🦠Microbiology Unit 11 – Mechanisms of Microbial Genetics

Key Concepts and Terminology

• Genome refers to the complete set of genetic material in an organism including genes and non-coding sequences
• Nucleotides are the building blocks of DNA and RNA consisting of a sugar, phosphate group, and nitrogenous base (adenine, guanine, cytosine, thymine in DNA; uracil in RNA)
• Central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins
• Plasmids are small, circular, self-replicating DNA molecules found in bacteria separate from the main chromosome
• Operons are functional units of bacterial genomes that include a promoter, operator, and one or more genes that are co-transcribed
• Horizontal gene transfer is the exchange of genetic material between organisms through mechanisms like conjugation, transduction, and transformation
• Recombinant DNA technology involves manipulating and combining DNA from different sources to create novel genetic sequences for various applications

DNA Structure and Function

• DNA is a double-stranded helical molecule composed of nucleotide monomers held together by phosphodiester bonds
  • Complementary base pairing (A-T and G-C) and hydrogen bonds between bases stabilize the double helix structure
• Antiparallel nature of DNA strands means they run in opposite directions with one strand oriented 5' to 3' and the other 3' to 5'
• DNA stores and transmits genetic information across generations serving as a blueprint for an organism's structure and function
• Genes are specific sequences of DNA that encode instructions for making functional products like proteins or RNA molecules
• Non-coding DNA regions have regulatory functions (promoters, enhancers) or structural roles (telomeres, centromeres)
• DNA packaging into chromatin and chromosomes allows the long molecules to fit inside cells and helps regulate gene expression
  • In prokaryotes, DNA is condensed into the nucleoid region without histones
  • In eukaryotes, DNA wraps around histone proteins to form nucleosomes, which further condense into higher-order structures

Bacterial Genome Organization

• Most bacteria have a single circular chromosome containing essential genes for survival and reproduction
• Bacterial genomes are compact with little non-coding DNA and genes arranged in operons for coordinated expression
• Plasmids are common in bacteria and often carry genes for adaptations like antibiotic resistance or metabolic functions
• Insertion sequences (IS) are short, mobile genetic elements that can move within a genome causing mutations and rearrangements
• Transposons are longer mobile elements that include genes for functions like antibiotic resistance in addition to IS for movement
• Integrons are genetic elements that can capture, store, and express mobile gene cassettes often involved in antibiotic resistance
• Bacterial genomes can evolve rapidly through horizontal gene transfer, mutations, and genome rearrangements in response to environmental pressures

DNA Replication in Prokaryotes

• Semiconservative replication produces two identical DNA molecules each with one original strand and one new complementary strand
• Replication initiates at a specific origin of replication (oriC) and proceeds bidirectionally until the replication forks meet at the terminus
• DNA helicase unwinds the double helix and separates the strands to provide single-stranded templates for replication
• Single-stranded binding proteins (SSB) stabilize the separated strands and prevent them from reannealing
• DNA primase synthesizes short RNA primers complementary to the single-stranded templates providing a starting point for DNA synthesis
• DNA polymerase III is the main enzyme that synthesizes new DNA strands by adding nucleotides complementary to the template in the 5' to 3' direction
  • The leading strand is synthesized continuously in the same direction as the replication fork
  • The lagging strand is synthesized discontinuously as short Okazaki fragments in the opposite direction
• DNA polymerase I replaces the RNA primers with DNA nucleotides and fills in gaps between Okazaki fragments
• DNA ligase seals the nicks between the newly synthesized DNA fragments creating a continuous strand

Gene Expression: Transcription and Translation

• Transcription is the synthesis of RNA from a DNA template catalyzed by RNA polymerase
  • Initiation involves RNA polymerase binding to a promoter sequence upstream of the gene and separating the DNA strands
  • Elongation occurs as RNA polymerase moves along the template strand in the 3' to 5' direction adding complementary nucleotides to the growing RNA chain
  • Termination happens when RNA polymerase reaches a terminator sequence and releases the newly synthesized RNA transcript
• Translation is the synthesis of proteins using the genetic code on an mRNA template read by ribosomes
  • The genetic code is a triplet code where three nucleotides (a codon) specify a particular amino acid or signal (start/stop)
  • Ribosomes consist of rRNA and proteins that catalyze the formation of peptide bonds between amino acids
  • tRNAs are adapter molecules with an anticodon that base pairs with the mRNA codon and carries the corresponding amino acid
  • Initiation involves the small ribosomal subunit binding to the start codon (AUG) on the mRNA with the help of initiation factors
  • Elongation occurs as the ribosome moves along the mRNA, matching codons with tRNA anticodons, and catalyzing peptide bond formation
  • Termination happens when the ribosome reaches a stop codon (UAA, UAG, UGA) and releases the completed polypeptide chain

Mutations and DNA Repair Mechanisms

• Mutations are changes in the DNA sequence that can alter gene function and phenotype
  • Point mutations involve single nucleotide changes like substitutions (one base replaced by another), insertions, or deletions
  • Frameshift mutations occur when insertions or deletions of nucleotides alter the reading frame of codons during translation
• Spontaneous mutations can arise from DNA replication errors, chemical modifications, or DNA damage from reactive molecules
• Induced mutations result from exposure to mutagens like UV radiation, chemicals, or viruses that increase mutation rates
• DNA repair mechanisms help maintain genome stability by fixing damaged or altered DNA sequences
  • Mismatch repair corrects errors made during DNA replication when incorrect nucleotides are incorporated
  • Base excision repair removes damaged bases (oxidized, alkylated) and replaces them with the correct nucleotide
  • Nucleotide excision repair removes bulky DNA lesions (thymine dimers) by cutting out a segment of the damaged strand and filling it in
  • Double-strand break repair fixes breaks in both strands of DNA through homologous recombination or non-homologous end joining

Genetic Transfer in Bacteria

• Conjugation is the direct transfer of genetic material (usually plasmids) between cells through a mating bridge (pilus)
  • The F factor (fertility factor) is a plasmid that carries genes for the formation of the conjugation pilus and DNA transfer
  • Hfr (high frequency of recombination) cells have the F factor integrated into the bacterial chromosome allowing transfer of chromosomal genes
• Transformation is the uptake of naked DNA from the environment by competent bacterial cells
  • Some bacteria (Streptococcus, Bacillus) are naturally competent while others (E. coli) can be made competent through chemical or electrical treatments
• Transduction is the transfer of bacterial genes via bacteriophages (viruses that infect bacteria)
  • In generalized transduction, any bacterial gene can be packaged into the phage capsid and transferred to another cell
  • In specialized transduction, only genes adjacent to the phage integration site on the bacterial chromosome are transferred
• Horizontal gene transfer contributes to bacterial evolution by allowing the rapid spread of beneficial genes (antibiotic resistance) through populations

Applications in Biotechnology and Medicine

• Recombinant DNA technology allows the creation of genetically modified organisms (GMOs) with novel traits
  • Restriction enzymes are used to cut DNA at specific sequences, and DNA ligase joins the fragments together
  • Vectors (plasmids, viruses) are used to introduce foreign DNA into host cells for replication and expression
• Genetically engineered bacteria are used to produce valuable products like insulin, growth hormones, and enzymes
• DNA sequencing technologies (Sanger, next-generation) have enabled rapid and cost-effective analysis of entire genomes
  • Comparative genomics helps identify conserved genes, regulatory elements, and evolutionary relationships between organisms
• Polymerase chain reaction (PCR) amplifies specific DNA sequences for applications like disease diagnosis, forensics, and research
• CRISPR-Cas9 is a powerful genome editing tool derived from bacterial adaptive immunity that allows precise modification of DNA sequences
  • CRISPR-based therapies are being developed to treat genetic diseases, infections, and cancers
• Synthetic biology aims to design and construct novel biological systems or organisms with specific functions using standardized genetic parts and engineering principles