Lecture Notes: BACTERIAL GENETICS Dr. E.

Padla

BACTERIAL GENETICS
Coverage: DNA Replication Genetic Variation Mutation IS and transposons Genetic Transfer Processes Genetic Recombination Gene Transfers R Plasmid Mediated Resistance

GENETICS
study of heredity and variation Gene - units of heredity Genome - total complement of genes Chromosome naked occupies 10% of cell volume about 1500X the cells length single, circular, tightly wound DNA exists in nucleoid replicate by binary fission
Cell Division Cell Division & DNA Replication Flow of Genetic Information

REPLICATION
DNA Replication semi-conservative Stages: Initiation initiated at ori C (origin of chromosomal replication) unwinding of double helix forming a replication fork primer synthesis Elongation DNA synthesis/polynucleotide chain elongation proceeds bi-directionally to a fixed terminus (ter C) Termination primer removal gap -filling

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Lecture Notes: BACTERIAL GENETICS Dr. E. Padla

Replication Fork Primer Synthesis Bi-Directional Replication Replication (Contd) LEADING STRAND SYNTHESIS Replication (Contd) - LAGGING STRAND SYNTHESIS Replication (Contd) LEADING/ LAGGING STRAND SYNTHESIS

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Replication Enzymes / Proteins helicase opens up the helix s-s binding proteins - stabilize the s-s arms of the replication fork topoisomerase II - nicks DNA to relieve tension from unwinding primase synthesizes RNA primer DNA polymerases DNA polymerase III elongates primer, produces Okazaki fragments DNA polymerase I excises RNA primer, fills gap Ligase - links Okazaki fragments
Comparison Of The Functional Characteristics Of E. coli DNA Polymerases

VARIATION
Phenotypic Variation metabolic changes in response to changing environment Genotypic Variation changes in genetic constitution
Mutation Gene transfers Transformation Transduction Conjugation

MUTATION heritable change in DNA sequence

Mutant - organism carrying mutated gene Wild-type - parent organism with normal gene

Classification o Spontaneous vs Induced o Selectable vs Non-Selectable o Base Substitutions/ Replacements o Insertions and Deletions o Those caused by Transposable Genetic lements (TGEs)

Mutation (Contd) Spontaneous Mutations occur without any apparent cause rare events

Lecture Notes: BACTERIAL GENETICS Dr. E. Padla

may be caused by replication errors

Induced Mutations those with identifiable cause (e.g. mutagens) physical agents chemical agents Mutation (Contd) Selectable Mutations confers selective advantage to the mutant Ex - mutation to drug resistance Non-Selectable Mutations mutant has no selective advantage over wild-type Ex - loss of color in a pigmented bacterium
Direct Selection for Mutants

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Mutation (Contd) Base Substitution/ Replacement involves substitution of one base for another kinds: Transition Transversion effects: silent mutation missense mutation non-sense mutation
Base Substitutions (contd) KINDS OF BASE SUBSTITUTION Possible Effects of a Point Mutation

Mutation (Contd) Insertions and Deletions addition or removal of one or more bases
Shifts in Reading Frame

Mutation (Contd) Reversion & Suppressor Mutation Reversion (back mutation) conversion of mutated gene back to its wild-type allele the 2nd mutation occurred at the site of original mutation Suppression (compensating mutation) conversion of mutant cell into one that is phenotypically identical to the wild-type 2nd mutation occurred at a locus different from the original; original mutation still retained
Back Mutations & Suppressor Mutations (Contd)

Lecture Notes: BACTERIAL GENETICS Dr. E. Padla

Mutation (Contd)

TRANSPOSABLE GENETIC ELEMENTS

o o o can transfer from one location to another, or between chromosome and plasmids not independent replicons have inverted terminal repeats types : insertion sequences transposons phage-associated transposons (Mu) TGEs (Contd) TYPES 1. Insertion Sequences simplest of the TGEs about 1000 bp carry no genes except those involved in transposition transposase
TGEs (Contd) INSERTION SEQUENCE Insertion Sequences

TGEs (Contd) TYPES 2. Transposons 10-fold longer than IS flanked by IS elements o IS - for transposability o central genes - codes for antibiotic resistance
TGEs (Contd) TRANSPOSON Resistance Phenotypes of Selected Transposons

TGEs (Contd) TYPES 3. Phage-Associated Transposons (e.g. bacteriophage Mu) exist in integrated state have gene required to make infectious phage particles are transposable phages considered a mutator phage
TGEs (Contd) BACTERIOPHAGE MU

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TGEs Transposition involves: transposase - catalyzes insertion into new site resolvase - involved in recombinational events accompanied by duplication of target site kinds: conservative transposition

Lecture Notes: BACTERIAL GENETICS Dr. E. Padla

o replicative transposition
Transposition

Fates of DNA Fragment After Transfer

GENETIC RECOMBINATION
genetic elements from two separate sources are brought together in one unit Classification: o Generalized recombination (homologous) o Site-Specific Recombination (non-homologous) o Illegitimate recombination (non-homologous)

Genetic Recombination - CLASSIFICATION 1. Generalized Recombination involves recombination (recA) genes requires sequence homology Ex. incorporation of homologous DNA via: transformation, transduction, conjugation 2. Site-Specific Recombination independent of (recA) genes requires little, if any, sequence homology recombination at highly preferred sites Ex. integration of phage at att site 3. Illegitimate Recombination independent of (recA) genes requires little, if any, sequence homology occur at random sites Ex. insertion of IS, transposons

GENE TRANSFERS
transfer of DNA from donor to recipient governed by chromosomal, viral, plasmid genes provide source of genetic diversity mechanisms o transformation o transduction o conjugation Gene Transfers (contd) Mechanisms transformation transduction conjugation

Lecture Notes: BACTERIAL GENETICS Dr. E. Padla

Gene Transfers (contd)

TRANSFORMATION
uptake of naked DNA by a recipient cell Transformation (Contd) Donor DNA derived from closely related strain size requirement is species-specific ds-DNA binds more effectively than ss-DNS Recipient Cell must be competent some are naturally competent; others are to be induced a transient state varies in Gram (+) and Gram (-) bacteria competence factors recognition sequences
Transformation

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Gene Transfers (contd)

TRANSDUCTION
o o bacteriophage-mediated transfer of DNA types: generalized transduction specialized transduction Bacteriophage Life Cycles Lytic Cycle: possessed by virulent phages leads to lysis of infected cell and release of new phages Lysogenic Cycle: possessed by temperate phages leads to integration of phage DNA into bacterial chromosome lysogeny prophage lysogen
Lytic Cycle Lysogenic Cycle Lysogenic Conversion

Transduction TYPES

Lecture Notes: BACTERIAL GENETICS Dr. E. Padla

Generalized Transduction mediated by virulent phages transducing phages carry random DNA fragments Specialized transduction mediated by temperatephages occurs as a result of lysogeny transducing phages carry selected segments of DNA

Generalized Transduction Specialized Transduction

Gene Transfers (contd)

CONJUGATION
transfer of DNA between two mating pairs requires cell-to-cell contact o involves F-plasmid encoded sex pilus o adhesins involves two mating types: Donor (male) and Recipient (female) Conjugation (Contd): Mating Types Donor (male): fertility (F) plasmid-carrying cell does not give an entire chromosome Recipient (female): lacks the F plasmid has receptor site (OmpA) for adherence F PLASMID small, circular, d-s DNA replicates independently of the chromosome conjugative o tra gene - sex pilus production F plasmid occurs in an autonomous or integrated states o F+ o Hfr o F F plasmid (Contd) F+ donors harboring an autonomous F plasmid F plasmid (Contd) Hfr donors with an integrated F plasmid F plasmid replicated with the chromosome can transfer large portions of donor genes at high frequencies
Formation of Hfr

F plasmid (Contd) F

Lecture Notes: BACTERIAL GENETICS Dr. E. Padla

donors with an autonomous F plasmid has segments of chromosomal DNA generated from imprecise excision of Hfr can transfer restricted portions of donor genes
Formation of F F Plasmid Transfer

F plasmid (Contd) F+ x Fconverts F- to F+ low transfer of donor chromosomal genes F plasmid (Contd) Hfr x Frecipient remains Fhigh transfer of certain donor chromosomal genes DNA Transfer with Hfr Order of Gene Transfer with Hfr F plasmid (Contd) F x Frecipient becomes F high transfer of donor genes on F low transfer of other donor chromosomal genes merodiploid formation

PLASMIDS
small, circular, d-s DNA capable of independent replication have an extra-chromosomal existence; may also integrate not essential to a cells survival contribute to phenotype of a cell confers selective advantage

Bacteria In Which Plasmids Have Been Detected

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Plasmids (contd) Properties Determined by Plasmids : Replication-Maintenance properties Host range narrow / broad Copy number low / high Incompatibility group compatible / incompatible Inc groups - DNA homology, pilus type, size

Lecture Notes: BACTERIAL GENETICS Dr. E. Padla

Resistance properties drug resistance resistance to heavy metal cations/anions Metabolic properties antibiotic & bacteriocin production Pathogenic properties toxin production Conjugal Properties Conjugative: often large plasmids (>35 kb) mediate their own transfer from cell to cell o tra genes code for transfer machinery Non-Conjugative: smaller plasmids (6-18 kb) can be transmitted by transduction, transformation conjugal transfer can be mobilized by a co-existing conjugative plasmid o mob genes mobilize their transfer
Conjugative & Non-Conjugative Plasmids Conjugative Plasmid Mobilization

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

Lecture Notes: BACTERIAL GENETICS Dr. E. Padla R PLASMIDS

consists of RTF & r determinants Resistance Transfer Factor (RTF) plasmid replication transfer by onjugation r determinants where all r genes are clustered acquired mobile genetic elements

R Plasmid Origin R Plasmid Evolution Resistance Phenotypes of Selected Transposons R Plasmids in Bacteria R Plasmid Transfer Pathways

Mechanisms Of R Plasmid-Mediated Resistance

Altering the target site of antibiotic Ex. erythromycin, lincomycin resistance Modifying the antibiotic so that it is no longer active Ex. chloramphenicol, penicillin, cephalosporin resistance Preventing the antibiotic from entering the cell Ex. Tetracycline, aminoglycoside resistance Specifying an enzyme that provides a substitute for a host-specified enzyme which is the target of the antibiotic Ex. Sulfonamide, trimetoprim resistance

Mechanisms Of R Plasmid-Mediated Resistance

ANTIBIOTIC Erythromycin Lincomycin Chloramphenicol Penicillns, Cephalosporins Tetracyclines Aminoglycosides Sulfonamides Trimetoprim RESISTANCE MECHANISM 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 ************************************************************************************** ************************************