גנטיקה של חיידקים

מוטציות Normal DNA

מוטציה 1 – שינוי בסיס ב-DNA C Missense Mutation

מוטציה 2 - שינוי בסיס ב-DNA T Nonsense Mutation

מוטציה 3 - תזוזת מסגרת הקריאה ATG CAT GCA TGC ATT TCC TGC TTA AAA 1. Addition Mutation AAT GCA TGC ATG CAT TTT CCT GCT TAA Reading Frame is Shifted 2. Deletion Mutation TGC ATG CAT GCA TTT CCT GCT TAA

העברת מטען גנטי בחיידקים העברת מטען גנטי – דרוש על מנת לשרוד. העברת מטען גנטי בחיידקים יכול להתרחש ב- 3 דרכים : Transformation - טרנספורמציה Transduction - טרנסדוקציה Cunjugation - קוניוגציה

הניסוי של גריפין Griffith’s Experiment 2 Strains of Streptococcus pneumoniae Virulent strain with a capsule – Pneumonia Avirulent strain without a capsule - no disease

Griffith’s Experiment הניסוי של גריפין חיידקים חיים בעלי קפסולה הוזרקו לעכברים העברים מתו מושבות חיידקים בעלי קפסולה בודדו מהחיידק המת.

Griffith’s Experiment 1. חיידקים חיים ללא קפסולה הוזרקו לעכברים 2. העכברים נשארו בריאים 3. מושבות של חיידקים ללא קפסולה בודדו מהעכברים.

Griffith’s Experiment חיידקים בעלי קפסולה, שהומתו ע"י חימום, הוזרקו לעכברים העכברים נשארו בריאים לא בודדו מובות חיידקים מהעכברים

Griffith’s Experiment חיידקים חיים ללא קפסולה, וחיידקים בעלי קפסולה, שהומתו ע"י חימום, הוזרקו לעכברים העכברים מתו מושבות של חיידקים בעלי קפסולה בודדו מהעכברים המתים.

A typical bacterial cell chromosome extra-chromosomal DNA

Transformation טרנספורמציה גנים עוברים מחיידק אחד לאחר כ “DNA ערום". Frederick Griffith (1928)

Transfer of genetic information Transformation and DNA Recombination Dead cells?

Transductionטרנסדוקציה DNA מועבר מחיידק אחד לשני ע"י וירוס. בקטריופאז' – וירוס התוקף תאי חיידקים בלבד.

בקטריופאג' או פאג' - Bacteriophage or phage טרנסדוקציה בקטריופאג' או פאג' - Bacteriophage or phage = וירוס התוקף חיידק lytic or virulent phage lysogenic or temperate phage Bacterial DNA transfer Generalized transduction Restricted transduction

Transduction Generalized transduction – lytic or lysogenic phage any suitably sized piece of DNA packaged

Transduction Bacteriophage or phage - bacterial viruses lytic or virulent phage lysogenic or temperate phage

Transduction Restricted transduction only genes adjacent to insertion site phage λ - gal or bio loci phage defective λdgal requires helper phage

Transduction

Conjugation One bacterium passes some DNA (in a plasmid) to another bacterium

bacterial conjugation/bacterial “sex”

conjugation

Bacterial Conjugation

Conjugation F+ x F- Hfr x F-

Conjugation Plasmid exit from genome: Hfr → F+ Hfr → F' recipient is a partial diploid

Mapping the bacterial genome Hfr transfer of bacterial genes

Mapping the bacterial genome

Conjugation Lederberg & Tatum, 1946 auxotrophs in E. coli auxotrophs mixed – 108 cells plated on minimal medium reversion vs. ? shown cell contact required

Conjugation Hayes, et. al., 1950s discovered family of mutants with a high frequency of recombination – Hfr mutants no conversion of F- in mating bacterial gene transfer high plasmid integrated into host genome specific locus ‘cross-over’

Conjugation Pilus needed for cell contact DNA synthesis needed for transfer rolling circle replication begins at origin (ori) one strand nicked

Conjugation Plasmid exit from genome: Hfr → F+ Hfr → F'

Plasmid pBR322

Transduction Bacteriophage or phage - bacterial viruses lytic or virulent phage lysogenic or temperate phage

Transduction Bacteriophage or phage - bacterial viruses lytic or virulent phage lysogenic or temperate phage Bacterial DNA transfer Generalized transduction Restricted transduction

Transduction Generalized transduction – lytic or lysogenic phage any suitably sized piece of DNA packaged

Evolution Survival/selection of the fittest Transfer of genetic information with selective advantage  survival and propagation of the recipients

Results of mutation and gene transfer antibiotic resistance

Basic genetic engineering

Microbial Genetics Genetic notation Three letter designation for each gene lac gal ara his lacZ, hisG, araC Genome sum total of all the genes of the organism all of the ‘chromosomes’ of the organism, the DNA bacteria are haploid Genotype vs. Phenotype Genotype: genetic makeup of the organism Phenotype: appearance; visible characteristics Does phenotype always reflect the genotype?

Microbial Genetics Gene - unit of inheritance structural gene - information for a protein regulatory gene - control protein formation operon – structural genes plus regulatory genes

Recombination bringing new gene combinations together Eukaryotes - crossing over during meiosis reciprocal exchange

Recombination bringing new gene combinations together Eukaryotes - crossing over during meiosis reciprocal exchange Prokaryotes - transfer of genes from one cell to another one-way transfer of genes DNA transferred: exogenote recipient DNA: endogenote partial diploid may be formed

Recombination bringing new gene combinations together Eukaryotes - crossing over during meiosis reciprocal exchange Prokaryotes - transfer of genes from one cell to another one-way transfer of genes DNA transferred: exogenote recipient DNA: endogenote partial diploid may be formed Transformation Conjugation Transduction Limited transfer: one gene to a few genes closely related cells

Transformation. Griffiths, 1928 - vaccine for pneumonia Transformation Griffiths, 1928 - vaccine for pneumonia Streptococcus pneumoniae smooth vs. rough strains ‘transforming principle’ Avery, McCarty & MacLeod, 1944

Transformation naked DNA released by cell binds to competent recipient cell competence is physiological state competence requires protein synthesis DNA binding protein on cell surface – receptor DNA must be dsDNA of suitable size (450-15,000 bp) one strand degraded ssDNA enters cell pairs with genome heteroduplex formed by recA

Heteroduplex DNA mediated by recA

Transformation

Conjugation Hayes, et. al., 1950s transfer one-way, not reciprocal donor and recipient strains (+ and – strains) donor: F+ recipient: F- mating of F+ x F- → 2 F+ F is F plasmid bacterial gene transfer rare

Transduction Restricted transduction only genes adjacent to insertion site phage λ - att locus gal or bio loci

Transduction Restricted transduction only genes adjacent to insertion site phage λ - att locus gal or bio loci phage defective λdgal requires helper phage

Transduction Restricted transduction only genes adjacent to insertion site phage λ - gal or bio loci phage defective λdgal requires helper phage

Fates of DNA Incorporation of genes transformation, conjugation, transduction

Fates of DNA Incorporation of genes transformation, conjugation, transduction Persist and replicate - clone of cells with the DNA self-replicating plasmids

Fates of DNA Incorporation of genes transformation, conjugation, transduction Persist and replicate - clone of cells with the DNA self-replicating plasmids Persist but not replicate abortive transduction

Fates of DNA Incorporation of genes transformation, conjugation, transduction Persist but not replicate abortive transduction Persist and replicate - clone of cells with the DNA self-replicating plasmids Degradation or host restriction

Fates of DNA

Host restriction restriction endonucleases cut at specific nucleotide sequences – 6-8 base pairs most generate ‘sticky ends’

Restriction enzymes EcoRI

Host restriction restriction endonucleases cut at specific nucleotide sequences – 6-8 base pairs most generate ‘sticky ends’ host DNA protected by methylation -G-A-A-T-T-C- -C-T-T-A-A-G-

Genetic engineering. Tools:. vectors - plasmids, bacteriophage Genetic engineering Tools: vectors - plasmids, bacteriophage restriction enzymes Products proteins - see Table 9.2 in text resistant plants

Conjugation Donor Definition: Gene transfer from a donor to a recipient by direct physical contact between cells Mating types in bacteria Donor F factor (Fertility factor) F (sex) pilus Recipient Recipient Lacks an F factor

Physiological States of F Factor Autonomous (F+) Characteristics of F+ x F- crosses F- becomes F+ while F+ remains F+ Low transfer of donor chromosomal genes F+

Physiological States of F Factor Integrated (Hfr) Characteristics of Hfr x F- crosses F- rarely becomes Hfr while Hfr remains Hfr High transfer of certain donor chromosomal genes F+ Hfr

Physiological States of F Factor Autonomous with donor genes (F’) Characteristics of F’ x F- crosses F- becomes F’ while F’ remains F’ High transfer of donor genes on F’ and low transfer of other donor chromosomal genes Hfr F’

Mechanism of F+ x F- Crosses Pair formation Conjugation bridge F+ F- DNA transfer Origin of transfer Rolling circle replication

Mechanism of Hfr x F- Crosses Pair formation Conjugation bridge Hfr F- DNA transfer Origin of transfer Rolling circle replication Homologous recombination

Mechanism of F’ x F- Crosses Pair formation Conjugation bridge F’ F- DNA transfer Origin of transfer Rolling circle replication

Conjugation Significance Gram - bacteria Gram + bacteria Antibiotic resistance Rapid spread Gram + bacteria Production of adhesive material by donor cells

Transposable Genetic Elements Definition: Segments of DNA that are able to move from one location to another Properties “Random” movement Not capable of self replication (not a replicon) Transposition mediated by site-specific recombination Transposase Transposition may be accompanied by duplication

Types of Transposable Genetic Elements Insertion sequences (IS) Definition: Elements that carry no other genes except those involved in transposition Nomenclature - IS1 Structure Importance Mutation Plasmid insertion Phase variation Transposase ABCDEFG GFEDCBA

Phase Variation in Salmonella H Antigens H1 gene H2 gene IS H1 flagella H2 flagella

Types of Transposable Genetic Elements Transposons (Tn) Definition: Elements that carry other genes except those involved in transposition Nomenclature - Tn10 Structure Composite Tns IS Resistance Gene(s) Importance Antibiotic resistance

The insertion of the IS-element is accompanied by a duplication of a short sequence around the insertion site. A proposed mechanism known as the cut and paste model for the occurence of this effect is illustrated opposite. A staggered single-strand cut is made in the target sequence followed by an insertion of the respective IS-elements. The cuts are subsequently sealed and as a result the target sequence is duplicated.

Plasmids Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon) Episome - a plasmid that can integrate into the chromosome

Classification of Plasmids Transfer properties Conjugative Nonconjugative Phenotypic effects Fertility Bacteriocinogenic plasmid Resistance plasmid (R factors)

Some Examples of Plasmid Encoded Genes Antibiotic resistance genes (enzymes that modify or degrade antibiotics) -- plasmids with these genes are generally called R factors Heavy metal resistance (enzymes that detoxify metals by redox reactions) Growth on unusual substrates (enzymes for hydrocarbon degradation, etc.) Restriction/modification enzymes (protect DNA, degrade unprotected DNA) Bacteriocins (proteins toxic to other bacteria lacking the same plasmid) Toxins Proteins that mediate plasmid transfer to uninfected strains (i.e. the tra genes) Genes which provide the cell with a pathogenic feature: such as the ability invade cells (Yersinia pestis) or produce toxins (E coli 0:157) plasmids of this type are often called virulence plasmids.