BACTERIAL GENETICS 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 Most are double stranded Template strand Coding strand Complementary bases paired by H-bonding (A-T; G-C) Length – kbp Distance between base pairs = 0.34 nm (3.4 x 10-7 mm)

GENETIC MATERIAL RNA Single-stranded Complementary bases – A-U; G-C Types: a. mRNA – for translation of DNA gene sequences b. rRNA – protein synthesis c. tRNA – protein synthesis

GENETIC MATERIAL RNA Single-stranded Complementary bases – A-U; G-C Types: a. mRNA – for translation of DNA gene sequences b. rRNA – protein synthesis c. tRNA – protein synthesis

PROKARYOTIC GENOME A replicon – DNA circles (chromosome + plasmid) which contain genetic information necessary for their own replication Single circular double stranded DNA molecule Usually haploid No histones – structure maintained by polyamines (spermine & spermidine)

BACTERIAL DNA REPLICATION Initiated at OriC Semi-conservative New DNA synthesis occurs at growing forks  proceed bidirectionally Leading strand – continuous (5’  3’) Lagging strand – Okazaki’s fragments

BACTERIAL DNA REPLICATION 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 Organization of biochemical pathways into operons Gene transcription regulated by repressor proteins  bind to operators Rate of protein synthesis by the ribosome can regulate transcription in prokaryotes Absence of nuclear membrane allow the ribosome to bind to the mRNA as it is being transcribed from the DNA

MUTATION Types of Mutation Spontaneous – occurs without apparent cause (e.g. polymerase mistakes) 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 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) Addition or insertion Deletion Substitution a. Transition – purine to purine; pyrimidine to pyrimidine b. Transversion – purine to pyrimidine and vice versa

MUTATION Effects of Mutation 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 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 Direct DNA repair Enzymatic removal of damage, such as pyrimidine dimers and alkylated bases 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 Recombinational or post-replication repair Retrieval of missing information by genetic recombination when both DNA strands are damaged SOS response Induction of many genes after DNA damage or interruption of DNA replication 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 Free DNA ds DNA adsorbs to the surface Entry of ss-DNA Look for area of homology The new gene is expressed

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: F+ - F plasmid transferred to recipient cell  recipient cell becomes F+ male F’ – if fragment of chromosomal DNA is incorporated into the plasmid  F’ male 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: Specialized – phage transfers particular genes, usually adjacent to their integration sites Generalized – selection of sequences is random; contain primarily bacterial DNA & little or no phage DNA

GENE TRANSFER TRANSDUCTION

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