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FE A. BARTOLOME M.D., DPASMAP Department of Microbiology Our Lad of Fatima University GENETIC MATERIAL Bacterial Genome • Total collection of genes carried by a bacterium both on its chromosome and on its extrachromosomal genetic element GENETIC MATERIAL DNA 1. Most are double stranded • Template strand • Coding strand 2. Complementary bases paired by H-bonding (A-T; G-C) 3. Length – kbp 4. Distance between base pairs = 0.34 nm (3.4 x 10-7 mm) GENETIC MATERIAL RNA 1. Single-stranded 2. Complementary bases – A-U; G-C 3. Types: a. mRNA – for translation of DNA gene sequences b. rRNA – protein synthesis c. tRNA – protein synthesis GENETIC MATERIAL RNA 1. Single-stranded 2. Complementary bases – A-U; G-C 3. Types: a. mRNA – for translation of DNA gene sequences b. rRNA – protein synthesis c. tRNA – protein synthesis PROKARYOTIC GENOME 1. A replicon – DNA circles (chromosome + plasmid) which contain genetic information necessary for their own replication 2. Single circular double stranded DNA molecule 3. Usually haploid 4. No histones – structure maintained by polyamines (spermine & spermidine) BACTERIAL DNA REPLICATION 1. Initiated at OriC 2. Semi-conservative 3. New DNA synthesis occurs at growing forks proceed bidirectionally • Leading strand – continuous (5’ 3’) • Lagging strand – Okazaki’s fragments BACTERIAL DNA REPLICATION 4. Enzymes: a. Helicase – unwinds the DNA at the origin b. Primase – synthesize primers to start the process c. DNA polymerase – copy the DNA; with proofreading functions d. Topoisomerase (gyrase) – relieves torsional strain e. DNA ligase REGULATION OF GENE EXPRESSION Mechanisms to adapt to changes in concentration of nutrients in the environment 1. Organization of biochemical pathways into operons 2. Gene transcription regulated by repressor proteins bind to operators 3. Rate of protein synthesis by the ribosome can regulate transcription in prokaryotes 4. Absence of nuclear membrane allow the ribosome to bind to the mRNA as it is being transcribed from the DNA MUTATION Types of Mutation 1. Spontaneous – occurs without apparent cause (e.g. polymerase mistakes) 2. Induced – caused by mutagen; increased frequency of mutation a. Physical agents (1) heat deamination of nucleotides (2) UVL pyrimidine dimer formation form cyclobutane ring between 2 pyrimidines (3) ionizing radiation form free radicals cause single strand breaks MUTATION Types of Mutation 1. Induced – caused by mutagen; increased frequency of mutation b. Chemicals (1) nucleotide base analogues mispairing (e.g. 5-bromouracil and thymine) (2) polycyclic flat molecules (e.g. EtBr) intercalates DNA inc. spacing of successive base pairs (3) DNA reactive chemicals (e.g. nitrous acid & alkylating agents) modify normal base into a chemically different structure MUTATION Types of Alterations in the DNA (as reflected in mRNA) 1. Addition or insertion 2. Deletion 3. Substitution a. Transition – purine to purine; pyrimidine to pyrimidine b. Transversion – purine to pyrimidine and vice versa MUTATION Effects of Mutation 1. Single codon change a. Silent mutation – change at the DNA level that does not result in any change of a.a. in the encoded protein • Occurs because >1 codon may encode an a.a. (e.g. val ala) b. Missense mutation – result in substitution of 1 a.a. for another c. Nonsense mutation – codon encoding an a.a. is changed to a stop codon (e.g. TAG) MUTATION Effects of Mutation 2. Numerous bases involved a. Frameshift mutation (+) change in reading frame premature truncation of protein b. Null mutation – with extensive insertion, deletion or gross rearrangement of chromosome structure completely destroy gene function MUTATION Repair Mechanisms 1. Direct DNA repair • Enzymatic removal of damage, such as pyrimidine dimers and alkylated bases 2. Excision repair • Excision of a DNA segment containing the damage, followed by synthesis of a new DNA strand • Damage recognized by endonuclease cut phosphodiester backbone on both sides of damage • Excision of DNA with damaged backbone carried out by exonuclease MUTATION Repair Mechanisms 3. Recombinational or post-replication repair • Retrieval of missing information by genetic recombination when both DNA strands are damaged 4. SOS response • Induction of many genes after DNA damage or interruption of DNA replication 5. Error-prone repair • Last resort of a bacterial cell before it dies • Fill in gaps when a DNA template is not available for directing an accurate repair GENE TRANSFER TRANSFORMATION • First mechanism of genetic transfer to be discovered • Involves the release of DNA into the environment by the lysis of some cells, followed by the direct uptake of the DNA by the recipient • Occurs among related species • Requires competence of recipient cells • Gram (+) and gram (-) bacteria H. influenzae, S. pneumoniae, Bacillus sp., Neisseria sp. GENE TRANSFER TRANSFORMATION + Donor cell Competent recipient cell The new gene is expressed + Free DNA Look for area of homology ds DNA adsorbs to the surface Entry of ss-DNA GENE TRANSFER CONJUGATION • DNA passed directly by cell-to-cell contact during the mating of the bacteria sex-like exchange • Results in a one way transfer of DNA from a donor (male) cell to a recipient (female) cell via the sex pilus • Depends on the presence or absence of a conjugative plasmid (e.g. F plasmid of E. coli) • F plasmid transfers itself convert recipient into F+ male • DNA transferred is single stranded GENE TRANSFER CONJUGATION Three forms of F: 1. F+ - F plasmid transferred to recipient cell recipient cell becomes F+ male 2. F’ – if fragment of chromosomal DNA is incorporated into the plasmid F’ male 3. Hfr – if F plasmid sequence is integrated into the bacterial chromosome cell called Hfr cell (high frequency of recombination cell) GENE TRANSFER GENE TRANSFER CONJUGATION F plasmid • E. coli, Bacteroides sp., Streptococci, Streptomyces, Clostridia R plasmid • does not use pili; transfer antibiotic resistance • Streptococci, Streptomyces, Clostridia GENE TRANSFER TRANSDUCTION • Mediated by bacteriophages picks up fragments of DNA & package them into bacteriophage particles DNA delivered to infected cells incorporated into bacterial genome • Types: 1. Specialized – phage transfers particular genes, usually adjacent to their integration sites 2. Generalized – selection of sequences is random; contain primarily bacterial DNA & little or no phage DNA GENE TRANSFER TRANSDUCTION