MB – Bacterial Genetics A.S.d.C. · MB – Bacterial Genetics A.S.d.C. Bacteria have chromosomes • In bacteria, observationalapproaches were not possible (eye-colour, wing shape,

MB – Bacterial Genetics A.S.d.C.

BACTERIALGENETICSAntibioticresistancedevelopsthroughnaturalselection:

Antibiotictargets:

Mechanismofresistance:


HorizontalGeneTransfer


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


• 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.


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


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


HOMOLOGOUSRECOMBINATION

1. InternalisedssDNArecruits*RecAprotein

2. RecApolymerisesonssDNA

3. RecApromotesahomologysearchalongchromosomalDNA

• RecAproteinisrequiredforrecombination

andDNArepairinbacteria

4.Strandexchange

a.Homologous

b.Heterologous

5.Replication(restriction/modificationsystemcompetesforaccessofunmethylatedDNA)


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


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?


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)


• 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


• FPlasmidcontainsgenesthatencodefor:

atype4secretionsystem,ananomachine,thatsynthesisesthepilus(Fpilus)

“surfaceexclusion”

stabilisationofmatingpairs

DNAtransfer

regulation

Fpilusissynthesisedbythedonorcell

bythetypeIVsecretionmachinery

Onceattachedtoarecipient,thecellscometogether.

DNAtransferthenoccurs.

DNAtransferdoesnotoccurthroughthepilus



• 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


• F-plasmidintegration:

-canstillinitiateconjugation

-canresultinbacterialchromosomemobilisation

(transferofchromosomalgenestotherecipientcell)

-initiatedatoriTsite

-rollingcirclereplication

• Transfermachinery:

-stilloperates;doesnotdistinguishbetweenFplasmidaloneorFplasmidintegrated

withinbacterialchromosome

• IntegratedF-plasmidandchromosomalgenesaretransferred

HFRCONJUGATION

• ConjugationandGenetransferbetweenHfrdonorandF-recipients

• justlikedescribedearlierwithFplasmid

• butwithbacterialchromosomesectionincluded

• recombinationbetweenchromosomalgenesmayoccurifhomologypresent

• theDNAstrandtransferredisverylongandoftenbreaks;theFplasmidwouldbethelastpiece

transferred,thereforerecipientsdonotbecomeF+


• LineartransportofHfrDNA

-directional

-canusethistomapgeneorder

-basedontransferfrequencytorecipient

-‘TimeofEntryMapping’by‘InterruptedMating’experiments

• Genesproximalanddownstreamof‘originoftransfer’

-morelikelytobetransferred.

-transferofproximallocus:(“a”)occursmorefrequentlyrelativetodistalloci(“b,c,d,…”).


• 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.


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’


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


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



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)


NONREPLICATIVETRANPOSITION

REPLICATIVETRANSPOSITION:

• Mobilegeneticelementsareeverywhere,theycarryimportantgenessuchasantibiotic

resistance;oftenfoundonplasmidsbutalsoonchromosomes.


• 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


Resultswillindicatewhichgenesarenotpresentinoutputpool

–theseareimportantforsurvivalinthehost.

Advantages–onlyafewanimalsareneededforsuchanexperiment.Thinkhowmanyyouwould

needofeachgenehadtobemutatedandthemutanttestedinananimal!

• Abilitytointegraterandomlymakesthemgoodgenetictools

• Anotherimportantuse–canbeengineeredtocarrynewgenes


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


VIRULENCEPHAGELYTICCYCLE

Contractionanddeliveryisacturallybeenusedbybacteriatoproduceasystemtoinjecttoxins

fromoneanother.TypeVIsecretionsystem-->secreteacrossmembers,toexchangetoxins.It

resemblesthestructureofaphage(thesheath).


BACTERIOPHAGELYFECYCLE

• >100phageparticlesreleasedfromabacterium

• aphage“lysate”isproducedinculture

• onagarplates,avisibleplaqueisseen

Twophagetypes:

• Virulentphages;

onlyundergolyticcyclee.g.,T4(previousslides)

alwayskillhostcell

• TemperatePhages;

Canundergolysogenicandlyticcycles

e.g.,λ

Canexistinhostforprolongedperiodsasprophage

Canintroducegeneticinformationtohost


Lyogeniccanbeinducedintolytic

• Alysogenisabacteriumthatcontainsaphageintegratedintoitschromosome

• Aprophageisphageintegratedintoitschromosome

Inalysogen:

• Bacteriumgrowshappily

• Mostphagegenesrepressed(exceptionisthe“repressor”)

• Nophageparticlesconstructed

• Phagenucleicacidreplicatesaspartofthehostchromosome

• BacteriophageLambda(λ)integration:

• CircularisationofDNA

• attP(Phagesite)&attB(Bacterialgenomesite)

• Sitespecificrecombination

RECOMBINATION,STRANDTRANSER


PHAGETRANSDUCTION:TWOTYPES

• DNAtransferfromdonorbacteriatorecipientbacteriaviabacteriophage

• Generalisedtransduction

-anypartofthebacterialchromosome

-occursduringlyticcycle

-randompackingofhostDNAintophageparticle

• Specialisedtransduction

-onlytransfersDNAadjacenttoprophageinsertion

-occursduringlysogenictolyticconversion

-inaccurateexcisionofprophagefromhostchromosome

GENERALISEDTRANSDUCTION

• Bacteriophage

-mustdigesthostDNA(nuclease)

-approx.1in104phageparticlescarryhostDNA

-knownas“transducingparticle”whenhostDNAispresent

• BacteriawithDonorDNArecombinedintochromosome:‘Transductant’

-DNAfragmentintegratesintochromosome

-homologousrecombinationusinghostRecAenzyme


• 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




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


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


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


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


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

Documents

MB – Bacterial Genetics A.S.d.C. · MB – Bacterial Genetics A.S.d.C. Bacteria have chromosomes • In bacteria, observationalapproaches were not possible (eye-colour, wing shape,