Microbial Genetics: Structure, Function, and Genetic Variation

Microbial Genetics

Structure of DNA

Deoxyribonucleic acid (DNA) is the hereditary material in all living organisms, encoding the instructions necessary for life. In prokaryotes, DNA is typically organized into a single circular chromosome, while eukaryotes possess multiple linear chromosomes.

• DNA Structure: DNA is a double helix composed of two antiparallel strands held together by hydrogen bonds between complementary nitrogenous bases: adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).

• Backbone: The backbone consists of alternating deoxyribose sugars and phosphate groups, connected by phosphodiester bonds.

• Directionality: Each strand has a 5’ (phosphate) end and a 3’ (hydroxyl) end, which is crucial for replication and transcription.

• Base Pairing: The proportion of G-C to A-T base pairs varies among organisms and affects DNA stability.

• Genes: A gene is a sequence of nucleotides that codes for a polypeptide or RNA molecule, determining an organism’s traits.

Example: If an organism has 18% thymine, it also has 18% adenine. The remaining 64% is divided equally between cytosine and guanine, so G+C content is 32%.

Plasmids

Plasmids are small, circular, double-stranded DNA molecules found in bacteria, separate from the chromosomal DNA. They replicate independently and often carry genes beneficial for survival but not essential for basic cellular functions.

• F (Fertility) Plasmid: Carries genes for conjugation (tra genes), enabling DNA transfer between bacteria.

• R (Resistance) Plasmid: Contains genes that confer resistance to antibiotics or toxic metals.

• Virulence Plasmids: Carry genes that enhance pathogenicity, such as toxin production.

DNA Replication in Prokaryotes

DNA replication is the process by which a cell duplicates its DNA, ensuring genetic information is passed to daughter cells. It is semiconservative, meaning each new DNA molecule consists of one parental and one newly synthesized strand.

• Key Enzymes:

  • Helicase: Unwinds the DNA double helix.

  • Single-Strand Binding Proteins: Stabilize unwound DNA.

  • Primase: Synthesizes RNA primers.

  • DNA Polymerase III: Synthesizes new DNA strands by adding nucleotides to the 3’ end.

  • DNA Polymerase I: Replaces RNA primers with DNA.

  • Ligase: Joins Okazaki fragments on the lagging strand.

• Leading vs. Lagging Strand: The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously in short segments called Okazaki fragments.

Gene Expression: Transcription and Translation

Gene expression involves two main processes: transcription (DNA to RNA) and translation (RNA to protein).

• Transcription: Synthesis of RNA from a DNA template by RNA polymerase. Steps include initiation, elongation, and termination (self-termination or Rho-dependent termination).

• Translation: Ribosomes assemble on mRNA and synthesize proteins by reading codons and matching them with amino acids via tRNA. Steps include initiation, elongation, and termination.

• Types of RNA:

  • mRNA (messenger RNA): Carries genetic information from DNA to ribosomes.

  • tRNA (transfer RNA): Brings amino acids to the ribosome during translation.

  • rRNA (ribosomal RNA): Structural and catalytic component of ribosomes.

Regulation of Genetic Expression

Cells regulate gene expression to conserve energy and resources. This occurs at both the transcriptional and translational levels.

• Operons: Clusters of genes under the control of a single promoter and operator. Types include:

  • Inducible Operons (e.g., lac operon): Usually off; turned on in the presence of a substrate (e.g., lactose).

  • Repressible Operons (e.g., trp operon): Usually on; turned off when the end product (e.g., tryptophan) is abundant.

• Translational Control: Small regulatory RNAs (miRNA, siRNA) can bind mRNA and prevent translation.

Mutations and Genetic Variation

Mutations are changes in the DNA sequence that can lead to genetic diversity. They may occur spontaneously or be induced by mutagens.

• Types of Mutations:

  • Point Mutations: Affect a single nucleotide (substitution, addition, deletion).

  • Frameshift Mutations: Addition or deletion of nucleotides that shifts the reading frame.

• Effects of Mutations:

  • Silent Mutation: No change in amino acid sequence.

  • Missense Mutation: Changes one amino acid.

  • Nonsense Mutation: Introduces a stop codon.

• Mutagens: Physical (ionizing, non-ionizing radiation) or chemical agents (base analogs, nucleotide-altering chemicals, frameshift mutagens) that increase mutation rates.

DNA Repair Mechanisms

• Light Repair (Photoactivation): Uses photolyase to repair pyrimidine dimers caused by UV light.

• Dark Repair: Endonuclease removes damaged DNA, which is then replaced by DNA polymerase and ligase.

Isolation and Detection of Mutants

• Direct Selection: Mutants are isolated by growing on selective media (e.g., antibiotic resistance).

• Indirect Selection (Replica Plating): Used to identify auxotrophs that cannot grow on minimal media.

• Ames Test: Screens chemicals for mutagenicity using Salmonella strains unable to synthesize histidine. Increased colony growth near a test agent indicates mutagenic activity.

Note: The Ames test is an indicator of mutagenicity, not direct carcinogenicity in humans.

Horizontal Gene Transfer

Horizontal gene transfer (HGT) is the movement of genetic material between organisms other than by descent. It increases genetic diversity in microbial populations.

• Transformation: Uptake of naked DNA from the environment by competent cells (e.g., Griffith’s experiment with Streptococcus pneumoniae).

• Transduction: Transfer of DNA via bacteriophages. Generalized transduction transfers random DNA segments; specialized transduction transfers specific genes adjacent to prophage integration sites.

• Conjugation: Direct transfer of DNA between bacteria via cell-to-cell contact, often mediated by F plasmids. High-frequency recombination (Hfr) strains can transfer chromosomal genes.

Key Definitions

• Introns: Non-coding sequences in eukaryotic genes, removed during RNA processing.

• Exons: Coding sequences that remain in mature mRNA.

• Auxotroph: Mutant organism requiring a specific nutrient not needed by the wild type.

• Prototroph: Wild-type organism that can synthesize all nutrients required for growth.

Comparison Table: Horizontal Gene Transfer Mechanisms

Mechanism	DNA Source	Transfer Method	Requirement
Transformation	Naked DNA	Uptake from environment	Competent cells
Transduction	Bacterial DNA	Bacteriophage-mediated	Phage infection
Conjugation	Plasmid or chromosomal DNA	Direct cell-to-cell contact	Conjugative plasmid (F factor)