Lecture 8 - Bacterial Genetics PDF

Lecture 8 Bacterial Genetics  Genetic Terminology Genetics - science of heredity What is a gene? How do they carry information? Heredity - replication and transmission How does genetic makeup make organisms what they are?  What is a Gene? Ge...

Lecture 8 Bacterial Genetics  Genetic Terminology Genetics - science of heredity What is a gene? How do they carry information? Heredity - replication and transmission How does genetic makeup make organisms what they are?  What is a Gene? Genome - genetic information of any organism, subject to change, may be due to damage, errors during replication, or horizontal gene transfer Chromosomes - organizational unit of genome Large molecule of DNA Carries genetic information of organism Prokaryotes have single circular chromosme (usually) Eukaryotes have many linear chromosomes GENES - segments of chromosomal DNA that code for functional products (Protein or RNA)  Genotype and Phenotype Genotype - genetic makeup of organism Represented by all genes of an organism Genes are only blueprints - bearers of information Only potential properties - need to be expressed Phenotype - expressed properties of the genotype Involves conversion of gene into functional product Functional product gives organism its characteristics Phenotype is manifestation of genotype  Mutations and Mutants Organisms isolated from environment with normally functioning genes – WILD TYPE STRAIN Sequence of genes can be altered – MUTATION – heritable, may be passed on to progeny Resulting strain and progeny termed a MUTANT Possible to identify mutants, useful for characterization of metabolic pathways and physiological processes Two commonly used tools, POSITIVE SELECTION and NEGATIVE SELECTION  Putting Mutations to Work Want a mutant that is resistant to penicillin mutagen Grow normal bacteria in presence of mutagen present Plate on solid media that contains penicillin or penicillin analog penicill Only bacteria that have acquired a mutation in that confers resistance to penicillin will survive resista nt Termed positive selection for desired mutation colonie s VERY powerful experimentally solid media containing  Negative Selection for Mutants Positive selection not always possible - loss of function mutations Want mutant that cannot synthesize histidine - no positive selection Grow bacteria in rich media with mutagen Plate on rich media Transfer colonies to velvet Plate on media containing and lacking histidine  How do Mutations Occur? Spontaneous mutations - errors during replication Rare in organisms with proofreading polymerase (DNA based organisms and some RNA viruses) Common in RNA based organisms that do not have proofreading polymerases (HIV) Chemical mutagenesis - chemical mutagens, nucleoside analogs, intercalating agents Radiation - UV light or DNA damaging (ionizing) radiation, X-rays, g- rays  Spontaneous Mutations r during DNA replication – heritable trait o misincorporation of nucleotide by DNA polymerase - rare One daughter cell has hybrid DNA Different sequence in each strand Next round of cell division produces two different DNA molecules One of the 4 grand daughter cells has acquired a mutation  Types of Mutations Most common type is base substitution AT GC or GC CG Can occur in gene - wrong amino acid may be incorporated into protein - missence mutation Redundancy in genetic code, mutation might not change amino acid incorporated - silent mutation May generate a stop codon - truncated gene product - shorter than normal - nonsense mutation  Types of Mutations Frameshift mutation - addition or removal of nucleotide Changes reading frame of gene All amino acids down stream are usually altered Also usually results in premature stop codon Results in truncated protein Dramatic affects to protein structure  Mutations - Chemical Mutagens Certain chemicals react with specific nitrogenous bases Reaction changes chemical structure of base Altered structure changes base pairing properties Mutation generated following replication of DNA  Mutations - Nucleoside Analogs Nulceoside - Nucleotide with out its phosphate Only ribose ring attached to nitrogenous base Nucleoside analogs are structurally similar to actual nucleosides Can be converted to nucleotide analogs and incorporated into DNA Base pairing properties are different Causes mutation during replication Base pairs with G Base pairs with C Mutations - Intercalating Agents Intercalating agents (ethidium bromide) insert between base pairs of DNA Significantly alters structure of DNA During replication, altered structure affects synthesis of DNA DNA polymerase can misincorporate nucleotides, add additional nucleotides, or delete nucleotides Extremely potent mutagens  Mutagenesis - UV and Ionizing Radiation UV radiation causes thymine dimers Alters structure of DNA Cause mutation during replication if not repaired g-radiation - causes double stranded breaks in DNA Cellular machinery attempts to put back together Can result in many types of mistakes DNA can be inverted, rearranged, or deleted All forms of mutations produce,  Drastic Measures - the SOS System Severe UV DNA damage results in activation of a extreme recombination repair system - SOS All genes involved repressed by LexA UV damage activates RecA, acquires ability to interact with LexA Interaction activates auto-protease activity of LexA LexA cleaves its self, genes required for SOS system expressed  Excision Repair – Error Free Mechanism for repair of UV damage and mismatched base pairs Distortion of double helix detected by repair machinery DNA on either side of damage nicked Single strand region containing damage removed DNA polymerase synthesizes new DNA DNA ligase seals gap Proofreading of DNA polymerase results in error free repair  Machinery Involved in Excision Repair ortion in double helix detected by UvrA mage recognition facilitates interaction with UvrB UvrB facilitates release of UvrA Release of UvrA allows recruitment of UvrC UvrC nicks DNA on either side of damage, UvrB/C released UvrD helicase unwinds and releases damaged DNA  Error Prone Repair Cleavage of LexA also induces expression of umuD, umuC, and dinB DinB encodes DNA polymerase IV, UmuC and UmuD assemble to form DNA polymerase V, very error prone – DNA mutases Capable of synthesizing DNA in absence of template instruction Results in high frequency of mutation Cell may die due to mutations introduced Cell definitely dies if nothing is done  Identification of Mutants the - Ames Test Uses Salmonella typhimurium strain that cannot make histidine Due to mutation in gene required for synthesis - requires back mutation to produce histidine, back mutation restores function to gene Grow strain in normal media and media containing suspected mutagen Only Platebacteria that have both cultures acquired on media a histidine lacking mutation can grow in absence of histidine If more colonies appear in presence of suspected mutagen, it is mutagenic  Ames Test can Identify Type of Mutation Caused by Mutagen Three types of S. typhimurium tester strains Each one has different mutation inactivating histidine biosynthetic gene: one frame shift, one missense, one inversion Strain harboring frame shift mutation requires frame shift mutation to restore function Strain with missence requires missense mutation to restore function Strain with inversion requires inversion mutation to restore function Strains that grow on histidine have acquired needed mutation Homologous Recombination in Prokaryotes Requires nicking of DNA followed by generation of single stranded DNA Single stranded DNA serves as donor Donor DNA initiates strand invasion of recipient DNA through homologus base pairing Creates Holliday Junction, junction migrates then resolves Results in generation of heteroduplex DNA Replication generates recombinants  Generating Single Stranded DNA Three rec components (RecBCD) bind double stranded DNA break RecBC has helicase function, RecD has exonuclease function Complex unwinds DNA, one strand degraded as strands separate Complex pauses at chi sequence, cleavage event occurs in strand being degraded RecD dissociates, RecBC continues to unwind DNA Generates single stranded DNA for strand invasion, bound by SSB to keep single stranded  Migration of Holiday Junction Requires Additional Factors A sufficient for invasion of single stranded DNA sufficient for migration of Holiday junction A and RuvB required for migration RuvA tetramer binds holiday junction, stabilizing function RuvB - ATP dependent DNA Helicase, responsible for migration of Holiday junction  Detecting Genetic Recombination Cultivate mutant culture in rich media to exponential growth Split culture into two aliquots, add exogenous DNA containing wild-type gene to one aliquot Grow both for equivalent amount of time, plate both on media lacking compound the mutant could not produce The no DNA sample reveals the spontaneous back mutation frequency DNA treated culture reveals frequency of recombination  Classical Experiment Detecting Griffith and Avery experiments with Streptococcus pneumoniae - 2 colony morphologys Recombination Smooth (S) - virulent, Rough (R) – avirulent Neither virulent when heat killed If viable (R) was mixed with heat killed (S) - resulted in restoration of virulence to (R) strains Colonies isolated are smooth – transformed, due to recombination  Competence Griffith and Avery were lucky – S. pneumonie naturally competent, able to take up foreign DNA spontaneously Many species of bacteria become naturally competent in specific phase of growth B. subtilis becomes naturally competent through quorum sensing Critical cell density results in accumulation of signaling peptide Uptake induces expression of several membrane proteins – autolysin, DNA binding protein, and nuclease Induces competence in about 20% of culture S. pneumoniae approach 100% competence  Transformation in Naturally Competent DNA attaches to DNA binding protein in membrane Organisms Some species take up double stranded DNA in tact Some species degrade one strand with a membrane associated nuclease, take up single strand DNA complexed by single stranded DNA binding protein if single strand taken up Facilitates interaction with RecA  Transformation in Naturally Competent RecA facilitates homologous base paring interactions and single Organisms strand invasion of chromosome Ruv proteins complete the process of strand invasion Resulting chromosome is a heteroduplex DNA replication followed by cell division results in one recombinant and one non-recombinant cell  Inducing Competence Some bacterial species do not become naturally competent Possible to “induce” competence with specific salts (LiCl) Mechanism of action unknown Believed to interact with membranes and destabilize Bridges nucleic acid and cell Facilitates internalization following heat shock Electroporation also effective  Transduction Generates Bacterial Diversity Bacteriophage infects bacterial cell, normally packages F DNA Occasionally packages fragment of chromosomal DNA Transferred to other bacteria, recombination integrates to chromosome Now have recombinant strain Generalized transduction Specialized Transduction Lysogenic bacteria integrate genetic material into bacterial chromosome Sometimes mistake is made when viral genome is excised from bacterial chromosome Some bacterial DNA is included with the viral DNA Virus infects other cell and lysogenizes Bacteria now contains genes from other bacterial strain, called SPECIALIZED TRANSDUCTION Sometimes has medical consequences (C. botulinum)  Plasmids Small autonomously replicating fragments of DNA All contain origin of replication – initiation of DNA synthesis Many contain genes that confer selective advantage (antibiotic resistance, use of alternate carbon sources) Variable in copy number (1-500 copies per cell), dependent on origin of replication Replication variable, some use mechanism similar to chromosome – form theta structure Conjugative plasmids use rolling circle replication, important for bacterial diversity  Plasmid Compatibility Many plasmids found in nature, fall into compatibility groups Grouping based on relation between mechanisms of initiating DNA synthesis Related plasmids harbor similar origins of replication, use similar machinery to regulate initiation of DNA synthesis Plasmids introduced to cell containing related plasmid results in inhibition of replication of new plasmid Usually results in elimination of new plasmid Some plasmids have no origin – integrate into chromosome, replicate when chromosome replicates – episome Sometimes plasmid eliminated from cell – process referred to as curing  Virulence Plasmids Bacillus anthracis – two essential factors required for virulence - capsule and toxin, each encoded on different plasmids Capsule - antiphagocytic polysaccharide layer Composed of poly-D-glutamic acid Inability to produce results in non-virulence Toxin - composed of three proteins, protective antigen (PA), edema factor (EF), lethal factor (LF) PA - binding unit of toxin, binds cells, inserts into membrane, creates channel of entry for EF and LF EF enters cell in presence of PA, exhibits adenylyl cyclase activity, responsible for influx of fluid at site of infection LF and PA constitute lethal toxin - major virulence determinant Entry into cell induces apoptosis - programmed cell death Necessary and sufficient for lethality in animal models  Resistance Plasmids Medically relevant – confers resistance to antibiotics – eg. R 100 Encode genes conferring resistance to mercury, sulfamethoxazole, streptomycin, chloramphenicol, and tetracycline Resistance genes acquired through two transposons Very problematic due to oriT and tra region of plasmid Confers ability to transfer plasmid from cell to cell Due to mechanism called conjugation  Conjugation Plasmid requires several features Must encode structural genes for pilus formation – sex pilus usually encoded in tra region Must encode genes that initiate transfer through sex pilus, usually encoded in tra region Must possess oriT – origin of replication used for transfer Most extensively investigated conjugative plasmid – F plasmid Cells harboring F plasmid termed donor cell (F+) Cells lacking F plasmid termed recipient cell (F-)  Transfer of F-plasmid Plasmid encoded gene products nick oriT Same gene products migrate around plasmid DNA strands separate Linear copy transferred through F-pilus to recipient cell Both copies converted to double stranded DNA Plasmid in recipient cell circularizes Recipient cell converted from F(-) to F(+)  Mechanism of Transfer Rolling circle replication generates single stranded donor DNA Single stranded donor DNA associates with plasmid encoded membrane proteins, facilitates transfer through F pilus DNA polymerase associates with single stranded donor DNA in recipient cell Primase associated with DNA polymerase synthesizes RNA primer Polymerase converts to double stranded DNA, ligase converts to closed circular form  Integration of F plasmid into Chromosome Mediated through homologous recombination Specific sites on F plasmid share homology with sequences in bacterial chromosome – termed IS (insertion sequence) Facilitates recombination event, results in integration of F-plasmid Several IS exist IS sequence facilitating recombination dictates location of integration and orientation Resulting strain now Hfr – high frequency recombination  Consequences of Integration F plasmid retains all genes, retains ability to initiate transfer to F– cells Initiation of transfer results in transfer of both plasmid DNA and chromosomal DNA Theoretically possible for Hfr strain to convert F– strain to Hfr Extremely rare event, due to lack of transfer of complete F plasmid region  Mapping of the Bacterial Chromosome Mixed bacterial strains with different genotypes – i.e. Hfr strain gal + thr +leu + lac + Strs , F– strain gal - thr – leu – lac – Strr Allowed conjugation to occur, disrupted at various time points – interrupted mating Detected recombination by plating on media lacking amino acids or containing galactose as a sole carbon source containing streptomycin Streptomycin kills Hfr strain F – strain only capable of growth if wild-type copy of gene recombined from Hfr donor Different prototrophies conferred at different interruption times  Transfer of Bacterial Genes to F Plasmid Multiple IS sequences exist in F plasmid and chromosome Facilitates excision of F plasmid from chromosome through homologous recombination Recombination between IS sequences not originally used for insertion event results in excision of chromosomal DNA along with F plasmid Resulting plasmid may retain all genes essential for conjugation and transfer Now transfers bacterial genes along with plasmid DNA Referred to as F' plasmid  Mutations and Complementation Recessive mutations allow for determination if mutations are in the same gene Mate homozygous recessive mutant parents (flies, not people!) If progeny are of mutant phenotype, both parents had a mutation in the same gene If the progeny have wild-type phenotype, mutations were in different genes Wild-type copy of gene from one parent COMPLEMENTED the mutant gene in the other parent Only works for recessive mutations  Genes are Also Referred to as Cistrons Need method to determine if mutations are in the same or different genes - CIS-TRANS TEST If mutation in is same allele, does not allow complementation to occur, mutation is in cis configuration (each copy mutated) Such mutations are said to be part of the same genetic unit or COMPLEMENTATION GROUP Mutations in different alleles exhibit wild-type phenotypes Are distinct genetic units, referred to as cistons due to analysis in cis-trans test  Impact of Transposons on Genome Often promote rearrangements of host genome - two mechanisms Transposition directly alters genome May delete or invert host sequences May move host sequences to new location May cause host machinery to rearrange genome Multiple copies of transposon provides sites of homology Provides sites for reciprocal recombination Many alterations possible (deletion, insertion, inversion, translocation)  Simple Transposons Referred to as INSERTION SEQUENCES (IS) - minimum transposable element Consist of single gene flanked by inverted repeats Gene encodes enzyme required for insertion into target DNA - TRANSPOSASE Length of of Selection inverted target repeat variable for different IS (4-13 base pairs) sequence variable Some IS completely random Other IS target semi-specific sequences Others semi-random  Composite Transposon Carry additional genes - not simply composed of genes necessary for transpositon Genes often confer resistance to drugs or other selective markers Central region contains selective marker - flanked by arms, IS elements Each arm potentially encodes complete and functional IS element IS element encoded by each arm usually the same IS element Two configurations of arms - direct repeat or inverted repeat Nomenclature - Tn followed by number (Tn10)  One Functional IS Required for Composite Transposons h IS elements of composite transposon may be functional n9 - functional copies of IS 1 at either end 1 organized as direct repeats ach copy of flanked by inverted repeats Other transposons have only 1 functional IS Tn10 and Tn5 - right IS functional Tn903 - both functional  Insertion into the Genome All transposons share similar mechanism Nine base pair target sequence cleaved, creates staggered cut Transposon joined to cleaved DNA, single stranded regions present Host DNA polymerase fills in single stranded region Host DNA ligase seals gaps Nature of target sequence variable Some transposons insert randomly  Implications of Mechanism of Insertion Staggered cut followed by insertion results in duplication of target sequence Size of target sequence variable, dependent on transposon Target may be sufficiently large to encompass gene or several genes Results in complete duplication of gene or genes Duplicated genesevolution Drives microbial mutate at higher frequency compared to single copy genes  Conservative vs. Replicative Transposition CONSERVATIVE TRANSPOSITION - DNA at original site of integration precisely conserved No mutation generated by excision of transposon Transposase involved significantly different from transposases catalyzing nonreplicative transposition Mechanism used similar to integrase, enzymes share significant similarity Excised transposon inserts at second target sequence No duplication due to transposition  Conservative vs. Replicative Transposition Two general mechanisms for transposition - replicative and nonreplicative REPLICATIVE TRANSPOSITION - two transposon encoded genes required TRANSPOSASE - acts on transposon initially inserted into genome Nicks DNA on either side of transposon RESOLVASE - resolves duplicated copy as second Essential site from original transposon for mechanism of transposition  Replicative Transposition Transposase mediates joining of donor and target sequences Generates 3´-OH - used by host machinery for DNA synthesis Synthesis duplicates transposon Ligase seals ends Two copies of transposon exist, donor and target DNA now joined Generates intermediate structure - COINTEGRATE  Replicative Transposition Transposase sufficient for formation of cointegrate Not capable of resolving and releasing donor and recipient DNA Requires second transposon gene product - RESOLVASE Catalyzes recombination event between duplicate copies of transposon Releases donor and target DNA  Transposon Mutagenesis Many transposons insert into the genome randomly or semi randomly Possible to conduct random or semi random mutagenesis Transform bacteria with transposon DNA, must have selectable marker – often antibiotic resistance Select for transposition events by inclusion of antibiotic in growth media Obtaining sufficient number of transposition events allows for saturation mutagenesis  Integrons Transopson like elements – acquire and express entire genes Differ from transoposons – integrate at highly specific sites Genes acquired expressed from promoter on integron Collection of genes referred to as cassettes Medically important due to acquisition of antibiotic resistance genes  Molecular Cloning ge due to staggered cut - leaves overhang - single stranded le of base pairing with other single stranded DNA d digested with HinDIII of interest has HinDIII sites on either side of gene plasmid and gene of interest has HinDIII overhangs teract through standard base pairing interactions attachment of gene to plasmid  Suitable Plasmids pBR322 historical cloning vector Moderate copy number (20 to 30 copies/cell) possible to induce high copy number with chloramphenicol (1000 – 3000 copies/cell) Facilitates large scale purification of supercoiled DNA Stable with up to 10 kb foreign DNA Contains two selectable markers Multiple restriction enzymes cut at single place in chromosome Readily introduced into chemically competent or electrocompetent cells  Cloning with pBR322 Digest pBR322 with restriction enzyme that cuts inside one of the selectable markers Cut DNA you wish to clone with same restriction enzyme Mix digested DNA together and ligate Transform bacteria with ligation mixture Select for resistance using uncut selectable marker Confirm insertion of desired DNA by screening for sensitivity to second antibiotic  Modern Cloning Vectors Historical cloning vectors requires two stages of screening to confirm insertion of desired DNA Modern cloning vectors allow detection of insertion of desired DNA with single step Plasmid contains engineered multi-cloning site (MCS) – multiple MCS enzymes cut within located only once lacZ gene, in frame Cloning of gene in MCS disrupts lacZ Possible to detect insertion of foreign DNA through blue white screening  Cloning with Phage l Significant region of l genome dispensable for function, deletion allows for replication, assembly of virion, and lysis Possible to delete non-essential region and replace with foreign DNA Useful if working with large fragments of DNA – 20 to 25 kb (genomic library construction) Plasmid based systems become unstable if insert is greater than 10 kb  Cloning with Phage l Digest l genome with enzyme to remove non-essential region Mix with desired DNA digested with same enzyme, ligate Mix ligation with preformed phage l heads Cos sites on l DNA facilitate attachment to and packaging by phage heads Completion of packaging allows for completion of assembly Assembled virion fully infective Efficiently delivers recombinant DNA to bacterial cell Recombinant DNA can be isolated from plaques on bacterial lawn  Targeted Gene Disruption Possible to disrupt specific gene using cells recombination machinery Clone gene of interest using plasmid, cut with restriction enzyme that cleaves at least once within gene (twice is better) Ligate to cassette with selectable marker, usually antibiotic resistanceplasmid, prevents replication in bacteria Linearize Transform bacteria with linearized plasmid Recombination machinery facilitates homologous recombination event, wild-type gene replaced with defective copy  Discovery of the CRISPR-Cas System Series of repeated sequences in bacterial genome discovered by Francisco Mojica in 1993, function unknown Independent analyses conducted by Mojica, Giles Vergnaud, and Alexander Bolotin in 1995 All reached conclusion that the region contained segments of DNA derived from bacteriophage All proposed this region confers resistance to bacteriophage infection Philippe Horvath demonstrated that the locus allows defense against bacteriophage infection Collectively analyses revealed that the locus has the bacteriophage DNA separated by Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR – expressed as single gene Analyses also revealed presence of four additional genes linked to the locus, tracr, Cas9, Cas1, and Cas2  Adaptation of the CRISPR System First exposure to a virus activates expression of Cas1 and Cas2 Cas1/Cas2 forms complex, binds and cleaves bacteriophage DNA, inactivates bacteriophage Also confers immunological memory Fragment of bacteriophage integrates into CRISPR gene adjacent to palindrome repeat Creates array of DNA segments for each different bacteriophage encountered called spacers Each spacer separated by palindrome repeat Each spacer confers protection against specific bacteriophage (4 shown in figure)  Expression of the CRISPR System Exposure to a bacteriophage induces expression of tracr, Cas9, and CRISPR array CRISPR array expressed as single long pre-crRNA, contains all spacers for all bacteriophage previously encountered Pre-crRNA processed into many crRNAs, each has palindrome sequence attached to individual spacer, each spacer specific for one bacteriophage Palindrome sequence complementary to sequence in tracrRNA, facilitates base pairing interactions tracrRNA also has second domain that folds into stem loop structure Stem loop structure on tracr serves as scaffold, binds Cas9 nuclease tracrRNA/crRNA/Cas9 complex competent to be delivered  Interference by the CRISPR System crRNA spacer region derived from bacteriophage genome Serves as guide to direct tracrRNA/crRNA/Cas9 complex to bacteriophage genome crRNA forms base pairing interactions with complementary sequence in bacteriophage genome Base pairing activates nuclease activity of Cas9, bacteriophage genome degraded, requires protospacer adjacent motif (PAM) Inactivates virus, proliferation prevented