Microbial Genetics

Genetic Material: DNA and Genome Organization

Microbial genetics focuses on the structure, function, and regulation of genetic material in microorganisms. The genome is the entire genetic complement of an organism, including all nucleotide sequences and genes. Genes carry instructions for the synthesis of RNA and proteins, which are essential for cellular function.

• Genotype: The complete set of genes in the genome.

• Phenotype: Physical features and functional traits resulting from gene expression.

Nucleotide Structure

Nucleotides are the monomers of nucleic acids, composed of a phosphate group, a pentose sugar (deoxyribose in DNA, ribose in RNA), and a nitrogenous base (Adenine, Guanine, Cytosine, Thymine, or Uracil).

• Phosphate group: Provides negative charge.

• Pentose sugar: Deoxyribose (DNA), Ribose (RNA).

• Nitrogenous bases: A, G, C, T (DNA); U replaces T in RNA.

Nucleic Acid Structure

Nucleotides are linked in a 5' to 3' manner, forming the backbone of nucleic acids. Energy for polymerization comes from tri/diphosphate building blocks.

Double Helix Structure

In most cells and viruses, DNA forms a double helix with two antiparallel strands. Hydrogen bonds form between complementary bases: C-G and A-T in DNA; U replaces T in RNA.

• Antiparallel: Strands run in opposite directions.

• Complementary: Base pairing ensures accurate replication.

Prokaryotic vs. Eukaryotic Genomes

Prokaryotic genomes are typically contained in circular chromosomes and plasmids, while eukaryotic genomes are found in linear nuclear chromosomes and extranuclear DNA (mitochondria and chloroplasts).

• Prokaryotes: Usually one circular chromosome, plasmids carry non-essential but advantageous traits.

• Eukaryotes: Multiple linear chromosomes in nucleus; extranuclear DNA is circular.

DNA Replication

Overview and Semiconservative Replication

DNA replication is an anabolic polymerization process requiring nucleotide monomers and energy. It begins with a double-stranded parental DNA helix, and each strand serves as a template for a new, complementary daughter strand. Replication is semiconservative: each new helix contains one original and one daughter strand.

Steps of DNA Replication

1. Remove DNA-associated proteins

2. Unwind and separate template strands (DNA helicase)

3. Synthesize RNA primers (Primase)

4. Extend primers with DNA polymerase III (synthesis in 3' direction)

5. Degrade RNA primers and fill gaps with DNA polymerase I

6. Rewind new hybrid strands

Leading vs. Lagging Strand Synthesis

DNA polymerase III synthesizes new DNA only in the 5' to 3' direction. Due to antiparallel strands, the leading strand is synthesized continuously toward the replication fork, while the lagging strand is synthesized discontinuously away from the fork in short fragments (Okazaki fragments).

• Leading strand: Continuous synthesis.

• Lagging strand: Discontinuous synthesis, filled in by DNA polymerase I and ligase.

Replication of Circular Chromosomes

In prokaryotes, replication begins at the origin of replication (ori) and proceeds bidirectionally around the circular chromosome. Topoisomerases relieve torsional strain during replication.

Gene Expression: Transcription and Translation

Transcription

Transcription is the process of copying DNA information within a gene into RNA nucleotide sequences. It occurs in three steps: initiation, elongation, and termination.

• Initiation: RNA polymerase binds to promoter with sigma factor.

• Elongation: RNA polymerase extends the transcript using triphosphate ribonucleotides.

• Termination: RNA polymerase releases the transcript at the terminator sequence.

Key Comparisons

• Transcription vs. DNA Replication: Transcription forms RNA from genes; replication forms DNA from chromosomes/plasmids.

• RNA polymerase vs. DNA polymerase: RNA polymerase synthesizes RNA and can unwind/rewind DNA; DNA polymerase synthesizes DNA and removes RNA primers.

• Fates of RNA: mRNA (codes for polypeptides), rRNA (forms ribosomes), tRNA (transfers amino acids).

Translation

Translation is the synthesis of polypeptides (proteins) through interpretation of mRNA nucleotide sequences. It occurs in three steps: initiation, elongation, and termination.

• Codon: Three-nucleotide unit in mRNA coding for a specific amino acid.

• Start codon: AUG (methionine).

• Stop codons: UAA, UAG, UGA (signal termination).

Transfer RNA (tRNA)

tRNA molecules carry specific amino acids and recognize mRNA codons via their anticodon region.

Ribosomal RNA (rRNA) and Ribosomes

rRNA combines with proteins to form ribosomes, which are the platform for protein synthesis. Prokaryotic ribosomes are 70S, smaller than eukaryotic 80S ribosomes.

Translation Steps

• Initiation: Ribosomal subunits assemble on mRNA at the ribosomal binding site (RBS), recruiting initiator tRNA.

• Elongation: Ribosome recruits amino acid-bound tRNAs according to mRNA sequence, forming peptide bonds.

• Termination: Release factors recognize stop codons, sever polypeptide, and ribosome dissociates.

Coupled Transcription and Translation

In prokaryotes, transcription and translation can occur simultaneously, allowing rapid gene expression.

Regulation of Gene Expression

Prokaryotic Gene Organization: Operons

Genes with similar functions are grouped into operons, regulated by common promoter and operator elements. Polycistronic mRNA carries information for several genes.

• Inducible operons: Activated by inducers (e.g., lactose operon).

• Repressible operons: Transcribed continually until deactivated by repressors (e.g., tryptophan operon).

Mutations and DNA Repair

Types of Mutations

Mutations are changes in the nucleotide base sequence of an organism's genome. They are rare due to DNA repair mechanisms and are usually deleterious, but can occasionally confer beneficial traits.

• Point mutations: Affect one or a few base pairs.

• Frameshift mutations: Alter the reading frame of codons.

Mutagens

• Radiation: Ionizing (X-rays) and nonionizing (UV light) cause DNA damage.

• Chemical mutagens: Nucleotide analogs, nucleotide-altering chemicals, frameshift mutagens.

Bacterial DNA Repair Mechanisms

• Direct repair: Enzymatic removal of damage.

• Excision repair: Removal and replacement of damaged DNA.

• Postreplication repair: Retrieval of missing information by recombination.

• SOS response: Induction of repair genes after DNA damage.

• Error-prone repair: Last resort, fills gaps with random sequences.

Genetic Recombination and Horizontal Gene Transfer

Genetic Recombination

Genetic recombination is the exchange of DNA segments with homologous sequences, resulting in recombinant cells with new nucleotide sequences.

• Vertical gene transfer: Genes passed to descendants.

• Horizontal gene transfer: Donor contributes part of genome to recipient; includes transformation, transduction, and conjugation.

Transformation

Uptake of DNA from the environment by competent cells, often from dead cells. If the DNA codes for a novel trait, the recipient can express it.

Transduction

Transfer of DNA from donor to recipient via a bacteriophage (virus). Generalized transduction transfers random DNA segments; specialized transduction transfers specific sequences, often toxin genes.

Bacterial Conjugation

Transfer of DNA between bacterial cells in physical contact, mediated by conjugation pili or adhesions. The ability to synthesize a pilus is conferred by the F plasmid (fertility factor).