Microbial Genetics

Structure and Function of the Genetic Material

Microbial genetics is the study of genes, how they carry information, how this information is expressed, and how genes are replicated. The genetic material of microorganisms is organized into chromosomes, which are structures containing DNA that physically carry hereditary information. Genes are segments of DNA that encode functional products, usually proteins, and the entire genetic content of a cell is called its genome. Genomics is the sequencing and molecular characterization of genomes.

• Genetic code: The set of rules by which a nucleotide sequence is converted to an amino acid sequence of a protein.

• Central dogma: Describes the flow of genetic information from DNA to RNA to protein.

Genotype refers to the genetic makeup of an organism, while phenotype is the expression of those genes.

Bacteria typically have a single circular chromosome made of DNA and associated proteins. For example, the Escherichia coli chromosome contains 4.6 million base pairs of DNA, which is highly supercoiled. The genome includes both protein-encoding genes and noncoding regions called short tandem repeats (STRs), which are repeating sequences of 2–5 base pairs.

The Flow of Genetic Information

Genetic information can be transferred vertically (from one generation to the next) or horizontally (between cells of the same generation). The main processes are:

• Expression: Genetic information is used within a cell to produce the proteins needed for cell function.

• Recombination: Genetic information can be transferred horizontally, creating new combinations of genes.

• Replication: Genetic information is transferred vertically to the next generation of cells.

DNA Replication

Structure of DNA

DNA forms a double helix with a backbone consisting of deoxyribose-phosphate. Two strands of nucleotides are held together by hydrogen bonds between adenine (A) and thymine (T), and between cytosine (C) and guanine (G). The strands are antiparallel, meaning they run in opposite directions. The order of the nitrogenous bases forms the genetic instructions of the organism. Because the two strands are complementary, each can serve as a template for the synthesis of a new strand during replication.

Mechanism of DNA Replication

DNA replication is a highly accurate process due to the proofreading capability of DNA polymerase. The process involves several key enzymes:

• Topoisomerase and gyrase: Relax the DNA strands.

• Helicase: Separates the DNA strands, creating a replication fork.

• DNA polymerase: Adds nucleotides to the growing DNA strand, always in the 5' to 3' direction. Initiation requires an RNA primer.

• Leading strand: Synthesized continuously.

• Lagging strand: Synthesized discontinuously, creating Okazaki fragments, which are later joined by DNA ligase.

Most bacterial DNA replication is bidirectional, and each offspring cell receives one copy of the DNA molecule.

RNA and Protein Synthesis

Types of RNA

RNA is a single-stranded nucleic acid with a ribose sugar and uracil (U) instead of thymine (T). The main types of RNA are:

• Ribosomal RNA (rRNA): Integral part of ribosomes.

• Transfer RNA (tRNA): Transports amino acids during protein synthesis.

• Messenger RNA (mRNA): Carries coded information from DNA to ribosomes.

Transcription in Prokaryotes

Transcription is the synthesis of a complementary mRNA strand from a DNA template. It involves three main steps:

• Initiation: RNA polymerase binds to the promoter sequence on DNA.

• Elongation: RNA polymerase adds ribonucleotides in the 5' to 3' direction.

• Termination: Transcription stops when the terminator sequence is reached.

Translation

Translation is the process by which mRNA is decoded to synthesize proteins. Codons, groups of three mRNA nucleotides, code for specific amino acids. There are 64 codons: 61 sense codons for amino acids, one start codon (AUG), and three stop codons (UAA, UAG, UGA). The genetic code is degenerate, meaning multiple codons can code for the same amino acid.

Translation begins at the start codon and ends at a stop codon. tRNA molecules transport amino acids to the ribosome and have anticodons that base-pair with mRNA codons. Amino acids are joined by peptide bonds.

In bacteria, translation can begin before transcription is complete because both processes occur in the cytoplasm.

Transcription in Eukaryotes

In eukaryotes, transcription occurs in the nucleus and translation in the cytoplasm. Genes are interrupted by noncoding regions (introns) and coding regions (exons). After transcription, introns are removed and exons are spliced together by small nuclear ribonucleoproteins (snRNPs).

Regulation of Bacterial Gene Expression

Gene Regulation Mechanisms

Some genes are constitutive (always expressed), while others are regulated and expressed only as needed. Regulation can occur at the pre-transcriptional level through repression (inhibiting gene expression) or induction (turning on gene expression).

• Repressors: Proteins that block transcription.

• Inducers: Molecules that initiate gene expression.

The Operon Model

An operon is a set of operator and promoter sites and the structural genes they control. The promoter is where RNA polymerase initiates transcription, and the operator controls transcription of structural genes.

In inducible operons (e.g., the lac operon), genes are not transcribed unless an inducer is present. In the absence of lactose, the repressor binds to the operator, preventing transcription. In the presence of lactose, allolactose (the inducer) inactivates the repressor, allowing transcription.

In repressible operons (e.g., the trp operon), genes are transcribed until turned off by a corepressor (excess tryptophan), which activates the repressor to bind the operator and stop transcription.

Mutation

Types of Mutations

A mutation is a permanent change in the DNA base sequence. Mutations can be neutral, beneficial, or harmful. Mutagens are agents that cause mutations, while spontaneous mutations occur without mutagens.

• Silent (neutral) mutation: Does not affect the protein's activity.

• Missense mutation: Base substitution results in a different amino acid.

• Nonsense mutation: Base substitution creates a stop codon.

• Frameshift mutation: Insertion or deletion of nucleotides shifts the reading frame, altering downstream amino acids.

Chemical and Physical Mutagens

Chemical mutagens include nitrous acid, nucleoside analogs, and frameshift mutagens (e.g., aflatoxin). Nucleoside analogs resemble normal bases but cause incorrect base pairing.

Radiation can also cause mutations. Ionizing radiation (X-rays, gamma rays) can break DNA backbones, while UV radiation causes thymine dimers, which can be repaired by photolyases or nucleotide excision repair.

Genetic Transfer and Recombination

Genetic Recombination

Genetic recombination is the exchange of genes between two DNA molecules, forming new gene combinations and contributing to genetic diversity. Crossing over involves the breaking and rejoining of DNA segments, allowing insertion of foreign DNA into the chromosome.

Gene transfer can be vertical (to offspring) or horizontal (between cells of the same generation). Horizontal gene transfer involves a donor cell and a recipient cell, resulting in a recombinant cell.

Plasmids and Transposons

Plasmids are self-replicating, circular DNA molecules found mainly in bacteria. They may carry genes for toxin production or antibiotic resistance (R factors). Transposons are mobile genetic elements that can move within and between DNA molecules.

Transformation in Bacteria

Transformation is the uptake of naked DNA from the environment by a bacterium. This was first demonstrated by Griffith's experiment with Streptococcus pneumoniae. Transformation can occur when bacterial cells die and release DNA, which is then taken up and incorporated by other bacteria.

Conjugation in Bacteria

Conjugation is the transfer of plasmids from one bacterium to another via direct cell-to-cell contact. In Gram-negative bacteria, this occurs via sex pili; in Gram-positive bacteria, a sticky substance holds cells together. Donor cells carry a conjugative plasmid (F factor), and recipients become F+ after receiving the plasmid. Hfr cells have the F factor integrated into their chromosome and can transfer chromosomal genes during conjugation.

Transduction in Bacteria

Transduction is the transfer of DNA from a donor to a recipient cell via a bacteriophage (virus that infects bacteria). Generalized transduction involves random bacterial DNA, while specialized transduction transfers specific genes.