Lecture Notes: BACTERIAL GENETICSDr. E. Padla

BACTERIAL GENETICSCoverage:DNA ReplicationGenetic Variation

MutationIS and transposons

Genetic Transfer ProcessesGenetic RecombinationGene Transfers

R Plasmid – Mediated Resistance

GENETICS study of heredity and variation

Gene - units of heredityGenome - total complement of genes

Chromosome naked occupies 10% of cell volume about 1500X the cell’s length single, circular, tightly wound DNA exists in nucleoid replicate by binary fission

Cell DivisionCell Division & DNA ReplicationFlow of Genetic Information

REPLICATION DNA Replication semi-conservative

Stages: Initiation

o initiated at ori C (origin of chromosomal replication)o unwinding of double helix forming a replication forko primer synthesis

Elongationo DNA synthesis/polynucleotide chain elongationo proceeds bi-directionally to a fixed terminus (ter C)

Terminationo primer removalo gap -filling

Lecture Notes: BACTERIAL GENETICSDr. E. Padla

Replication Fork Primer SynthesisBi-Directional Replication Replication (Cont’d) – LEADING STRAND SYNTHESISReplication (Cont’d) - LAGGING STRAND SYNTHESISReplication (Cont’d) – LEADING/ LAGGING STRAND SYNTHESIS

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

o DNA polymerase III– elongates primer, produces Okazaki fragmentso DNA polymerase I – excises RNA primer, fills gap

Ligase - links Okazaki fragments

Comparison Of The Functional Characteristics Of E. coli DNA Polymerases VARIATIONPhenotypic Variation

metabolic changes in response to changing environmentGenotypic Variation

changes in genetic constitution

MutationGene transfers

TransformationTransductionConjugation

MUTATION heritable change in DNA sequence

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

Classification o Spontaneous vs Inducedo Selectable vs Non-Selectableo Base Substitutions/ Replacementso Insertions and Deletionso Those caused by Transposable Genetic lements (TGEs)

Mutation (Cont’d)Spontaneous Mutations occur without any apparent cause rare events may be caused by replication errors

Lecture Notes: BACTERIAL GENETICSDr. E. Padla

Induced Mutations those with identifiable cause (e.g. mutagens) physical agents chemical agents Mutation (Cont’d)Selectable Mutations confers selective advantage to the mutantEx - mutation to drug resistance

Non-Selectable Mutations mutant has no selective advantage over wild-typeEx - loss of color in a pigmented bacterium Direct Selection for Mutants

Mutation (Cont’d)Base Substitution/ Replacement involves substitution of one base for another kinds:

o Transitiono Transversion

effects:o silent mutationo missense mutationo non-sense mutation

Base Substitutions (cont’d) – KINDS OF BASE SUBSTITUTIONPossible Effects of a Point Mutation Mutation (Cont’d)Insertions and Deletions addition or removal of one or more bases Shifts in Reading Frame Mutation (Cont’d)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 (Cont’d)

Lecture Notes: BACTERIAL GENETICSDr. E. Padla

Mutation (Cont’d)TRANSPOSABLE GENETIC ELEMENTS can transfer from one location to another, or between chromosome and plasmids not independent replicons have inverted terminal repeats types :

o insertion sequenceso transposonso phage-associated transposons (Mu)

TGEs (Cont’d) – TYPES 1. Insertion Sequences

simplest of the TGEs about 1000 bp carry no genes except those involved in transposition

transposase TGEs (Cont’d) – INSERTION SEQUENCEInsertion Sequences TGEs (Cont’d) – TYPES 2. Transposons

10-fold longer than IS flanked by IS elements

o IS - for transposabilityo central genes - codes for antibiotic resistance

TGEs (Cont’d) – TRANSPOSONResistance Phenotypes of Selected Transposons TGEs (Cont’d) – 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 (Cont’d) – BACTERIOPHAGE MU

TGEsTransposition involves:

o transposase - catalyzes insertion into new siteo resolvase - involved in recombinational events

accompanied by duplication of target site kinds:

o conservative transpositiono replicative transposition

Transposition

Lecture Notes: BACTERIAL GENETICSDr. E. PadlaFates 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 - CLASSIFICATION1. 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 transformationo transductiono conjugation

Gene Transfers (cont’d)Mechanisms transformation transduction conjugation

Lecture Notes: BACTERIAL GENETICSDr. E. PadlaGene Transfers (cont’d)TRANSFORMATION uptake of “naked” DNA by a recipient cell

Transformation (Cont’d)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”

o some are naturally competent; others are to be inducedo a transient stateo varies in Gram (+) and Gram (-) bacteria

competence factors recognition sequences

Transformation Gene Transfers (cont’d)TRANSDUCTION bacteriophage-mediated transfer of DNA types:

o generalized transductiono specialized transduction

Bacteriophage Life CyclesLytic 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 CycleLysogenic CycleLysogenic Conversion Transduction – TYPESGeneralized Transduction Specialized transduction mediated by virulent phages mediated by temperatephages transducing phages carry random DNA fragments occurs as a result of lysogeny

transducing phages carry selected segments of DNA

Generalized TransductionSpecialized Transduction

Lecture Notes: BACTERIAL GENETICSDr. E. PadlaGene Transfers (cont’d)CONJUGATION transfer of DNA between two mating pairs requires cell-to-cell contact

o involves F-plasmid encoded sex piluso adhesins

involves two mating types: Donor (male) and Recipient (female)

Conjugation (Cont’d): 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 Hfro F’

F plasmid (Cont’d)F+ donors harboring an autonomous F plasmid F plasmid (Cont’d)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 (Cont’d)F’ 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 (Cont’d)

Lecture Notes: BACTERIAL GENETICSDr. E. PadlaF+ x F-

converts F- to F+ low transfer of donor chromosomal genes F plasmid (Cont’d)Hfr x F-

recipient remains F- high transfer of certain donor chromosomal genes

DNA Transfer with HfrOrder of Gene Transfer with Hfr F plasmid (Cont’d)F’ x F-

recipient 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 cell’s survival contribute to phenotype of a cell confers selective advantage

Bacteria In Which Plasmids Have Been Detected Plasmids (cont’d)Properties Determined by Plasmids : Replication-Maintenance properties

o Host range narrow / broad

o Copy number low / high

o Incompatibility group compatible / incompatible Inc groups - DNA homology, pilus type, size

Resistance propertiesdrug resistance resistance to heavy metal cations/anions

Metabolic propertiesantibiotic & bacteriocin productionPathogenic properties

toxin production

Conjugal Properties

Lecture Notes: BACTERIAL GENETICSDr. E. Padla

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 persistenceo loss of target structure

Genetico chromosomal (mutations, replication errors)o extra-chromosomal (R-plasmids)

R Plasmid-Determined Resistance in Enteric Bacteria

R PLASMIDS consists of RTF & r determinants

Resistance Transfer Factor (RTF) plasmid replication transfer by onjugation

r determinantswhere all r genes are clusteredacquired mobile genetic elements

R Plasmid Origin R Plasmid Evolution Resistance Phenotypes of Selected Transposons

Lecture Notes: BACTERIAL GENETICSDr. E. PadlaR Plasmids in BacteriaR 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 activeEx. chloramphenicol, penicillin, cephalosporin resistance

Preventing the antibiotic from entering the cellEx. Tetracycline, aminoglycoside resistance

Specifying an enzyme that provides a substitute for a host-specified enzyme which is the target of the antibioticEx. Sulfonamide, trimetoprim resistance

Mechanisms Of R Plasmid-Mediated Resistance

ANTIBIOTIC RESISTANCE MECHANISMErythromycinLincomycin

Methylation of 23S rRNAs of ribosomes

ChloramphenicolPenicillns, Cephalosporins

Detoxification by chloramphenicol transacetylaseb-lactamase

TetracyclinesAminoglycosides

Prevents intracellular accumulationUptake inhibited

SulfonamidesTrimetoprim

Sulfonamide-resistant dihydropteroate synthetaseTrimetoprim-resistant dihydrofolate reductase

ANTIBIOTIC RESISTANCE CAN BE CONTAINED …..But only by the most careful monitoring of strainsAnd prudent use of antibiotics

**************************************************************************************************************************