Bacterial Genetics

Bacterial DNA and RNA

Single circular dsDNA chromosome, supercoiled, ~1 mm long. No nuclear membrane, no histones. V. cholerae has two chromosomes.

Watson-Crick double helix. A pairs with T (2 H-bonds), G pairs with C (3 H-bonds). A+T:G+C ratio fixed per species.

Replication: bidirectional from origin (E. coli), rolling-circle in conjugation and phages. Enzymes — helicase (unwinds), DNA gyrase/topoisomerase (relieves supercoiling), DNA polymerase III (main synthesis, 5’→3’), DNA ligase (joins fragments).

RNA differs from DNA: ribose instead of deoxyribose, uracil instead of thymine. Three types — mRNA, rRNA, tRNA.

Gene Expression

Gene = DNA segment coding one polypeptide.

Codon = 3 bases on mRNA coding one amino acid. 61 sense codons, 3 stop codons (UGA, UAG, UAA). Start codon AUG = methionine (fMet in bacteria).

Anticodon = complementary 3 bases on tRNA.

Transcription: DNA → mRNA (RNA polymerase).

Translation: 4 steps — initiation, elongation, translocation, termination.

Plasmids

Extrachromosomal, circular, dsDNA. Not essential. Up to 40+ copies/cell. Independent replication. Episome = plasmid integrated with chromosome. Curing = plasmid loss (acridine, radiation, thymine starvation, heat).

Types by function:

• F (fertility) plasmid — sex pilus, conjugation
• R (resistance) plasmid — antibiotic resistance
• Col plasmid — bacteriocins
• Virulence plasmid — toxins, adhesins
• Metabolic plasmid — unusual substrate digestion, urease, N2 fixation

Mutation

Random, heritable, nucleotide sequence change. Frequency 10⁻²–10⁻¹⁰/division. Spontaneous or induced (mutagen — UV, alkylating agents, 5-bromouracil, acridine dyes).

Types:

• Transition: purine↔purine or pyrimidine↔pyrimidine
• Transversion: purine↔pyrimidine
• Silent — same amino acid
• Neutral — different but functionally equivalent amino acid
• Missense — different amino acid
• Nonsense — stop codon formed
• Frameshift — insertion/deletion not multiple of 3, shifts reading frame
• Reverse mutation — restores wild phenotype: true reversion (exact sequence back), equivalent reversion (different codon, same amino acid), suppressor mutation (second gene compensates)

Detection: gene sequencing (current standard). Older: fluctuation test (proves spontaneous mutation), replica plating (detects auxotrophs), Ames test (tests mutagenicity/carcinogenicity).

Horizontal Gene Transfer — 4 methods

1. Transformation
2. Transduction
3. Lysogenic conversion
4. Conjugation

Transformation

Uptake of naked/free DNA from medium, integration into chromosome. Seen in: Streptococcus, Bacillus, Haemophilus, Neisseria, Acinetobacter, Pseudomonas.

Needs competent bacteria (log phase, competence factors). S. pneumoniae — any DNA. H. influenzae — only related species DNA.

Steps: dsDNA binds DNA-binding protein → nicked by nuclease → one strand degraded → other strand + competence protein internalized (energy needed) → single strand integrates into chromosome.

Griffith experiment (1928): live noncapsulated + heat-killed capsulated pneumococci → mouse dies (transformation). Avery, MacLeod, McCarty (1944) — confirmed DNA is transforming material.

Transduction

Bacterial DNA transfer via bacteriophage.

Phage cycles: Lytic (multiply → lyse host → release phages). Lysogenic (phage DNA = prophage, integrated, replicates with host, no harm).

Generalized transduction: packaging error during lytic cycle — host DNA packaged instead of phage DNA. Transducing phage injects DNA but cannot lyse (no phage genome). Donor DNA fate: abortive (70-90%, non-integrated but expressed), stable (integrated), unstable (degraded).

Restricted/specialized transduction: prophage excises imprecisely, carries adjacent bacterial DNA. Studied in lambda phage of E. coli. Two outcomes: whole transducing genome integrates as prophage (needs helper phage), OR crossover exchanges donor/recipient DNA.

Role: transfers plasmid resistance genes (e.g. penicillin resistance in staph), genetic engineering tool.

Lysogenic conversion

Prophage = extra genetic element, confers new properties (e.g. toxin genes). C. diphtheriae — diphtheria toxin from lysogenic phage; curing phage = nontoxigenic.

Phage-coded toxins: Diphtheria toxin, Cholera toxin, E. coli verocytotoxin, Streptococcal pyrogenic exotoxin A & C, Botulinum toxin C & D.

Difference from transduction: phage DNA itself = new gene (lysogenic conversion) vs phage = vehicle for bacterial genes (transduction).

Conjugation

Donor (male, F+) → recipient (female, F-) via conjugation tube. Discovered by Lederberg and Tatum (1946).

F+ × F-: F factor (fertility plasmid, tra genes for sex pilus) replicates by rolling circle, copy transferred → recipient becomes F+. “Maleness” is infectious. Chromosomal transfer rare.

Hfr (high frequency recombination): F factor integrated into chromosome (episome). Transfers chromosomal genes with high frequency, but connection usually breaks before whole genome transfers → recipient stays F-.

F’ conjugation: F factor reverts from integrated to free, carries chromosomal DNA piece = F’ factor. Transfer to F- = sexduction; recipient becomes F’.

R factor (resistance factor) = RTF (resistance transfer factor, mediates transfer) + r determinants (each = resistance to one drug). If RTF and r separate — resistance non-transferable but host stays resistant.

Col factor = plasmid coding bacteriocins, transferred by conjugation. Colicin (coliform bacteriocin), pyocin (Pseudomonas), diphthericin (C. diphtheriae).

Fate of donor DNA: degraded by host nuclease OR integrated by recombination (replacement piece — transformation; extra piece — other methods).

Transposition

Transposons (“jumping genes”/mobile genetic elements) — move chromosome↔chromosome, plasmid↔plasmid, chromosome↔plasmid. No DNA homology needed (differs from recombination). Not self-replicating (unlike plasmids) — depend on host DNA. Discovered by Barbara McClintock (1940s, maize), Nobel Prize 1983. Found in bacteria, viruses, eukaryotes.

Insertion sequence: simplest, 1-2 kbp, transposase gene flanked by inverted repeats (form stem-loop).

Composite transposon: larger, extra genes (resistance/toxin) between two insertion sequences.

Artificial Gene Transfer

Genetic engineering = deliberate genome modification via recombinant DNA technology.

Steps: restriction enzyme cuts DNA → Southern blot isolates desired fragment (electrophoresis → membrane transfer → probe detection → extraction) → ligated to vector (DNA ligase) → vector into bacteria (transformation/electroporation, rarely transduction) → cloning (culture, protein expression).

Applications: vaccine production (Hep B, HPV), diagnostic antigens (ELISA), therapeutic proteins (growth hormone, insulin, interferons, IL-2, TNF, factor VIII), transgenic animals (transfection), gene therapy.

Vector = DNA piece for foreign gene insertion + stable maintenance + cloning. 4 types: plasmids, bacteriophages, cosmids, artificial chromosomes.

Blotting Techniques

• Southern blot — DNA detection
• Northern blot — RNA detection
• Western blot — protein detection
• Eastern blot — Western blot variant, detects post-translational modifications

The Bacterial Chromosome

Bacteria carry their hereditary information as a single haploid chromosome — a supercoiled, circular, double-stranded DNA molecule about 1 mm long, folded into an irregular cytoplasmic region called the nucleoid. There is no nuclear membrane, no nucleolus, and no histone packaging of the kind eukaryotic chromatin uses. A few species depart from this pattern: some carry a linear chromosome, and Vibrio cholerae carries two separate circular chromosomes.

DNA itself follows the Watson–Crick double-helix model — two complementary strands of sugar-phosphate backbone carrying the bases adenine, guanine, thymine and cytosine, held together by base pairing (A–T via two hydrogen bonds, G–C via three). Because pairing is obligatory, the A+T:G+C ratio is fixed for a given species but varies widely between species — a property that is itself sometimes used taxonomically.

Replication in bacteria proceeds bidirectionally from a single origin, unwinding at a moving replication fork (the pattern seen in E. coli), though the rolling-circle mechanism takes over during conjugation and during phage reproduction. The machinery is enzymatic: helicase unwinds the duplex, topoisomerase (DNA gyrase in E. coli) relieves the supercoiling this generates, DNA polymerase III carries out the bulk of new-strand synthesis in the 5′→3′ direction while polymerases I and II mainly handle repair, and DNA ligase seals the fragments together.

Bacterial RNA differs from DNA in two respects — it uses ribose instead of deoxyribose, and uracil replaces thymine. Three RNA species do the work of expressing a gene: messenger RNA carries the coding sequence, ribosomal RNA builds the translation machinery, and transfer RNA delivers amino acids.

From Gene to Protein

A gene is simply the stretch of DNA that encodes one polypeptide. Expression proceeds through transcription (DNA copied into mRNA by RNA polymerase) and translation (mRNA decoded by ribosomes into protein).

The genetic code is read three bases at a time as a codon on the mRNA. Of the 64 possible codons, 61 are sense codons specifying one of the 20 amino acids — since there are more codons than amino acids, most amino acids have more than one codon (a redundancy called degeneracy). The remaining three (UGA, UAG, UAA) are stop codons with no amino-acid assignment; they simply terminate translation. AUG is the near-universal start codon, coding for methionine in eukaryotes and for a modified form, N-formylmethionine, in bacteria. The tRNA anticodon is the three-base sequence that pairs with a codon to deliver the matching amino acid.

Translation itself runs through four phases: initiation (the ribosome assembles on the mRNA and the first tRNA locks onto the start codon), elongation (successive tRNAs deliver amino acids that are joined into a growing chain), translocation (the ribosome steps to the next codon), and termination (a stop codon triggers release of the finished polypeptide).

Plasmids

Plasmids are small, double-stranded, circular DNA molecules that replicate independently of the chromosome in the bacterial cytoplasm (and occasionally in yeast). They are dispensable — a cell can gain or lose one without dying — and a single cell may carry anywhere from one copy up to forty or more.

A plasmid that integrates into the chromosome and thereafter replicates along with it is called an episome; the process of losing a plasmid, whether spontaneously or by deliberate treatment (acridine dyes, radiation, thymine starvation, elevated temperature), is called curing.

Plasmids can be grouped by behavior and by function:

• Conjugative vs non-conjugative — conjugative plasmids can transfer themselves to another cell via conjugation; non-conjugative plasmids cannot.
• Compatible vs incompatible — two plasmids that share the same replication or partition machinery compete for it, and one is eventually lost; only genuinely compatible plasmids can coexist stably in one cell.
• By function — fertility (F) plasmids carry the tra genes that build the sex pilus and drive conjugation; resistance (R) plasmids carry antibiotic-resistance genes; Col plasmids code for bacteriocins; virulence plasmids code for toxins and adherence factors (E. coli’s heat-labile/heat-stable toxins, siderophore production, the K88 adherence antigen); and metabolic plasmids equip the host to digest unusual substrates (toluene, salicylate, camphor), fix nitrogen, or synthesize urease.

Because they move DNA readily between cells, plasmids are the workhorse vectors of genetic engineering — genes can be inserted into them artificially and carried into a new host for protein production or gene therapy.

Mutation

A mutation is a random, heritable change in the nucleotide sequence, occurring at a frequency of roughly 10⁻² to 10⁻¹⁰ per cell per division and affecting chromosomal DNA far more often than plasmid DNA. Mutations arise either spontaneously, with no external trigger, or are induced by a mutagen — physical agents such as UV radiation (which particularly damages cytosine and thymine) or chemical agents such as alkylating agents, 5-bromouracil, and acridine dyes.

Most mutations go unnoticed because they are silent, lethal, or affect a function no one is looking for; they become visible only when they alter something observable — such as an E. coli mutant that has lost the ability to ferment lactose, easily spotted on MacConkey agar. Practically, mutation matters most in two places: the emergence of drug resistance, and the deliberate attenuation used to make live vaccines.

Mutations can be classified at different levels:

• Single base-pair substitutions — a transition swaps one purine for the other (A↔G) or one pyrimidine for the other (C↔T); a transversion swaps a purine for a pyrimidine or vice versa. At the codon level, the same substitution can be silent (new codon, same amino acid), neutral (different but functionally equivalent amino acid), missense (a genuinely different amino acid), or nonsense (the new codon becomes a premature stop codon, truncating the protein).
• Insertions or deletions — when the number of bases added or removed is not a multiple of three, every codon downstream is shifted out of frame; this frameshift mutation is usually highly damaging because it scrambles the entire reading frame from that point onward.
• Reverse mutations — a second mutation that restores the original phenotype. A true reversion restores the exact original sequence; an equivalent reversion produces a different codon that happens to code for the same original amino acid; a suppressor mutation restores the phenotype through a compensating change in a different gene entirely.

Mutants are detected either by gene sequencing (the current method of choice) or by older phenotypic methods: the fluctuation test demonstrates that mutations arise spontaneously rather than being induced by the selective agent itself, replica plating identifies auxotrophic mutants that fail to grow without a specific nutrient, and the Ames test screens a chemical for mutagenic (and by extension, potential carcinogenic) activity.

Horizontal Gene Transfer

Unlike vertical transmission to daughter cells at division, horizontal gene transfer moves genetic material sideways, between bacteria that are not parent and offspring. Four mechanisms exist: transformation, transduction, lysogenic conversion, and conjugation.

Transformation

Transformation is the uptake of naked, free DNA released into the surrounding medium (typically by lysis of another bacterium) and its incorporation into the recipient’s own chromosome. It has only been demonstrated naturally in a handful of genera — Streptococcus, Bacillus, Haemophilus, Neisseria, Acinetobacter, and Pseudomonas.

Uptake depends on the recipient being competent — actively dividing in log phase and expressing competence factors. Some competent species (S. pneumoniae) will take up DNA from any source; others (H. influenzae) only take up DNA from closely related species because they lack the broader competence machinery.

Mechanistically: a long double-stranded DNA fragment binds a DNA-binding protein on the competent cell’s surface and is nicked by a nuclease; one strand is degraded by the recipient’s own exonucleases; the surviving single strand, bound to a competence-specific protein, is actively internalized (an energy-dependent step); and that single strand is finally integrated into the host chromosome in place of the corresponding homologous region.

The classic demonstration is Griffith’s 1928 experiment: mice injected with live noncapsulated pneumococci survived, mice injected with heat-killed capsulated pneumococci survived, but mice injected with a mixture of the two died — the live, harmless strain had been transformed into a lethal capsulated strain by capsular genes released from the dead bacteria. Avery, MacLeod, and McCarty later identified DNA itself as the transforming material.

Transduction

Transduction is the transfer of bacterial DNA from one cell to another carried by a bacteriophage. Phages replicate through either a lytic cycle (multiply, lyse the host, release progeny phages) or a lysogenic cycle (the phage DNA integrates into the host chromosome as a prophage and is quiescent, replicating passively along with the bacterial genome).

Generalized transduction occurs when phage assembly goes wrong during a lytic infection: instead of packaging its own DNA, a daughter phage accidentally packages a random fragment of host DNA. This transducing phage still injects that DNA into the next bacterium it infects, but because it carries no phage genome it cannot trigger a new lytic cycle. Inside the recipient, the transferred host DNA has three possible fates: most commonly (70–90%) it survives without integrating (abortive transduction); it may integrate stably into the recipient chromosome; or it may simply be degraded.

Restricted (specialized) transduction happens when a prophage excises imprecisely from the chromosome, dragging along a piece of adjacent bacterial DNA — studied most thoroughly in lambda phage of E. coli. The resulting hybrid genome (phage + bacterial DNA) can either integrate wholesale as a new prophage (if a helper phage is already present) or exchange material with the recipient chromosome by crossover.

Transduction is a real route by which resistance genes spread — plasmid-borne penicillin resistance in staphylococci moves this way — and has also been explored as a genetic-engineering tool for correcting inborn metabolic errors.

Lysogenic Conversion

While a phage genome sits integrated as a prophage, it behaves as an extra piece of the host chromosome and can confer entirely new properties on the bacterium — most importantly, coding for a toxin. The textbook example is Corynebacterium diphtheriae: diphtheria toxin is encoded by a lysogenic phage, and a strain cured of that phage becomes nontoxigenic. Other phage-coded toxins include cholera toxin, E. coli verocytotoxin, streptococcal pyrogenic exotoxins A and C, and botulinum toxins C and D. The distinction from transduction is conceptual: in lysogenic conversion the phage genome itself is the new genetic element conferring the property, whereas in transduction the phage is merely the vehicle carrying someone else’s (bacterial) genes.

Conjugation

Conjugation transfers genetic material from a donor (“male”) bacterium to a recipient (“female”) through direct contact via a conjugation tube, discovered by Lederberg and Tatum in 1946.

A donor cell carries the F factor (fertility factor), a conjugative plasmid encoding the genes for sex pilus formation and self-transfer; a cell lacking it is F⁻. The F pilus draws donor and recipient together and forms the conjugation tube; the F factor then replicates by the rolling-circle mechanism, sending a copy across into the recipient, which is completed to double-stranded DNA there. The recipient thereby converts from F⁻ to F⁺ and can itself go on to conjugate — maleness is, in this sense, infectious. Chromosomal genes are only rarely co-transferred during ordinary F⁺ × F⁻ mating.

When the F factor integrates into the bacterial chromosome (behaving as an episome), the resulting cell is called Hfr (high frequency of recombination), because it transfers chromosomal genes to a recipient far more efficiently than an ordinary F⁺ cell does. Only a portion of the chromosome (plus part of the F factor) usually transfers before the connection breaks, so the F⁻ recipient in an Hfr mating does not become F⁺.

If the integrated F factor later reverts to its free state, it can excise imprecisely and carry a fragment of adjacent chromosomal DNA out with it, becoming an F′ factor. Conjugation of an F′ donor with an F⁻ recipient — called sexduction — transfers that chromosomal fragment along with the F factor, and the recipient becomes F′.

Conjugation is the principal route by which multidrug resistance spreads: the R factor (resistance factor) consists of a resistance transfer factor (RTF, which mediates conjugative transfer, functionally analogous to the F factor) plus one or more resistance determinants (r), each conferring resistance to a specific drug class. If RTF and the r determinants dissociate into separate plasmids, the host remains resistant but can no longer transfer that resistance to others. Conjugation likewise transfers Col factors, the plasmids coding for bacteriocins — antibiotic-like proteins that kill other bacteria, called colicins when made by coliforms, with analogues such as pyocin (Pseudomonas) and diphthericin (C. diphtheriae) in other genera.

Whatever the route of entry, transferred donor DNA is ultimately either degraded by host nucleases or integrated by recombination — as a replacement for the corresponding host sequence (typical of transformation) or as an additional piece (typical of the other mechanisms).

Transposition

Transposons (transposable elements, or “jumping genes”) are segments of DNA capable of moving between chromosome and chromosome, plasmid and plasmid, or chromosome and plasmid, in a cut-and-paste fashion — a process called transposition. Unlike ordinary recombination, transposition requires no sequence homology between the transposon and its new insertion site, and unlike plasmids, transposons cannot replicate independently — they depend entirely on the replication of whatever chromosome or plasmid currently carries them. First described by Barbara McClintock in maize in the 1940s (Nobel Prize, 1983), transposons occur across bacteria, viruses, and eukaryotic genomes alike.

The simplest form, an insertion sequence, is 1–2 kb long: a central transposase gene flanked at both ends by inverted repeat sequences. Because the two flanking repeats are complementary to each other in reverse order, each strand can fold back on itself into a stem-loop structure. A composite transposon is larger and carries extra genes — commonly for antibiotic resistance or toxin production — sandwiched between two insertion sequences.

Gene Transfer by Artificial Methods

Genetic engineering is the deliberate, laboratory-directed alteration of an organism’s genome, carried out through recombinant DNA technology: bacterial DNA is cut with restriction endonucleases into fragments; the fragment carrying the gene of interest is isolated (Southern blot — electrophoretic separation, transfer to a nitrocellulose membrane, detection with a labelled DNA probe, and extraction of the identified band); that fragment is ligated into a vector; the recombinant vector is introduced into a new bacterial host, usually by transformation (electroporation) and occasionally via a phage vector by transduction; and the transformed bacteria are cultured to yield large quantities of the desired gene product.

Applications include producing vaccine antigens (hepatitis B, HPV), producing the antigens used in diagnostic immunoassays, manufacturing therapeutic proteins (human growth hormone, insulin, interferons, interleukin-2, tumour necrosis factor, factor VIII), creating transgenic animals by introducing foreign DNA into an animal genome (transfection), and gene therapy — replacing a defective gene with a normal copy in a patient with a genetic disease.

A vector for this purpose is simply a piece of DNA that can carry a foreign fragment, replicate stably, and be maintained for cloning; the four major classes are plasmids, bacteriophages, cosmids, and artificial chromosomes (bacterial or yeast).

Blotting techniques transfer DNA, RNA, or protein from a gel onto a membrane for specific detection: Southern blot detects DNA (via a nucleic-acid probe), Northern blot detects RNA (same principle, RNA probe), Western blot detects protein (via immunoassay), and Eastern blot is a variant of Western blot used to detect post-translational modifications (lipid, carbohydrate, phosphorylation) on a protein.

Hand-drawing notes (no rendered diagrams for this topic)

Conjugation — F+ × F-, Hfr × F-, F′ × F-. Draw three side-by-side panels, each showing two cells joined by a conjugation tube (pilus). Panel 1 (F+ × F-): donor’s F factor (small circle) replicates and a copy crosses the tube; recipient becomes F+. Panel 2 (Hfr × F-): the F factor is shown integrated into the chromosome (large circle) in the donor; only a fragment of chromosome + partial F factor crosses before the tube breaks; recipient stays F-. Panel 3 (F′ × F-): the F factor carries a small extra loop of chromosomal DNA attached to it; this whole unit crosses; recipient becomes F′. Label each donor cell’s genotype and each recipient’s resulting genotype explicitly — the single most common exam error is stating that Hfr × F- mating converts the recipient to F+, when in fact the connection typically breaks first and the recipient stays F-.

Transduction — generalized vs restricted. Two parallel flowcharts. Generalized: lytic cycle → packaging error → host DNA (not phage DNA) packaged into capsid → injected into new host → three-way branch labelled abortive / stable / unstable. Restricted: prophage integrated at a fixed site → imprecise excision drags adjacent host gene along → transducing phage → two-way branch labelled “whole genome integrates (needs helper phage)” / “crossover exchange.” Label which phage this is classically studied in (lambda, for restricted transduction) since exam questions frequently ask this by name.

Transformation. A single linear sequence: naked dsDNA released by lysed donor → binds DNA-binding protein on competent recipient → nicked by nuclease → one strand degraded → other strand (with competence protein) enters cell → integrates into chromosome, replacing the homologous region. Label the energy-dependent step explicitly (uptake of the single strand), since it is commonly asked which step requires energy.

Griffith’s experiment. Four mice in a row: (1) live noncapsulated pneumococci → survives; (2) heat-killed capsulated pneumococci → survives; (3) live noncapsulated + heat-killed capsulated together → dies; (4) [optional control] live capsulated alone → dies. Label experiment year (1928) and the researchers who later identified DNA as the transforming principle (Avery, MacLeod, McCarty, 1944) as a separate caption, not part of the diagram itself.

Not rendered

Plasmid types, mutation classification, and R-factor structure are already fully captured as tables in notes.md/lnr.md — a diagram would only redraw information a table already states clearly, which does not clear the bar for a rendered figure under this pipeline’s diagram policy.

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