Premed · Premed · Microbiology
Lecture 9: Microbial Genetics -- Mutation and Recombination
Microbiology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the structure and organization of bacterial genomes
- Explain the process of DNA replication in bacteria
- Define and classify types of mutations
- Describe the mechanisms of DNA repair
- Explain genetic recombination and its significance in bacterial evolution
- Distinguish between vertical and horizontal gene transfer
Lecture Content
I. The Bacterial Genome
The typical bacterial genome consists of a single, circular, double-stranded DNA chromosome, though notable exceptions exist: Borrelia and Streptomyces possess linear chromosomes, and Vibrio cholerae carries two chromosomes. Genome sizes range from about 0.5 to 10 megabases (Mb), with E. coli possessing approximately 4.6 Mb encoding roughly 4,300 genes.
The chromosome resides in the nucleoid, a condensed region of the cytoplasm that is not enclosed by a membrane. The DNA is compacted by negative supercoiling introduced by DNA gyrase and relaxed by topoisomerase I. Nucleoid-associated proteins (NAPs) such as HU, IHF, H-NS, and Fis further organize and compact the chromosome.
Plasmids are small, circular, extrachromosomal DNA elements that replicate independently using their own origins of replication. They carry accessory genes conferring selective advantages -- antibiotic resistance, virulence factors, metabolic enzymes, and heavy metal resistance -- without being essential for growth under normal conditions. Plasmids vary in copy number (high-copy versus low-copy), and those belonging to the same incompatibility group cannot coexist stably within the same cell.
II. DNA Replication in Bacteria
Bacterial DNA replication is semi-conservative, with each daughter molecule containing one parental strand and one newly synthesized strand. In E. coli, replication initiates at a single origin of replication (oriC), where DnaA protein binds and opens the double helix at an AT-rich region. DnaB helicase then unwinds the DNA, and DnaG primase synthesizes short RNA primers to initiate synthesis.
Replication proceeds bidirectionally from oriC, with two replication forks moving in opposite directions. DNA polymerase III, the main replicative enzyme, synthesizes DNA exclusively in the 5' to 3' direction. On the leading strand, synthesis is continuous in the direction of fork movement. On the lagging strand, synthesis is discontinuous, producing Okazaki fragments of 1,000--2,000 nucleotides, each initiated by a new RNA primer. The proofreading activity of DNA Pol III (3' to 5' exonuclease) reduces the error rate to approximately 10^-7 per base pair.
DNA polymerase I removes RNA primers via its 5' to 3' exonuclease activity and fills the resulting gaps. DNA ligase seals the nicks between adjacent Okazaki fragments. Topoisomerases relieve the torsional strain generated ahead of the replication fork; DNA gyrase (topoisomerase II), which introduces negative supercoils, is the target of fluoroquinolone antibiotics. Single-strand binding (SSB) proteins stabilize and protect single-stranded DNA intermediates. Replication terminates when the two forks meet at the terminus region (ter), where the linked daughter chromosomes are resolved by topoisomerases and recombinases.
III. Mutations
A mutation is a heritable change in the DNA sequence. Spontaneous mutations arise from intrinsic errors during replication and from spontaneous chemical changes to bases, including tautomeric shifts (rare base forms that mispair), depurination (loss of a purine base), deamination (for example, conversion of cytosine to uracil, causing a C-G to T-A transition), oxidative damage (such as 8-oxoguanine mispairing with adenine), and replication slippage at repetitive sequences leading to insertions or deletions.
Induced mutations are caused by external agents known as mutagens. Chemical mutagens include base analogs (5-bromouracil, 2-aminopurine) that incorporate into DNA and cause mispairing, alkylating agents (nitrosoguanidine, EMS) that alter base pairing by adding alkyl groups, intercalating agents (acridine orange, ethidium bromide) that insert between base pairs and cause frameshift mutations, and deaminating agents (nitrous acid) that chemically modify bases. Radiation can also induce mutations: UV light produces thymine dimers that block replication, while ionizing radiation causes double-strand breaks and base damage. Transposable elements represent another source of induced mutations, as their insertion can disrupt gene function.
A. Types of Point Mutations
Base substitutions replace one nucleotide with another. A transition exchanges a purine for a purine or a pyrimidine for a pyrimidine, while a transversion exchanges a purine for a pyrimidine or vice versa. The consequences for the encoded protein vary. A silent (synonymous) mutation does not change the amino acid due to codon degeneracy. A missense mutation incorporates a different amino acid, which may or may not affect protein function depending on the chemical properties of the substitution and its location in the protein. A nonsense mutation creates a premature stop codon (UAG, UAA, or UGA), truncating the protein. Frameshift mutations result from insertions or deletions of nucleotides not in multiples of three, shifting the reading frame and altering all downstream codons, which usually produces a nonfunctional protein.
B. Large-Scale Mutations
Larger genomic changes include deletions (loss of a DNA segment), insertions (addition of DNA, often by transposable elements), inversions (reversal of a DNA segment), and duplications (copying of a segment).
C. Mutant Phenotypes
Mutations generate phenotypic diversity. Auxotrophic mutants require a growth factor that the wild-type (prototroph) can synthesize. Antibiotic-resistant mutants may have altered drug targets, upregulated efflux pumps, or produce enzymes that inactivate the drug. Conditional mutants (such as temperature-sensitive mutants) produce a protein that functions at one temperature but not another. Collectively, mutations provide the raw material upon which natural selection acts, driving evolution.
<image>A figure illustrating types of point mutations and their effects on protein coding. Panel A: A wild-type DNA sequence with its corresponding mRNA codons and amino acid sequence. Panel B: Silent mutation -- one base changed but the same amino acid encoded (codon degeneracy shown). Panel C: Missense mutation -- one base changed leading to a different amino acid (highlighted in red). Panel D: Nonsense mutation -- base change creates a premature stop codon (UAG), with the truncated protein shown. Panel E: Frameshift mutation -- single nucleotide insertion shifts all downstream codons, resulting in a completely altered amino acid sequence and premature stop. Each panel clearly labeled with the mutation type and outcome.</image>
IV. DNA Repair Mechanisms
Cells employ multiple repair systems to maintain genomic integrity. Proofreading by the 3' to 5' exonuclease activity of DNA Pol III corrects errors during replication, improving accuracy roughly 100-fold. Mismatch repair (MMR) corrects errors that escape proofreading: MutS detects the mismatch, MutL and MutH are recruited, and MutH cleaves the newly synthesized (unmethylated) strand at a nearby GATC site, after which an exonuclease removes the erroneous segment and DNA Pol III resynthesizes it. Defects in MMR produce a mutator phenotype with elevated mutation rates.
Base excision repair (BER) handles damaged bases. DNA glycosylases recognize and excise abnormal bases (for example, uracil-DNA glycosylase removes uracil resulting from cytosine deamination), an AP endonuclease cleaves the backbone at the resulting abasic site, and DNA Pol I fills the gap. Nucleotide excision repair (NER) handles bulky lesions such as thymine dimers and cross-links. The UvrABC endonuclease system scans the DNA (UvrA-UvrB), makes incisions flanking the damage (UvrC), and UvrD helicase removes the damaged segment of approximately 12 nucleotides, after which DNA Pol I fills and ligase seals.
Photoreactivation is a direct repair mechanism in which photolyase uses visible light energy to split thymine dimers. The SOS response is activated by severe DNA damage that generates extensive single-stranded DNA regions. RecA protein binds the single-stranded DNA and stimulates autocleavage of the LexA repressor, derepressing roughly 40 SOS genes, including error-prone DNA polymerases (Pol IV and Pol V) that allow translesion synthesis -- bypassing lesions at the cost of introducing mutations. This adaptive mutagenesis may generate beneficial mutations under stress conditions.
V. Genetic Recombination
Homologous (general) recombination requires extensive sequence similarity between two DNA molecules and is essential for integrating horizontally transferred DNA. In E. coli, the RecBCD pathway initiates when the RecBCD enzyme enters at a double-strand break, unwinds and degrades DNA until it encounters a Chi (crossover hotspot instigator) sequence, then generates a 3' single-stranded tail. RecA protein coats this single-stranded DNA and promotes strand invasion into a homologous duplex, forming a heteroduplex region. Branch migration extends this region, and the resulting Holliday junction is resolved by the RuvABC complex: RuvA recognizes the junction, RuvB provides the motor for branch migration, and RuvC is an endonuclease that cleaves the junction, producing recombinant DNA molecules.
Site-specific recombination occurs at defined short sequences and is mediated by dedicated recombinases. A classic example is the integration of bacteriophage lambda DNA into the E. coli chromosome at the attB and attP sites by lambda integrase. Site-specific recombination also drives phase variation, such as flagellar antigen switching in Salmonella.
<image>A step-by-step diagram of homologous recombination via the RecBCD pathway. Panel A: Double-strand break at one end of a linear DNA fragment (e.g., from conjugation or transduction). Panel B: RecBCD enzyme enters at the break, unwinds and degrades DNA until it encounters a Chi sequence. Panel C: RecA protein coats the resulting 3' single-stranded tail and promotes strand invasion into a homologous region of the recipient chromosome, forming a D-loop. Panel D: Branch migration extends the heteroduplex; a Holliday junction is formed. Panel E: RuvABC resolves the Holliday junction by cleavage, producing two recombinant DNA molecules. Each step labeled with the key enzymes involved.</image>
VI. Transposable Elements
Insertion sequences (IS elements) are the simplest transposons, typically 700--2,000 bp long, containing only the transposase gene flanked by inverted repeats. Transposase catalyzes excision and insertion, creating short direct repeats of target DNA (target site duplication) upon insertion.
Composite (compound) transposons have a central region carrying accessory genes -- often antibiotic resistance determinants or toxin genes -- flanked by IS elements. Examples include Tn5 (kanamycin resistance) and Tn10 (tetracycline resistance). Complex transposons do not rely on flanking IS elements but encode their own transposase and resolvase; Tn3, which carries ampicillin resistance, is a well-known example.
Transposition can proceed by two mechanisms. Conservative (cut-and-paste) transposition excises the element from its original location and inserts it at a new site. Replicative transposition copies the element, leaving one copy at the original site while inserting a copy at the new location.
Transposons have profound effects on bacterial genomes: they cause insertional inactivation of genes, generate chromosomal rearrangements, and serve as vehicles for spreading antibiotic resistance genes among bacteria. Integrons are related genetic elements that capture and express gene cassettes (often encoding antibiotic resistance) through site-specific recombination catalyzed by an integrase, making them major contributors to multidrug resistance.
