of three; shifts the reading frame and is usually the most damaging point mutation.
• Auxotroph - mutant needing a growth factor the prototroph makes; found by replica plating, never by direct selection.
• Luria-Delbrück - fluctuation in resistant-colony counts proves mutations are spontaneous and pre-existing.
• Ames test - His⁻ *Salmonella* reversion assay with S9 liver fraction; screens chemicals for mutagenicity (OECD TG 471).
• Thymine dimer - UV (254 nm) lesion repaired by photolyase (photoreactivation) or by UvrABC excision repair.
• SOS response - RecA-triggered cleavage of LexA induces repair genes and error-prone Pol V, giving UV-induced mutations.
• Transformation - uptake of naked DNA; only process abolished by DNase; needs competence.
• Conjugation - contact-dependent transfer via sex pilus and tra genes; F⁺×F⁻ spreads the plasmid, Hfr×F⁻ transfers chromosomal genes.
• Interrupted mating - maps genes in minutes of transfer time; *E. coli* map is 100 minutes long.
• Generalised vs specialised transduction - any gene by a lytic phage (P1, P22) versus only att-adjacent genes by a temperate phage (λ gal, bio).
• lac operon - negatively controlled by LacI plus positive cAMP-CAP control; allolactose/IPTG inducer; explains diauxic growth.
• trp operon - repressible by tryptophan as co-repressor, plus attenuation through the leader peptide.
• Horizontal gene transfer - conjugative plasmids, transposons, integrons and phages spread antibiotic resistance between species.

Formulas and examples

Example 1 - mutation frequency. A culture is diluted and 0.1 mL spread on nutrient agar (10⁶ dilution) gives 180 colonies; 0.1 mL undiluted (containing 1.8 × 10⁸ cells) on rifampicin agar gives 36 resistant colonies.

• Total viable count = 180 × 10⁶ × 10 = 1.8 × 10⁹ CFU/mL.
• Resistant count = 36 × 10 = 3.6 × 10² CFU/mL.
• Mutation frequency = 3.6 × 10² / 1.8 × 10⁹ = 2 × 10⁻⁷, i.e. 1 resistant mutant per 5 million cells.

Example 2 - transformation efficiency. 50 ng of pUC19 is used to transform 200 µL of competent cells; 100 µL of the recovery mix is plated on ampicillin agar and gives 340 colonies.

• Colonies from the whole 200 µL = 340 × 2 = 680 transformants.
• DNA used = 50 ng = 0.05 µg.
• Efficiency = 680 / 0.05 = 1.36 × 10⁴ transformants per µg - acceptable for chemical competence (10⁴-10⁶/µg); electroporation reaches 10⁸-10¹⁰/µg.

Example 3 - interrupted mating map. In an Hfr × F⁻ cross, markers first appear among recombinants at: thr 8 min, leu 9 min, pro 15 min, lac 23 min, gal 30 min.

• Order from oriT = thr - leu - pro - lac - gal.
• Distance lac to gal = 30 − 23 = 7 min; thr to gal = 22 min.
• On a 100-minute, 4,600 kbp map, 1 min ≈ 46 kbp, so lac-gal ≈ 322 kbp.

Example 4 - co-transduction mapping. P1 lysate from a wild-type donor transduces a *leu⁻ ara⁻* recipient. Of 500 Leu⁺ transductants, 160 are also Ara⁺; of 500 Leu⁺ transductants in a second cross, 45 are also Thr⁺.

• Co-transduction with ara = 160/500 × 100 = 32%; with thr = 45/500 × 100 = 9%.
• Higher co-transduction means closer linkage, so ara is nearer leu than thr is.

Example 5 - MOI and uninfected cells. 2 × 10⁷ λ phage are added to 1 × 10⁸ *E. coli* cells.

• MOI = 2 × 10⁷ / 1 × 10⁸ = 0.2.
• P(0) = e^−0.2 = 0.819, so about 81.9% of cells escape infection and ~18.1% are infected - low MOI is used deliberately so that most infected cells receive a single phage.

Where it is used

• Vaccine and biologics manufacture: engineered *E. coli* and *Pichia* strains at Serum Institute of India, Bharat Biotech, Biological E and Biocon for recombinant hepatitis-B antigen, insulin, streptokinase and monoclonal antibodies; roles in strain development, upstream process and QC.
• Molecular diagnostics: PCR, qPCR and sequencing in NABL ISO 15189 labs (Dr Lal PathLabs, Metropolis, SRL, ICMR-VRDL network) for TB (CBNAAT/Xpert MTB-RIF detecting rpoB mutations), HIV viral load, HPV and dengue.
• Antimicrobial resistance surveillance: detecting mecA, bla-NDM-1, ESBL, vanA and carbapenemase genes under the ICMR AMR Surveillance Network and the National Action Plan on AMR; informing hospital antibiotic policy.
• Industrial strain improvement: mutation and selection for higher yields of penicillin, streptomycin, citric acid, amylase and lactic acid; UV/NTG mutagenesis followed by screening in fermentation R&D.
• Agriculture and environment: *Bt* toxin genes and Bt cotton approvals through GEAC; *Rhizobium* and biofertiliser strain genetics; plasmid-encoded degradative pathways for oil-spill and effluent bioremediation.
• Food and regulatory testing: PCR-based pathogen detection for FSSAI compliance, GMO detection in imported food, and biosafety documentation for IBSC/RCGM approvals.

Interview questions

• Q: Why are bacteria such convenient genetic models? A: They are haploid with one small genome, so a mutant phenotype shows immediately; they divide in 20-30 minutes, grow on defined media, and large populations allow selection of very rare events.
• Q: What is the difference between a plasmid and an episome? A: A plasmid replicates independently of the chromosome; an episome is a plasmid that can also integrate into the chromosome and replicate with it, as the F factor does.
• Q: How would you prove that gene transfer in a given cross is transformation and not conjugation? A: Add DNase to the mixture - transformation is abolished because free DNA is degraded, while conjugation, which needs cell contact, is unaffected; a filter separating the cells also blocks conjugation only.
• Q: Why does the recipient in an Hfr × F⁻ cross usually stay F⁻? A: Transfer begins at oriT and the F-plasmid tra region is transferred last, so the mating is almost always interrupted before the F genes enter.
• Q: Distinguish generalised and specialised transduction. A: Generalised transduction by a lytic phage such as P1 or P22 can carry any chromosomal fragment through a packaging error; specialised transduction by a temperate phage such as λ carries only genes adjacent to its integration site after imprecise excision.
• Q: Explain diauxic growth in terms of the lac operon. A: Glucose keeps cAMP low, so cAMP-CAP cannot activate the lac promoter; only after glucose is exhausted does cAMP rise, the operon is induced, and growth resumes on lactose after a lag.
• Q: What does an O^c mutation tell you about the operator? A: The operator is a DNA site, not a diffusible product, so an O^c mutation is *cis*-dominant - it affects only the genes on the same DNA molecule, unlike lacI⁻ which is *trans*-recessive.
• Q: What is attenuation and why does the lac operon not use it? A: Attenuation terminates transcription of a biosynthetic operon when the end-product amino acid is plentiful, sensed through ribosome speed on a leader peptide; lac is a catabolic operon with no such amino-acid-charging signal.
• Q: What is the principle of the Ames test and why is S9 added? A: Reversion of a His⁻ *Salmonella* strain to His⁺ on histidine-free medium measures mutagenicity; rat-liver S9 microsomal fraction supplies cytochrome P450 enzymes so that chemicals needing metabolic activation are also detected.
• Q: How does the SOS response increase mutation rate? A: DNA damage activates RecA, which promotes LexA self-cleavage, inducing repair genes and the low-fidelity polymerases Pol IV and Pol V that replicate past lesions - allowing survival at the cost of errors.
• Q: How does a transposon differ from a plasmid in moving genes? A: A transposon cannot replicate independently; it moves within or between DNA molecules by transposase-mediated non-homologous recombination and must reside in a replicon such as a plasmid or chromosome.
• Q: Why is blue-white screening used, and what makes a colony white? A: The vector carries lacZα; an insert in the polylinker disrupts it, so no β-galactosidase is made, X-gal is not cleaved and the recombinant colony stays white while non-recombinants turn blue.
• Q: Which Indian bodies approve recombinant DNA work? A: The institutional IBSC for routine work, RCGM under DBT for research and small-scale activities, and GEAC under MoEFCC for environmental release, under the 1989 GMO Rules framed under the Environment (Protection) Act, 1986.

Common mistakes

• Treating conjugation as "bacterial sexual reproduction" with gamete fusion - it is one-way DNA transfer, and no zygote or fusion occurs.
• Saying generalised transduction moves whole chromosomes; a phage carries only one headful, roughly 1-2% of the genome.
• Confusing an inducer with a substrate: IPTG induces the lac operon but is not metabolised, which is exactly why it is used.
• Calling lacI⁻ and O^c the same thing - lacI⁻ is recessive and complemented in *trans*, while O^c is *cis*-dominant.
• Forgetting the denominator in mutation-frequency problems, or reporting mutants per plate instead of per total viable cells.
• Expecting to isolate auxotrophs by direct selection; they must be found by replica plating or penicillin enrichment because they cannot grow on the selective minimal plate.
• Confusing photoreactivation (light-dependent photolyase, direct reversal) with excision repair (UvrABC, light-independent).
• Reporting transformation counts without normalising to µg of DNA, so efficiencies cannot be compared between runs.

Related topics

• Bacterial growth and metabolism · Bacterial structure and classification · Virology and bacteriophages · Molecular biology: DNA replication, transcription and translation · Recombinant DNA technology and bioprocessing · Antimicrobial resistance and antibiotic mechanisms