DLS-HSI medicine 2016

Subject: MICROBIOLOGY and PARASITOLOGY Topic: BACTERIAL GENETICS Lecturer: DR. ELEANOR PADLA Date of Lecture: June 13, 2013 READ AT YOUR OWN RISK! GENETICS study of heredity and variation defines inheritance and variability Gene units of heredity Genome total complement of genes Chromosome naked (prokaryotes have no nuclear membrane) occupies 10% of cell volume about 1500X the cells length single, circular, tightly wound DNA (bacterial genes are haploid, they only have single copies of the gene) exists in nucleoid, NOT nucleus replicate by binary fission (asexual mode of reproduction) termed as replicon, it has information for its own replication FLOW OF GENETIC INFORMATION Central Dogma: Replication Transcription Translation (DNA) (RNA) (proteins) Replication copying of double stranded DNA Transcription DNA to mRNA (via RNA polymerase) Translation mRNA to protein (carried out by ribosomes) REPLICATION DNA Replication semi-conservative (In bacteria, DNA Replication is semi conservative, which means the parent strand separates in two strands, each individual replicates, retaining (conserving) one parental strand and forming a newly synthesized strand.) STAGES OF DNA REPLICATION Initiation initiated at ori C (origin of chromosomal replication) unwinding of double helix forming a replication fork helicase which opens up the helix single-stranded binding proteins (SSBP) stabilize the single-stranded arms of the replication fork; prevents reannealing topoisomerase II nicks DNA to relieve tension from unwinding primer synthesis RNA primase synthesizes RNA primer; the RNA primer has to be synthesized at the 5 -end of the replicating arm of the DNA, it is where DNA polymerase will attach new polynucleotides. No DNA polymerase can initiate polymerization, it can only catalyze the addition of new polynucleotide. If there is no RNA primer, there is no polymerization. Elongation o DNA synthesis/polynucleotide chain elongation o proceeds bi-directionally to a fixed terminus (ter C) DNA polymerase III elongates primer, produces Okazaki fragments Leading strand (5 to 3) elongates continuously with the addition of polynucleotides Lagging strand (3 to 5) elongates in discontinuous manner via the addition of polynucleotides; elongation is discontinuous because DNA polymerase III is not able to fully synthesize in 3 to 5 direction Okazaki fragments short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication Termination o primer removal DNA polymerase I excises RNA primer, fills gap o gap -filling Ligase links Okazaki fragments DNA POLYMERASES 5 to 3 polymerization I II III Yes Yes Yes 3 to 5 exonuclease (proofreading) Yes (more active) Yes Yes 5 to 3 exonuclease (primer removal) Yes No Yes Proofreading ensures no error in polymerization. There is only one in a billion chances that there will be an error. DNA polymerase I is the leading in primer removal. DNA polymerase III is the leading in polymerization.

VARIATION Phenotypic Variation metabolic changes in response to changing environment nonheritable usually temporary and reversible Genotypic Variation changes in genetic constitution heritable usually permanent Mutation Gene transfers o Transformation o Transduction o Conjugation MUTATION heritable change in DNA sequence Mutant organism carrying mutated gene Wild-type parent organism with normal gene Classification of mutations: o Spontaneous vs Induced o Selectable vs Non-Selectable o Base Substitutions/ Replacements o Insertions and Deletions o Those caused by Transposable Genetic Elements (TGEs) Spontaneous Mutations occur without any apparent cause rare events (1 in 1 billion); bacterial replication is high fidelity may be caused by replication errors Induced Mutations more common those with identifiable cause (e.g. mutagens) physical agents (e.g., UV light, X-rays) chemical agents (e.g., nitrous acids) Selectable Mutations confers selective advantage to the mutant growth over the parent gene Example: mutation to drug resistance Non-Selectable Mutations mutant has no selective advantage over wild-type Example: loss of color in a pigmented bacterium Base Substitution/ Replacement synonymous to point mutation involves substitution of one base for another Kinds: o Transition purine to purine or pyrimidine to pyrimidine substitution o Transversion purine to pyrimidine or pyrimidine to purine substitution Review of Biochem! Purines: Guanine and Adenine Pyrimidines: Cytosine, Uracil, Thymine Effects: o Silent mutation resulting amino acid same with the original; normal o Missense mutation resulting amino acid differs from the original; faulty o Non-sense mutation resulting substitution is a stop codon; no protein is formed (Stop codons are UAG, UGA, UAA) Insertions and Deletions addition or removal of one or more bases Microinsertion/microdeletion: one to two bases Macroinsertion/macrodeletion: three or more bases Frameshift Usually caused by microinsertion/deletion Occurs when insertion/deletion results in bases indivisible by 3 Results in nonfunctional protein

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Reversion & Suppressor Mutation Reversion (back mutation) conversion of mutated gene back to its wild-type allele the second mutation occurred at the site of original mutation rarely happens; needs a very specific and almost improbable event Suppression (compensating mutation) conversion of mutant cell into one that is phenotypically identical to the wild-type second mutation occurred at a locus different from the original; original mutation still retained Two wrongs make a right applies here TRANSPOSABLE GENETIC ELEMENTS jumping genes can transfer from one location to another, or between chromosome and plasmids not independent replicons; they need to be associated with a chromosome or plasmid which is self-replicating have inverted terminal repeats (palindromes) Types : o Insertion sequences o Transposons o Phage-associated transposons (Mu) 1. Insertion Sequences (IS) simplest of the transposable genetic elements about 1000 base pairs carry no genes except those involved in transposition transposase enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism Transposons composite type 10-fold longer than IS flanked by IS elements o IS for transposability o central genes code for antibiotic resistance Phage-Associated Transposons (e.g. bacteriophage Mu) bacteriophage is a type of virus that infects bacteria exist in integrated state; part of viral genome of bacteriophage have gene required to make infectious phage particles are transposable phages considered a mutator phage; it is originally called a virus that causes mutation requires sequence homology (exogenote and endogenote should be related for recombination to occur) Example: incorporation of homologous DNA via transformation, transduction, conjugation Site-Specific Recombination (non-homologous) independent of (recA) genes requires little, if any, sequence homology recombination at highly preferred sites Example: integration of phage at att site Illegitimate Recombination (non-homologous) independent of (recA) genes requires little, if any, sequence homology occur at random sites Example: insertion of IS, transposons

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GENE TRANSFERS transfer of DNA from donor to recipient governed by chromosomal (transformation), viral (transduction), and plasmid (conjugation) genes provide source of genetic diversity Mechanisms: o transformation o transduction o conjugation TRANSFORMATION Governed by chromosomal genes uptake of naked DNA by a recipient cell Donor DNA (exogenote) o derived from closely related strain o size requirement is species-specific o ds-DNA binds more effectively than ss-DNS Recipient Cell o must be competent can take up foreign DNA some are naturally competent; others are to be induced a transient state; competence only occurs in the end of log phase in microbial growth o varies in Gram (+) and Gram (-) bacteria competence factors proteins that facilitate entry of DNA recognition sequences TRANSDUCTION governed by viral genes bacteriophage-mediated transfer of DNA Types: o generalized transduction o specialized transduction GENERALIZED TRANSDUCTION mediated by virulent phages transducing phages carry random DNA fragments contains phage particles and bacterial chromosome from the recipient cell SPECIALIZED TRANSDUCTION mediated by temperate phages transducing phages carry selected segments of DNA contains purely phage particles occurs as a result of lysogeny instead of only excising itself, like the phage DNA, it carries a portion of the bacterial DNA

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TRANSPOSITION involves: o Transposase catalyzes insertion into new site o Resolvase involved in recombinational events accompanied by duplication of target site creates a single-stranded break Kinds: o conservative transposition transposable element jumps into another site (cut-paste) o replicative transposition transposable element is duplicate; one remains in the original site, the other is transferred to another site (copy-paste) Fates of DNA fragment after transfer, when it enters the cell: 1. degradation by endonuclease since the DNA is considered foreign 2. stabilization since some of the DNA fragments may get into the cell and assume a circular form (i.e., plasmid) 3. integration by recombination GENETIC RECOMBINATION genetic elements from two separate sources are brought together in one unit Endogenous: recipient cells genome Exogenous: donor cells genome Classification: o Generalized recombination (homologous) o Site-Specific Recombination (non-homologous) o Illegitimate recombination (non-homologous) 1. Generalized Recombination (homologous) involves recombination (recA) genes [wraps itself around two recombining elements] displacement of the endogenous and insertion of exogenote

BACTERIOPHAGE LIFE CYCLES a. Lytic Cycle possessed by virulent phages leads to lysis of infected cell and release of new phages b. Lysogenic Cycle possessed by temperate phages leads to integration of phage DNA into bacterial chromosome Lysogeny process by which the phage DNA is integrated into the bacterial chromosome Prophage integrated phage DNA; latent dormant, nonmultiplying Lysogen bacteria carrying the latent prophage

Lysogenic Conversion change in property of recipient cell due to dictates of the gene carried by the phage changing the phenotype because of lysogeny

MICROBIOLOGY and PARASITOLOGY: BACTERIAL GENETICS

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CONJUGATION transfer of DNA between two mating pairs requires cell-to-cell contact o involves F (fertility factor)-plasmid encoded sex pilus which serves as the conjugation tube o adhesins involves two mating types: Donor (male) and Recipient (female) o Donor (male): F+ fertility (F) plasmid-carrying cell does not give an entire chromosome, it transfers the F o Recipient (female): F lacks the F plasmid has receptor site (OmpA) for adherence of the conjugation tube (sex pilus) F PLASMID small, circular, double-stranded DNA replicates independently of the chromosome conjugative o tra (transfer) gene - for sex pilus production F plasmid occurs in an autonomous or integrated states o F+ o Hfr o F F+ Hfr F o Non-Conjugative: smaller plasmids (6-18 kb) can be transmitted by transduction or transformation without tra gene conjugal transfer can be mobilized by a coexisting conjugative plasmid mob genes mobilize their transfer

Evidence of Plasmid- Mediated Phenotypes Plasmid instability and curing Plasmid loss of protoplasting Plasmid transfer Plasmid isolation R-PLASMID MEDIATED ANTIBIOTIC RESISTANCE Genetic Determinants of Resistance chromosomal genes plasmid genes Drug Resistance Non-genetic o bacterial persistence o loss of target structure Genetic o chromosomal (mutations, replication errors) o extra-chromosomal (R-plasmids) R PLASMID-DETERMINED RESISTANCE IN ENTERIC BACTERIA Sulfonamycin (Sulfonamide) Streptomycin Tetracycline Choramphenicol
Exposure to antibiotic led to the rise of the R plasmid/drug resistance Some resistance gene predate antibiotic therapy; meaning, before the antibiotics were developed, some bacteria already had the resistance for the substances

donors harboring an autonomous F plasmid found in the cytoplasm donors with an integrated F plasmid F plasmid replicated with the chromosome can transfer large portions of donor genes at high frequencies donors with an autonomous F plasmid has segments of chromosomal DNA generated from imprecise excision of Hfr can transfer restricted portions of donor genes

F PLASMID TRANSFER F+ x F converts F- to F+; resulting into two F+ after conjugation low transfer of donor chromosomal genes Hfr x F recipient remains F high transfer of certain donor chromosomal genes

R PLASMIDS IN BACTERIA consist of RTF & r determinants o Resistance Transfer Factor (RTF) plasmid replication transfer by conjugation o r determinants where all r genes are clustered acquired mobile genetic elements R Plasmid Origin RTF + r derminants = r plasmid R Plasmid Evolution Addition of more mobile genetic elements No homology needed Resistance phenotypes Selected transposons Resistance Phenotypes of Selected Transposons R Plasmid Transfer Pathways MECHANISMS OF R PLASMID-MEDIATED RESISTANCE MECHANISMS EXAMPLES
Altering the target site of antibiotic Modifying the antibiotic so that it is no longer active Preventing the antibiotic from entering the cell Specifying an enzyme that provides a substitute for a hostspecified enzyme which is the target of the antibiotic erythromycin, lincomycin resistance chloramphenicol, penicillin, cephalosporin resistance Tetracycline, aminoglycoside resistance Sulfonamide, trimetoprim resistance

usually long but not all genes will be transferred, just fragments genes that are able to convert F- into F+ are not included in the transfer

F x F recipient becomes F high transfer of donor genes on F low transfer of other donor chromosomal genes merodiploid formation (partially diploid) PLASMIDS small, circular, d-s DNA capable of independent replication (replicon) have an extra-chromosomal existence; may also integrate not essential to a cells survival contribute to phenotype of a cell confers selective advantage Properties Determined by Plasmids : Replication-Maintenance properties o Host range: narrow / broad o Copy number: low / high o Incompatibility group: compatible / incompatible Complete incompatibility: genes cannot exist in the same cell Incompatible groups: DNA homology, pilus type, size Resistance properties o drug resistance o resistance to heavy metal cations/anions Metabolic properties o antibiotic and bacteriocin production Pathogenic properties o toxin production Conjugal Properties o Conjugative: often large plasmids (>35 kb) mediate their own transfer from cell to cell tra genes code for transfer machinery

MECHANISMS OF R PLASMID-MEDIATED RESISTANCE ANTIBIOTIC RESISTANCE MECHANISM

Erythromycin Lincomycin Chloramphenicol Penicillins, Cephalosporins Tetracyclines Aminoglycosides Sulfonamides Trimetoprim Methylation of 23S rRNAs of ribosomes Detoxification by chloramphenicol transacetylase b-lactamase Prevents intracellular accumulation Uptake inhibited Sulfonamide-resistant dihydropteroate synthetase Trimetoprim-resistant dihydrofolate reductase

ANTIBIOTIC RESISTANCE CAN BE CONTAINED But only by the most careful monitoring of strains And prudent use of antibiotics
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