MB – Bacterial Genetics A.S.d.C. · MB – Bacterial Genetics A.S.d.C. Bacteria have chromosomes...
Transcript of MB – Bacterial Genetics A.S.d.C. · MB – Bacterial Genetics A.S.d.C. Bacteria have chromosomes...
MB – Bacterial Genetics A.S.d.C.
BACTERIALGENETICSAntibioticresistancedevelopsthroughnaturalselection:
Antibiotictargets:
Mechanismofresistance:
MB – Bacterial Genetics A.S.d.C.
HorizontalGeneTransfer
MB – Bacterial Genetics A.S.d.C.
Bacteriahavechromosomes
• Inbacteria,observationalapproacheswerenotpossible(eye-colour,wingshape,shapeofa
pea,etc)
• However:Onegene-OneenzymeHypothesis(BeadleandTatum,1941)
providedlinkbetweenbacterialphysiologyandgenetics
• IrradiatedNeurosporacrassa(afungus)• Isolatedmutantsunabletosynthesiseparticular
nutrientorvitamin(sodefectiveinasyntheticstep)
• Suchmutantsweretermed‘Auxotrophs’
• Prototrophsarewild-typeforthatcharacteristic
Conclusion:
-genesproduceenzymes
-producedlinearprogressionforbiochemicalpathways
-pathwayconservationwasgoodphenotypetomonitor
-butsegregationmustbediscrete
-(i.e.mustfollowonegene=oneenzymehypothesis)
• Howisgeneticvariabilityintroducedandinheritedinbacteria?
• Mostorganismsfollowed‘Darwinian’Rules:
• Genomechangesoccurrandomly/spontaneously
• Changesnotdrivenbytheenvironment
• Ifchangesarebeneficialforsurvivalinanenvironment,thenmorelikelytobepassed
ontoprogeny
• Bacteriawerethoughttofollow
“directed”change/“adaptive-mutation”hypothesis-“Lamarckism”
o Mutationsinducedbyselection
o Adaptationoforganismtoenvironment
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• SalvadorLuriaandMaxDelbruck(1943)
E.coliresistancetobacteriophageT1Bacteriacanbegrownonanagarplatetoformalawn
Ifphagearepresenttheywillkillbacteriaresultinginaplaque
Observation:shortlyafterthemajorityofbacteriaarekilledbyphage(seenasaplaque),
phage-resistantcolonieswillappear.
Question:
Arethesemutationsinducedbythephage?
Oraretheyalreadypresentinthepopulation?
Afundamentalquestioningenetics
• GrewaliquidcultureofE.coli• Aliquotedintoaseriesoftubesandgrewagaincreatingidenticalindependentsub-culturesto
bemonitoredseparately
• Eachsub-culturewasplatedonagarwithphageandresistancetophagecounted
Now:
Ifmutationwastheresultofexposuretothephage,
(inducedmutation;A)frequencyofresistanceineachcultureshouldbesimilar
Ifmutationwasspontaneous,B,thefrequencyofmutationshouldbeveryvariable.
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BACTERIALTRANSFORMATION
Transformation-isolatedDNAmoleculesaretakenupfromexternalsurroundingsandincorporatedintothegenome
• Processissolelyencodedbytherecipientbacteria
(unliketransductionandconjugation)
• Allrequiredproteinsencodedwithinrecipientcoregenome
• Mosttransformablebacteriadonotpermanentlyexpresstheproteinsrequiredfor
transformation
• Bacteriaaredescribedascompetentwhentheyareabletoundergotransformation
• Onlyafractionofthebacteriainapopulationbecomecompetent
Firstspeciestobediscoveredastransformable*:
StreptococcuspneumoniaeGram+ve(Griffiths)
• approx.80speciesnowknowntobetransformable*
• bothGram+veandGram–vespecies
SomeNaturallytransformablespecies
• Bacillussubtilis(Gram+ve)
• Streptococcuspyogenes(Gm+ve)• Neiserriagonorrheoa(Gm–ve)• Haemophilusinfluenza(Gm–ve)
WHYBETRANSFORMABLE?
• Nutrition:
-Nucleotidesource?
-Somebacteriabecomecompetentatstationaryphase
-Inefficient,moreefficienttocatalysecompleteDNAbreakdowninextracellular
environment
-Usenormalimportchannelstorecoverthenutrients
• Genomemaintenance/repair:
-UptakeofpotentiallyhomologousDNAallowsdamagerepair?
-Consistentwith“self”DNAuptakerequirement
-SomeevidencethatcompetencesystemsareinducedbyDNAdamage
• Genomediversification:
-DNAuptakeincreasesgeneticdiversity
-Increasingdiversityduringstress(e.g.starvation)maximiseslikelihoodofsurvivors
• DisadvantagesofDNAuptake?
-energycost
-newgenescouldbepotentiallyharmful
MECHANISMSOFTRANSFORMATION:1.Bacteriadevelop“competence”
2.CellsbinddoublestrandedDNAintheenvironment
3.MovementofDNAovercellmembrane/cellwall
4.ConversiontosinglestrandedDNA
5.Intracellularfate:
-maintainedinthegenomeasaplasmidor
-homologousrecombinationintogenomeor
-degradation
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1.Bacteriadevelopcompetencenaturally
• usuallyasacultureencountersstress
e.g.nutrientlimitation
• cellscoordinatelyexpressanewsetofgenes,
i.e.aregulonisactivated• cellssynthesisenewproteins
• constructaproteinstructureonthecellwall,
the“Compilus”,andothersurfaceproteins
• Compilusisrelatedtoanotherstructurefoundonbacteria-type4pili(T4P)
• DNAbindinganduptake:Gm+vespecies
CaptureofexogenousDNAbythetransformationpilus*(ComGCpilus).
BindingofdsDNAbytheDNAbindingproteinComEA.
RecognitionofthedsDNAbythenucleaseEndAandConversiontossDNA
TransportofthessDNAstrandbyComEC,drivenbytheATP-dependenttranslocaseComFA
• DNAbindinganduptake:Gm-vespecies
PilQsecretomechannelenablespilustocrossoutermembrane,binddsDNA(specific
sequences)andtransportitintoperiplasm.
BindingofdsDNAbytheDNAbindingprotein(ComE).
TransportoftheDNAstrandacrossinnermembranebyComA(ComFA?)
Endonucleaseprobablyexistsbutnotfoundyet
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HOMOLOGOUSRECOMBINATION
1. InternalisedssDNArecruits*RecAprotein
2. RecApolymerisesonssDNA
3. RecApromotesahomologysearchalongchromosomalDNA
• RecAproteinisrequiredforrecombination
andDNArepairinbacteria
4.Strandexchange
a.Homologous
b.Heterologous
5.Replication(restriction/modificationsystemcompetesforaccessofunmethylatedDNA)
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NATURALTRANSFORMATION-SUMMARY
• ComplexprocessthatrequiresdedicatedDNAuptakeandintegrationsystems
• Ancientprocess;butonlyafractionofbacterianowappearcapableofnaturaltransformation
• Involvedincreatinggeneticdiversity,repairingDNAandpotentiallyanutrientsource.
-geneticdiversityincludesadditionofnewgenes(e.g.AMR),deletionofgenesand
replacementofgeneticalleles.
• Ahighlyregulatedprocess
• Mechanismcandifferbetweenbacterialspecies
• Normallysystemis“off”
• Environmentalandcellularcuesrequiredforinduction
ARTIFICIALTRANSFORMATION
• Manybacterialspeciescanbetransformedartificially,e.g.E.coli,Salmonella,Staphylococcus• Usescirculardouble-strandedDNA
(naturaltransformationprefersdslinearDNA)
• Artificialtransformation:
-isrelativelyinefficient
-isakeytoolingenecloning(molecularbiology,syntheticbiology)
Electroporation:-electricfield(10-20kV/cm)changesmembranepermeabilityproperties
dsDNAthenenterscell bacterialmembranerepairrestoresnatural
permeability
Chemicaltransformation:
-CaCl2treatmentofcells(usuallyE.coli)at4°C-otherdivalentcationsusedinclude
Mg2+,Mn
2+,Rb
2+
-causesmembranepermeabilitychange-allowplasmidentry
1. Makebacteriacompetent,e.g.withCaCl2
AddDNAandincubateonicefor30min
-DNAcanbindtothebacterialsurface
2. Heatshockthebacteria/DNAmixture(42⁰C≈30sec)
-changesmembranepermeability
-allowsuptakeofDNA
3. Putonicefor30mins
-restoresnormalbacterialmembraneproperties
4. Addrichbrothtocellsandincubateat37⁰C≈1hour
-allowsexpressionofselectivemarkere.g.Amp
5. Plateonselection/screeningmedium
-Tovisualisetransformants
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ELECTROPORATION-replacesSteps1,23withHighvoltage
• Geneticbackgroundoftransformedbacteria
-Restrictionsystem–why?
• Preparationofartificiallycompetentbacteria
-Manipulationsthataltermembranepermeability
• QualityofthetransformingDNA
-Ionchelators,saltcontaminants,concentration,damagedDNA
• SizeofthetransformingDNA
-Largeplasmidsaretakenuplessefficiently
• Artificialtransformationisstillfairlyinefficient.
-e.g.108transformantspermgDNA;
-mostbacteriainasampleareNOTtransformed,e.g.1in1000aretransformed
Selection\screenisrequiredtoidentifytransformantsfromnon-transformants
• SELECTION
Mostbacteriawillnotbetransformed,soyoumustselectfortranformants
-usuallyanantibioticresistantgeneisencodeinaplasmidvector.
• SCREENING
TransformantswillcontaineithervectoraloneORvectorplusinsert
-howwillyoutellthedifference?
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CONJUGATION
• Contrastswithverticalgene
transfer(.i.e.inherited)
• Allowsbacteriatogain
geneticdiversity
(entirelynewtraitsrather
thanalteringexistingones)
• Threemechanisms:
• (Natural)Transformation
• Transduction
• Conjugation
• DEMONSTRATIONOFBACTERIALMATING
Used2strains,eachcontainingauxotrophicmutants
Q:canmutantassortmentoccurbyco-incubationofthesestrains?
Basicmatingstrategy:A+B-xA-B+
®A+B+,
(AandB=differentaminoacidrequirements)
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• Thereforeeither(a)exchangeofgeneticmaterial(mating)
or(b)cross-feedingwasoccurring
• GENERALCHARACTERISTICSOFCONJUGATION
DNAtransferfromonecelltoanotherbymeansofcell-to-cellcontact
OccursmostlyinGram–vebacteria
SomeGram+vespecies:(Streptomyces,Streptococcus,Clostridia)
Prevalencesuggestsadvantageoustothespecies
Maybemoreprevalentthanwasthoughtpreviously
Also:
Usuallytransfersplasmids,ratherthanchromosome
DNAtransferisunidirectional:donortorecipient(WilliamHayes,1953)
THEFFACTOR
• Strainsthatwerefoundtotransfergenesbyconjugation
weredesignatedaspossessinga‘Fertility’factor(F)
• Ffactor:
• ConferredabilitytodonateDNA
• Canbelostandregainedeasily
• StrainscarryingFaredonorsanddesignatedF+
• StrainslackingFarerecipientsanddesignatedF-
• Fisaconjugativeplasmidwhichencodesthemachineryforconjugation
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• FPlasmidcontainsgenesthatencodefor:
atype4secretionsystem,ananomachine,thatsynthesisesthepilus(Fpilus)
“surfaceexclusion”
stabilisationofmatingpairs
DNAtransfer
regulation
Fpilusissynthesisedbythedonorcell
bythetypeIVsecretionmachinery
Onceattachedtoarecipient,thecellscometogether.
DNAtransferthenoccurs.
DNAtransferdoesnotoccurthroughthepilus
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• WHICHCELLSCANBERECIPIENT?
Thehostrangeisdefinedbyseveralfactors:
-1Recognitionofrecipientbydonor
-2SuccessfulcompletionofDNAtransfer
-3Replicationoftransferredplasmid
Plasmid Conjugationhostrange1 Transferhostrange2 Replicationhost
range3
F Narrow(Gram-veenterics broad narrow
RP4 Broad(Gram-veandGram
+ve)
broad broad
• Hostrangeofsomeconjugalplasmidsisverybroad:
-Includestransferto:
-bacteria,yeast,plantcells,andmammaliancells
-However,plasmidreplicationisoftenlimited
RFACTORS:
Resistance(R)factor:
-Plasmidsthatencodemultipleantibioticresistances
-originallydescribedinShigellainthe1950s
-notnecessarilyconjugativeplasmids
-butmanyareconjugativeandcanrapidlyspreadbetweendiversebacterialspecies
HFRSTRAINS
• InsomeE.colistrains,theFplasmidcanbeintegratedintothechromosome
• Occursduetohomologousrecombinationbetweeninsertionalsequences(IS)(seeL37)on
plasmidandchromosome
• MultipleISsitespresentthroughoutbacterialgenomes
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• F-plasmidintegration:
-canstillinitiateconjugation
-canresultinbacterialchromosomemobilisation
(transferofchromosomalgenestotherecipientcell)
-initiatedatoriTsite
-rollingcirclereplication
• Transfermachinery:
-stilloperates;doesnotdistinguishbetweenFplasmidaloneorFplasmidintegrated
withinbacterialchromosome
• IntegratedF-plasmidandchromosomalgenesaretransferred
HFRCONJUGATION
• ConjugationandGenetransferbetweenHfrdonorandF-recipients
• justlikedescribedearlierwithFplasmid
• butwithbacterialchromosomesectionincluded
• recombinationbetweenchromosomalgenesmayoccurifhomologypresent
• theDNAstrandtransferredisverylongandoftenbreaks;theFplasmidwouldbethelastpiece
transferred,thereforerecipientsdonotbecomeF+
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• LineartransportofHfrDNA
-directional
-canusethistomapgeneorder
-basedontransferfrequencytorecipient
-‘TimeofEntryMapping’by‘InterruptedMating’experiments
• Genesproximalanddownstreamof‘originoftransfer’
-morelikelytobetransferred.
-transferofproximallocus:(“a”)occursmorefrequentlyrelativetodistalloci(“b,c,d,…”).
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• INTERRUPTEDMATINGEXPERIMENTS(SEXINABLENDER)
ISOLATIONOFF'
• Remember:Fplasmidcanbothintegrateandexcisefromthegenome(a)(b)
• ImproperexcisionofintegratedFcanoccur,resultinginF’(Fprime)
• F’usuallycontainschromosomalgenes
(viaillegitimaterecombination)(c)(d)
TheseF’plasmids:
-behavelikeF-plasmid(i.e.autonomouslyreplicating,conjugation-competent)
• canincorporatehostgenomicfragments(replacingsomeplasmidsequences)
-arestableplasmid,withintegrationfairlyrarebecauseofthealteredISsites.
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InF’xF-cross,F’remainsF’andF-becomesF’
-recipientbacteriumnowknownasamerodiploidorpartialdiploid(e)
-becauseofpresenceof2copiesofaparticulargene(chromosomalandF’)
• Hfrconjugation
-HfrstrainformedviaF+integrationwithinbacterialchromosome
-cantransferhostchromosome
-TransferoriginatesatoriTofinsertedFplasmid
-Transferofentirechromosomeisveryrare(requires100minutes)
-TheacceptorstrainremainsF-
-WasveryusefulinestablishingageneticmapofE.coligenome
(interruptedmating)
• F’strains
-arise,viaillegitimaterecombination,fromexcisionofintegratedFplasmid
-flankinghostsequencesareincludedintheresultingF’plasmid
-canreplicateandtransferDNAjustlikeF+
-TransferredDNAisaplasmidinrecipient,butcarriesaportionofthedonorgenome
-F’xF-=F’andF’
MB – Bacterial Genetics A.S.d.C.
TRANSPOSITION
• BarbaraMcClintock:
-colourvariationinmaize(Zeamays)1940s
-dogmawasthatgeneswerestable
-sheusedstainingandmicroscopytotrackchromosomes
-9thchromosomeregularlysufferedbreakagesatthesameposition
• Geneticbasis
• ‘Activator(Ac)’and‘Dissociator(Ds)’
• Dswasunstableregiononchromosome9
• Dscouldchangeposition
• DsbreakagerequiredasecondelementAc(alsomobile)
PhenotypesofkernelsthatledtoMcClintock’sdiscoveryofchromosomebreakageatDs.I:dominantinhibitoryalleleoftheCgene;C:fullanthocyaninpigmentationwhentogetherwith
thewild-typeBzalleleoftheBronzegene;bz:recessivealleleoftheBronzegene,bronzepigmentationwithC.ThechromosomeconstitutionofthekernelsshowninA–DisIBz/Cbz(neglectingendospermtriploidy).(A)ColorlessIBz/Cbzphenotype.(B)Randombreakageof
chromosome9resultsinlossoftheIalleletorevealfullypigmentedCBzsectors,followedbylossoftheBzalleletorevealthebronze-coloredCbzphenotype.(C)Chromosomebreakageata
DstransposonlocatedproximaltoboththeCandBzlociresultsinsimultaneouslossofboththe
IandBzalleles,givingonlythecolorlessandbronzephenotypes.Thecoloredrimsresultfrom
complementationbetweenthewild-typeCalleleinCbztissueandthewild-typeBzalleleintissuecontainingtheinhibitoryIalleleoftheCgene.(D)AlteredphenotypeproducedbychromosomebreakageatDsaftertranspositiontoanewsitejustproximaltotheCgeneatthedistalendofchromosome9.TheinitialchromosomebreakatDseliminatestheIallele,revealingpigmentedCBzsectors.Thecirclehighlightsatwinsectorarisingfromadicentricchromatid
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formedatthecleavagesiteandsubsequentrandombreakageofthedicentricduringcell
division,givingrisetoadjacentpatchesofCBzandCbztissue.(E)Phenotyperesultingfrom
chromosomebreakageattheDsjustproximaltotheCgeneinakernelhavingthegeneticconstitutionCDs/c,whereCisthedominantallele(pigmentedaleurone)andcistherecessiveallele(colorlessaleurone).TheC→cvariegationresultsfromchromosomebreakageatDsandsubsequentlossoftheCallele.(F)Phenotypeofanunstablemutationarisingbytranspositionof
DsintotheCgene,causingamutationtothecolorlesscallele.Thec→CvariegationiscausedbysomatictranspositionofDsoutofthegene,restoringthecoloredphenotypeoftheCallele.
TRANSPOSABLEELEMENTSANDTRANSPOSONS
TwotypesofelementscanbeinsertedintobacterialDNA:
• Insertionsequences(ISsequences)
-simplesttransposableelement
• Transposons
-simpleorcomposite
Mechanismoftransposition
-replicativeornon-replicative
InsertionofISsequencesandtransposonscanaltergeneexpression:
-activatenearbygenesinthegenome;
-causemutations;
-addnewgenestothegenome
INSERTIONSEQUENCES(IS)
• simplestMobileGeneticElement;alsotermedISelements;
• shortinlength:~750bp–2,000bp
• onlyencodeelementsnecessaryfortransposition
• invertedrepeatsandtransposase
• transposase
-enzymerequiredformobilisationandinsertion
• invertedrepeats(IR)
-10–50bp
-recognisedbytransposase
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TRANSPOSONS
• LargerthanISelements(severalkb)
• Morecomplex–theycarry‘passenger’DNA
Mechanismoftransposition:
• Transposition-themovementofatransposableelement
• Twostages
• Excision
• Insertion
• Twodifferentmethods
• Non-replicative/conservative(transposonremovedfromoldsite,e.g.Tn10)
• Replicative(newcopyoftransposableelementgenerated,e.g.Tn3)“copyandpaste”
(althoughthat’snotatermwidelyused)
MB – Bacterial Genetics A.S.d.C.
NONREPLICATIVETRANPOSITION
REPLICATIVETRANSPOSITION:
• Mobilegeneticelementsareeverywhere,theycarryimportantgenessuchasantibiotic
resistance;oftenfoundonplasmidsbutalsoonchromosomes.
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• Shigellaishighlyrelatedto,andevolvedfrom,E.coli• Acquiredalarge221kbvirulenceplasmid
• Largevirulenceplasmid
ISmaterialrepresents46%
oftheplasmid
26fulllengthISelements;
andextensivescarsfromancestralrearrangements
• Abilitytointegraterandomlyinalargegenomemakesthemgoodgenetictools
• Animportantuse-insertionalinactivation-disruptgenefunction
Question:abacterialgenomehas3000genes.
Whichgenesareessentialforaspecificprocess,
e.g.formationofbiofilm,growthinananimalhost?
Obviouslymanygenesareessentialforgrowthin“normal”labconditions,butaretherespecific
genesthatarerequiredforgrowthinvivo?
Constructapooloftransposonmutantsinthebacterium,suchthateverybacteriahasan
insertion,butatadifferentlocation.
3,000genes;maybe10,000insertions,>1pergene.
Canmakepoolsof>100,000mutants
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Resultswillindicatewhichgenesarenotpresentinoutputpool
–theseareimportantforsurvivalinthehost.
Advantages–onlyafewanimalsareneededforsuchanexperiment.Thinkhowmanyyouwould
needofeachgenehadtobemutatedandthemutanttestedinananimal!
• Abilitytointegraterandomlymakesthemgoodgenetictools
• Anotherimportantuse–canbeengineeredtocarrynewgenes
MB – Bacterial Genetics A.S.d.C.
PHAGETRANSDUCTION
BACTERIOPHAGE:
• Mostabundantlifeform*inbiosphere
-estimated**that10xmorephageexistthanbacteria;1030bacteria;10
31phage
• Virusesofbacteria
-mostbacteriaaresusceptibletophage
• Nomenclature:T4,l,P22etc
• Structure:
• NucleicAcid(DNA/RNA)
• Proteins–capsidhead,core,sheathetc
PHAGE-MEDIATEDTRANSFEROFGENETICMATERIAL
• 2forms:
-generalised
-specialised
• Discovery(1951):
Salmonellatyphimurium
-mixed2auxotrophicmutantstogether
-obtainedprototrophicstrain!
RepeatedwithaU-tubewithfilter:
-Noprototrophicformation
-Notconjugation
-Alteredporesizeoffilter:smallporesdidstopprototrophformation
Concludedgenetictransferwasmediatedbybacteriophage
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VIRULENCEPHAGELYTICCYCLE
Contractionanddeliveryisacturallybeenusedbybacteriatoproduceasystemtoinjecttoxins
fromoneanother.TypeVIsecretionsystem-->secreteacrossmembers,toexchangetoxins.It
resemblesthestructureofaphage(thesheath).
MB – Bacterial Genetics A.S.d.C.
BACTERIOPHAGELYFECYCLE
• >100phageparticlesreleasedfromabacterium
• aphage“lysate”isproducedinculture
• onagarplates,avisibleplaqueisseen
Twophagetypes:
• Virulentphages;
onlyundergolyticcyclee.g.,T4(previousslides)
alwayskillhostcell
• TemperatePhages;
Canundergolysogenicandlyticcycles
e.g.,λ
Canexistinhostforprolongedperiodsasprophage
Canintroducegeneticinformationtohost
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Lyogeniccanbeinducedintolytic
• Alysogenisabacteriumthatcontainsaphageintegratedintoitschromosome
• Aprophageisphageintegratedintoitschromosome
Inalysogen:
• Bacteriumgrowshappily
• Mostphagegenesrepressed(exceptionisthe“repressor”)
• Nophageparticlesconstructed
• Phagenucleicacidreplicatesaspartofthehostchromosome
• BacteriophageLambda(λ)integration:
• CircularisationofDNA
• attP(Phagesite)&attB(Bacterialgenomesite)
• Sitespecificrecombination
RECOMBINATION,STRANDTRANSER
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PHAGETRANSDUCTION:TWOTYPES
• DNAtransferfromdonorbacteriatorecipientbacteriaviabacteriophage
• Generalisedtransduction
-anypartofthebacterialchromosome
-occursduringlyticcycle
-randompackingofhostDNAintophageparticle
• Specialisedtransduction
-onlytransfersDNAadjacenttoprophageinsertion
-occursduringlysogenictolyticconversion
-inaccurateexcisionofprophagefromhostchromosome
GENERALISEDTRANSDUCTION
• Bacteriophage
-mustdigesthostDNA(nuclease)
-approx.1in104phageparticlescarryhostDNA
-knownas“transducingparticle”whenhostDNAispresent
• BacteriawithDonorDNArecombinedintochromosome:‘Transductant’
-DNAfragmentintegratesintochromosome
-homologousrecombinationusinghostRecAenzyme
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• GeneralisedTransductioncanprovidelinkageinformation
• About50additionalgenescanbecarriedinaphage
• “2mins”ofchromosome
• Fine-scalemapping
• ‘Cotransductionofgenes’
• dependsondistance
• genesclosetogetheraremorelikely
• tobepackagedtogether,henceahighercotransductionfrequency
Twogeneticmarkers(A,B)arepackagedontosinglegeneralisedtransducingfragment
• Typically,phenotypeofonegeneticmarkerisselected
-transductantsthatinheritedonemarkerwillbescreenedforsecondinheritedmarker
• Co-transductionfrequency:
-ratiooftransductantsthatco-inherited(A+andB
+)/bytotalnumberofA
+
transductants
LINKAGEMAPPINGOFBACTERIALGENESBYTRANSDUCTION
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SPECIALISEDTRANSDUCTION
WhencellsensesdangeritproducesalotofRecAtoprotectitsDNA.PhagecansenseRecA
anditexcicesffromthegenome,butwhenitexcicesitdoesnotdoitalwayscorrectly,
sometimesittakeswithittheGalorothergenes.
Repressionrelieved:
-Lyticcyclekicksoff
-Prophageexcision
Excisionofphagefromchromosome:
-viaasite-specificrecombinationprocess
-‘integrase’and‘excisionase’-1excisionper10
6iisincorrectandtakeswithitsomehostDNA
Resultis‘specialisedtransducingphage’
-canstillinfect
-mayhaveadefectivegenome
-mightneedawild-typehelpertoinfect
-carriesgenesadjacenttooriginalsiteofintegration
Onlyspecificportionsofbacterial
chromosomearetransduced
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SURVIVINGINTHEBACTERIALJUNGLE
Intheirnaturalenvironment,bacteriahavetocompetefor
spaceandnutrientstosurvive.Theydothisby:
• productionofantimicrobialcompounds(e.g.bacteriocins)
tokillotherspecies
• secretionofenzymestoaidutilisationofnutrientse.g.
bgalactosidasetometaboliselactose
• modificationofthephysicalenvironment
e.g.productionofpolymerstoaidattachmenttosurfaces
• usingmotilitymechanismstofindnewhabitat
• fratricide(bacteriakilltheirsiblings)
Howeverbacteriacanalsocooperate
• quorumsensing–theentirepopulationofaspeciesproduces
moleculesthatenablethepopulationtorespondina
coordinatedmanner(althoughcertainbacteriacan“cheat”)
• mutualism–productionofcompoundsintotheenvironmentthat
benefittheentirecommunity,e.g.siderophoresthatsequester
ironwhichcanthenbetakenupbybacteria
UptakeofDNAcanbegoodforacell!
• increasesgenepool
• increasesmetabolicdiversityandfunctionsthecellcancarryout
• antibioticresistance
• moregenes,themorecontrolacellcanhave
UptakeofDNAcanbebadforabacterium!
• themoreDNAinacell,themoreenergyneededtoreplicateit
• cellshavetoensurefaithfulreplication
• errorscanbecostly,leadingtocelldeath
• foreignDNAcanbeharmful
• mutantgenesmayreplacefunctioninggenes
• DNAmightinsertintofunctionalgenesoroperons
DefenseagainstinvadingDNA:R-M
Restrictionandmodification(R-M)ofDNA
1.Restrictionendonuclease(RE)–cleavesunmethylatedDNA
2.Methlytransferase(MTase)–methylatesthecellsDNAattherecognitionsiteoftheREto
preventrestriction
MB – Bacterial Genetics A.S.d.C.
Restriction-Modification(R-M)Systems(R-M)
Atleast3000differentrestrictionendonucleases(REs)exist,
withover300specificities
Manymore–1000s–probablyexist
FoundinBacteriaandArchaea
Almostallbacterialappeartoproduce
REsandMTases.
90%ofsequencedgenomescontainsatleastoneR-Msystem
Bacteriahaveevolvedotherwaystodefendthemselves:
• SwitchONandOFFphagereceptors
• Secretepolysaccharidesthatlimitaccesstoareceptor
• Expressmembraneproteinsthatinterferewithnucleicacidinjection
• Suicide(abortiveinfection)–cellsacrificesitselftopreventspreadofviralDNA
• Othermechanismscontinuetobediscovered
BUT–phagesarepersistent!
• theycanovercomesomeofthesemechanisms!
usedifferentreceptors;producedegradingenzymes;
mutatetheirproteinstoavoidtriggeringabortiveinfection
MB – Bacterial Genetics A.S.d.C.
PHAGETHERAPY
Discoveredover100yearsago
Phageasantimicrobialswereinvestigatedlongbeforeantibiotics
werediscovered
GeorgeEliava(1892-1937)–amicrobiologistfromGeorgia;
trainedatPasteurInstitute,PariswithFelixD'Herelle
(co-discoveredbacteriophage)
FoundedanInstituteinTbilisi,
focusingonphagetherapy.
However,asthegoldeneraofantibioticsdevelopedinthe1950-70s,
interestinphagetherapydeclined.
Pointstoconsider:
• Antibioticsarechemicals,notlifeforms;theydonotgrowandmutate.
Naturalsectioncanresultinalterationsto
phageaswellastothebacterialhost.
Potentially,bacteriacouldbecomeresistanttophage,
butphagecouldthenevolvetoinfectthis“resistant”strain,andsoon.
• Phagearehighlyspecifictocertainbacterialspecies,T4infectsE.coli,andwillnotkillofflargenumbersofourownmicrobiota.
Naturehasevolvedphagetokillaspecificstrainofbacteria.
Canwedesignphagetotargetspecificstrainsofpathogens?
3. Genomemetasequencingrevealsmany,manymorephage
intheenvironmentthanoriginallythought.
e.g.sequencingofeverythingintheoralcavityrevealsphagetranscripts
arephageactiveduringperiodontaldisease?
couldsuchcommunitiesbeasourceofnewphageforpotentialtherapies?
1990s–genomesequencingrevealed
manybacterialandarchaealspecieshadunusualrepetitivesequences
Short(24-40bp)repeatsequences;palindromic;invertedrepeatsattheend
Spacersare20-50bp;notrepetitive
Arrangedinclustersorarrays
ClusteredRegularlyInterspacedShortPalindromicRepeats
MB – Bacterial Genetics A.S.d.C.
3components:
DNArepeatsequences
Spacersregions,notallidentical
Associatedgenes–Casgenes-containnucleaseandhelicasedomains
1.SpacersequencesmatchDNAfromphagethatnormallyinfectthebacterium
e.g.spacersinStreptococcusthermophilusmatchphagethatinfectthisspecies
2.Bacterialstrainsthathaveaspacerderivedfromaphageareresistanttothatphage
3.TheCRISPRlocusistranscribed(RNAisproduced)
Hypothesis:CRISPRisanimmunitysystemofprokaryotes–preventsforeignDNAfrom
invading
CRISPR-Casisanadaptiveimmunitysystemfoundinbacteriaandarchaeatodefendform
invadinggeneticmaterial
Itisanadaptivesystem-allowsbacteriatoadapttonewphagesequences
Mechanismisthrough:
• captureofforeignDNAandstorageinarrays
• usingguideRNAstorecogniseinvadingDNA
• usingnucleasestocleaveinvadingDNA