👨‍👩‍👦‍👦General Genetics Unit 13 – Genetics of Bacteria and Viruses

Basic Concepts in Bacterial and Viral Genetics

• Bacteria and viruses are microorganisms that have genetic material in the form of DNA or RNA
• Bacterial genomes are typically circular, double-stranded DNA molecules located in the cytoplasm (nucleoid)
• Viral genomes can be DNA or RNA, single-stranded or double-stranded, and linear or circular depending on the virus type
• Bacteria reproduce asexually through binary fission, resulting in clonal populations with identical genetic material
• Viruses are obligate intracellular parasites that require host cells for replication and propagation
• Genetic variation in bacteria and viruses arises through mutations, recombination, and horizontal gene transfer
• Understanding bacterial and viral genetics is crucial for developing strategies to combat infectious diseases and harness their potential in biotechnology

DNA Structure and Replication in Microorganisms

• Bacterial DNA is typically a circular, double-stranded molecule with a single origin of replication (oriC)
• DNA replication in bacteria is bidirectional, starting at the oriC and proceeding in both directions until the replication forks meet
• The bacterial DNA replication process involves DNA polymerase III, which synthesizes the leading and lagging strands
  • The leading strand is synthesized continuously in the 5' to 3' direction
  • The lagging strand is synthesized discontinuously as Okazaki fragments, which are later joined by DNA ligase
• DNA gyrase and topoisomerases play essential roles in relieving the supercoiling tension generated during DNA replication
• Some viruses (e.g., double-stranded DNA viruses) replicate their genomes using host cell machinery
• Other viruses (e.g., retroviruses) employ unique replication strategies, such as reverse transcription of RNA into DNA
• Rolling circle replication is a mechanism used by some viruses and plasmids to rapidly amplify their genetic material

Gene Expression and Regulation in Bacteria

• Bacterial genes are organized into operons, which are clusters of genes that are co-transcribed into a single mRNA molecule
• The lac operon in Escherichia coli is a well-studied example of gene regulation in bacteria
  • It consists of three structural genes (lacZ, lacY, and lacA) and is regulated by the lac repressor (lacI)
  • In the absence of lactose, the lac repressor binds to the operator region, preventing transcription of the lac operon
  • When lactose is present, it binds to the lac repressor, causing a conformational change that allows transcription to proceed
• Transcription in bacteria is initiated by RNA polymerase at promoter regions upstream of the genes
• Sigma factors are proteins that associate with RNA polymerase and help recognize specific promoter sequences
• Attenuation is a mechanism of transcriptional regulation that involves the formation of alternative RNA secondary structures, which can terminate transcription prematurely
• Riboswitches are regulatory RNA elements that bind specific metabolites and alter gene expression by affecting transcription or translation
• Small regulatory RNAs (sRNAs) in bacteria can modulate gene expression by base-pairing with target mRNAs, affecting their stability or translation efficiency

Bacterial Genetic Recombination

• Bacterial genetic recombination is the exchange of genetic material between two DNA molecules, leading to the generation of new combinations of alleles
• Transformation is the uptake of exogenous DNA from the environment by competent bacterial cells
  • Some bacteria (e.g., Streptococcus pneumoniae) are naturally competent, while others require specific conditions to become competent
• Conjugation is the transfer of genetic material between bacterial cells through direct cell-to-cell contact
  • Conjugative plasmids (e.g., F plasmid) encode the necessary genes for the formation of a conjugation pilus and DNA transfer
  • The transferred DNA is typically a single-stranded copy of the plasmid, which is then replicated in the recipient cell
• Transduction is the transfer of bacterial DNA from one cell to another via bacteriophages (bacterial viruses)
  • Generalized transduction occurs when any bacterial DNA is packaged into the phage capsid and transferred to another cell
  • Specialized transduction involves the transfer of specific bacterial genes adjacent to the prophage integration site
• Site-specific recombination systems, such as the lambda phage integration/excision system, allow for precise integration or removal of genetic elements at specific sites in the bacterial genome
• Homologous recombination in bacteria is mediated by the RecA protein and involves the exchange of genetic material between two similar DNA sequences

Viral Genome Organization and Replication

• Viral genomes can be composed of DNA or RNA, which can be single-stranded (ss) or double-stranded (ds)
  • Examples include ssDNA (e.g., parvovirus), dsDNA (e.g., adenovirus), ssRNA (e.g., influenza virus), and dsRNA (e.g., rotavirus)
• Viral genomes can be linear or circular, and some viruses have segmented genomes (e.g., influenza virus)
• The size of viral genomes varies widely, ranging from a few kilobases to over a megabase
• Viruses with DNA genomes typically replicate in the host cell nucleus using host cell machinery
  • Some DNA viruses (e.g., herpesviruses) encode their own DNA polymerases and other replication enzymes
• RNA viruses replicate in the host cell cytoplasm using virus-encoded RNA-dependent RNA polymerases (RdRps)
  • Positive-sense RNA viruses (e.g., poliovirus) can directly serve as mRNA for translation and as a template for genome replication
  • Negative-sense RNA viruses (e.g., influenza virus) must first have their genome transcribed into positive-sense RNA by the viral RdRp
• Retroviruses (e.g., HIV) have an RNA genome that is reverse transcribed into DNA by the viral reverse transcriptase enzyme
  • The resulting DNA provirus integrates into the host cell genome and is transcribed by host cell machinery
• Viral genome replication strategies often involve the production of multiple copies of the genome or subgenomic RNAs to facilitate efficient viral protein synthesis and virion assembly

Bacteriophages: Bacterial Viruses

• Bacteriophages, or phages, are viruses that specifically infect and replicate within bacterial cells
• Phages play important roles in bacterial evolution, ecology, and the transfer of genetic material between bacterial populations
• Lytic phages (e.g., T4 phage) replicate and lyse the host cell, releasing new phage particles
  • Lytic phages have a virulent life cycle and cause the rapid death of the infected bacterial cell
• Lysogenic phages (e.g., lambda phage) can integrate their genome into the host bacterial chromosome, forming a prophage
  • The prophage is replicated along with the bacterial genome and passed on to daughter cells during cell division
  • Environmental stressors or other signals can trigger the excision of the prophage and initiate the lytic cycle
• Phage therapy is the use of bacteriophages to treat bacterial infections as an alternative or complement to antibiotics
• Phage display is a technique that uses bacteriophages to express and screen for peptides or proteins with desired properties (e.g., antibody fragments)

Genetic Tools and Techniques in Microbiology

• Plasmids are extrachromosomal, self-replicating DNA molecules that are widely used as vectors for cloning and expressing genes in bacteria
  • Examples include pBR322 and pUC19, which contain antibiotic resistance genes and multiple cloning sites
• Restriction enzymes are bacterial endonucleases that recognize and cleave specific DNA sequences, allowing for the precise manipulation of DNA fragments
• DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between the 3' hydroxyl and 5' phosphate groups of adjacent DNA nucleotides
• Polymerase chain reaction (PCR) is a technique used to amplify specific DNA sequences using primers, dNTPs, and a thermostable DNA polymerase (e.g., Taq polymerase)
• DNA sequencing technologies (e.g., Sanger sequencing, next-generation sequencing) enable the determination of the precise nucleotide sequence of DNA fragments or entire genomes
• CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that have been harnessed for genome editing and regulation in various organisms
• Reporter genes (e.g., green fluorescent protein, luciferase) are used to study gene expression and regulation in bacteria and other microorganisms
• Transposons are mobile genetic elements that can move from one location to another within a genome, and they are used for mutagenesis and genetic screening in bacteria

Applications and Implications in Medicine and Biotechnology

• Understanding bacterial genetics is crucial for developing new antibiotics and combating the rise of antibiotic resistance
  • Identifying essential bacterial genes and pathways can lead to the discovery of novel drug targets
• Vaccines against bacterial pathogens can be developed using attenuated strains, purified antigens, or recombinant DNA technology
• Viral vectors, such as adenoviruses and lentiviruses, are used in gene therapy to deliver therapeutic genes to target cells
• Recombinant protein production in bacteria (e.g., Escherichia coli) is widely used to manufacture biopharmaceuticals, enzymes, and other valuable proteins
• Genetically engineered bacteria can be used for bioremediation, the process of using microorganisms to degrade or detoxify environmental pollutants
• Bacteriophages have potential applications in food safety, such as the control of foodborne pathogens in food production and processing
• CRISPR-Cas systems derived from bacteria are being explored for the development of novel antimicrobials and the manipulation of microbial communities
• Synthetic biology approaches using bacterial genetics enable the design and construction of novel biological systems with desired functions (e.g., biosensors, biofuels)