MB447 Microbial Genetics Chapter 5 Finals Coverage 2024-2025 PDF

MB447 - MICROBIAL GENETICS CHAPTER 5 | FINALS COVERAGE 3rd Year, 1st Semester | A.Y. 2024 - 2025 | BS Microbiology | BASILLA, ADB In bacteria, genetic recombination occurs after CHAPTER 5.1...

MB447 - MICROBIAL GENETICS CHAPTER 5 | FINALS COVERAGE 3rd Year, 1st Semester | A.Y. 2024 - 2025 | BS Microbiology | BASILLA, ADB In bacteria, genetic recombination occurs after CHAPTER 5.1 ❖ transformation, transduction, or conjugation. GENE TRANSFER IN BACTERIA ❖ To detect exchange of DNA, recombinant cells Microbes use mechanisms other than mutation to must phenotypically differ from both parents create genetic variability Mutations are subject to selective pressure SITE-SPECIFIC RECOMBINATION Each mutant form that survives becomes an ★ Important in insertion of viral genome into host allele, an alternate form of a gene. chromosomes ★ There is only a small region of homology Recombination is the process in which one or more between inserted genetic material and host nucleic acids are rearranged or combined to produce a chromosome new nucleotide sequence (recombinants) ★ Recombination occurs at specific target sites in 1. Genetic Recombination DNA molecules 2. Transformation ★ Process mediated by recombinase enzymes 3. Transduction 4. Conjugation 5. Hfr Strains and Chromosome Mobilization TRANSPOSABLE ELEMENTS ★ Segments of DNA that move about the genome Recombination at the MOLECULAR LEVEL in a process called transposition Three types ➔ Can be integrated into different sites in 1. Homologous recombination the chromosome 2. Site-specific Recombination Mobile DNA: Transposable Elements 3. Transposition Discrete segments of DNA that move as a unit from one location to another within other DNA molecules are transposable elements Two main types of transposable elements in Bacteria are insertion sequences and transposons ○ Both carry genes encoding transposase ○ Both have short inverted repeats at ends required for transposition iNSERTION SEQUENCES & TRANSPOSONS ★ Insertion sequences (IS) ➔ Simplest transposable element HOMOLOGOUS RECOMBINATION ➔ ~1000 nucleotides long ★ Most common ➔ Inverted repeats are 10 to 50 base pairs ★ Usually involves a reciprocal exchange ➔ Only gene is for the transposase between a pair of DNA molecules with similar ➔ Found in chromosomes and plasmids nucleotide sequences of Bacteria and Archaea and in some ★ RecA proteins carry out the process bacteriophages ★ Double-strand break occurs between ★ Transposons molecules, allowing exchange to be mediated ➔ Larger than IS but have same essential Effect of homologous recombination on genotype components ❖ To generate new genotypes, the homologous ➔ Transposase moves any DNA between sequences must be related by distinct (e.g., inverted repeats diploid eukaryotic cell). BASILLA, ADB | 1  ➔ Genes inside vary widely (e.g., antibiotic ★ Copy is inserted in target DNA resistance in Tn5 and Tn10) ➔ Complex behavior may be observed UTILITY OF TRANSPOSON MUTAGENESIS ★ Powerful tool for creating mutants MECHANISMS OF TRANSPOSITION ○ Transposons with antibiotic resistance ★ Transposase recognizes, cuts, ligates DNA are used ★ When inserted, a short sequence in target at ○ Transposon is introduced on a plasmid integration site is duplicated that cannot be replicated in the cell ★ Conservative: transposon is excised from one ★ Cells capable of growing on selective medium location and reinserted at a second location likely acquired transposon (Tn5) ★ Most insertions will be in genes that encode ○ Copy number is constant = one proteins ★ Replicative: a new copy of transposon is ★ Next step: screen for loss of function to produced and inserted at a second location determine insertion site ○ Number of transposons present doubles CHAPTER 5.2 GENE TRANSFER IN BACTERIA I. Transformation is the genetic transfer process by which free DNA is incorporated into a recipient cell and brings about genetic change Competence in transformation ❖ Competent: a cell that can take up DNA and be transformed; genetically determined ❖ In naturally transformable bacteria, competence is regulated, and competence-specific proteins uptake and process DNA CONSERVATIVE TRANSPOSITION Quorum sensing in Bacillus subtilis ★ Also called cut-and-paste transposition Quorum sensing, chitin sensing, ★ Transposase catalyzed excision catabolite repression in Vibrio cholerae ★ Cleavage of new target site and ligation into High-efficiency natural transformation site is rare in Bacteria ❖ In other strains, specific procedures are necessary to make cells competent (e.g., treatment of E.coli with high Ca⁺² and chilling) ❖ Electricity can be used to force cells to take electroporation II. Transduction transfer of DNA from one cell to another by bacteriophage Two modes 1. Generalized transduction: DNA from any portion REPLICATIVE TRANSPOSITION of the host genome is packaged inside the ★ Two genes coding for enzymes virion ○ Transposase ➔ Donor genes cannot replicate ○ Resolvase independently ★ Original transposon remains at parental site in ➔ Will be lost without recombination DNA 2  2. Specialized transduction: DNA from a specific ★ Viral replication is under control of the bacterial region of the host chromosome is integrated host chromosome. directly into the virus genome ★ Upon induction, viral DNA sometimes exercises ➔ May be integrated during lysogeny, or incorrectly and takes adjacent host genes homologous recombination may occur along with it. ★ Occurs in many Bacteria and at least one ★ Limit to amount of host DNA that can replace Archaea phage DNA, but helper phage can assist ➔ Multiple-antibiotic-resistance genes in Salmonella, Shiga-like toxins in E.coli, GENE TRANSFER AGENTS (GTAs) virulence factors in Vibrio cholerae, ★ Defective bacteriophages that transfer DNA photosynthetic genes in cyanobacteria between prokaryotic cells ○ Result from prokaryotes hijacking UPTAKE AND INTEGRATION OF DNA IN defective viruses specifically for DNA TRANSFORMATION exchange ○ Resemble tiny tailed bacteriophages ★ Natural transformation starts with reversible ○ Contain random small pieces of host DNA binding that becomes irreversible DNA ★ Competent cells bind up to 1000x more DNA ○ Do not contain genes encoding own than non competent cells production Linear DNA first bound by DNA-binding ○ Do not produce viral plaques protein similar to pilus ○ Isolated from many Bacteria and some Either ds fragment taken in or nuclease methanogenic Archaea degrades one strand and other is taken ○ May have evolved as mechanism for in protected gene dispersion RecA integrates DNA ★ In contrast, plasmid DNA must remain ds and circular for replication Bacterial Transformation is the uptake of free DNA from the Environment GENERALIZED TRANSDUCTION ★ Virtually any gene can be transferred to the recipient (transductant). ★ Sometimes host DNA accidentally packaged into phage, forming transducing particle that is METHODS FOR DETECTING GENE TRANSFER AGENTS defective and cannot lead to viral lytic infection ★ Upon lysis, transducing particles and normal virions released ★ Recipients of transducing particles may recombine DNA. ★ Low efficiency: one in 10⁶-10⁸ cells transduced LYSOGENY AND SPECIALIZED TRANSDUCTION ★ Extremely efficient transfer ★ Selective and transfers only small part of bacterial chromosome ★ Phage genome is integrated at specific sites (e.g., Lambda in E. coli: next to galactose utilization genes). 3  CONJUGATION High rates of genetic recombination between ★ Conjugation (mating): horizontal gene transfer genes on the donor (Hfr) and recipient (F*) that requires cell-to-cell communication chromosomes ➔ Plasmid-encoded Integration provides a mechanism for ➔ Occurs between closely related or mobilizing a genome. distantly related cells ➔ Donor cell: contains conjugative Presence of the F plasmid results in three distinct plasmid changes ➔ Recipient cell: does not contain plasmid Ability to synthesize F pilus ➔ Other genetic elements (e.g., other Mobilization of DNA for transfer to another cell plasmids or chromosome) may be Alteration of surface receptors so that cell can mobilized (transferred during no longer be a conjugation recipient conjugation) ★ F (“fertility”) plasmid ➔ ~99 kbp circular DNA molecule ➔ Contains genes that regulate DNA replication ➔ Also contains transposable elements that allow the plasmid to integrate into the host chromosome ➔ Contains tra genes that encode transfer functions (synthesis of sex pilus and type IV secretion system for DNA transfer) Integration of F plasmid and chromosome ➔ Pili allow specific pairing through mobilization receptor contact, pulling cells together, ❖ Insertion sequences (IS; mobile genetic and DNA transferred through junction. elements) are present in both the F plasmid and E. coli chromosome providing regions of sequence homology. ❖ Homologous recombination results in F plasmid integration into chromosomes. ❖ After integration, tra functions normally and strain synthesizes pili. When the recipient encountered, part of plasmid and chromosomal genes transferred. ➔ many Hfr strains possible GENE TRANSFER IN ARCHAEA Horizontal gene transfer in Archaea The Formation of Hfr Strains and Chromosome ❖ Most Archaea contain a single circular Mobilization chromosome. F plasmid is an episome (can integrate into the ❖ Genetic manipulation of Archaea lags behind host chromosome). Bacteria. Cells possessing a nonintegrated F plasmid are Most well-characterized Archaea are called F+. extremophiles growing under high salt Cells possessing an integrated F plasmid are or high temperature. called Hfr (high frequency of recombination). ❖ Most known antibiotics do not affect Archaea, so choice of selectable markers is limited. 4  What do we know so far? ★ No single species is a model organism for Archaea. ★ More genetic work has been done on some extreme halophiles (e.g., Halobacterium and Haloferax). ★ Various transfer mechanisms and several plasmids found ★ Transposon mutagenesis well-developed in some methanogens ★ Natural competence for transformation in some methanogens and hyperthermophiles ★ Plasmid exchange in Thermococcus through envelope budding ★ Transduction and gene transfer agents very rare Conjugation in Archaea ★ Sulfolobus conjugates ○ Pili are not formed. ○ Unidirectional DNA transfer ○ Genes are not similar to those in gram-negative Bacteria. ○ mechanism likely different than Bacteria ★ Most Sulfolobus can exchange DNA by cell aggregation occurring when pili synthesis is triggered by UV radiation. ★ Specialized structures, such as nanotubes in Thermococcus, cytoplasmic bridges 5