Means of perrenation -sporulation-and-morphogenesis.pdf

Means of perrenation -sporulation-and-morphogenesis

1. Bacterial Endospore
By –Dr. Ekta Khare

2. Endospores
•Anumberofgram-positivebacteriacanformaspecialresistant,dormant
structurecalledanendospore.
•Anendosporeisadormant,tough,andnon-reproductivestructure.
•Thename"endospore"issuggestiveofasporeorseed-likeform,butitisnota
truespore(i.e.,notanoffspring).Itisastripped-down,dormantformtowhich
thebacteriumcanreduceitself.
•Endosporesdevelopwithinvegetativebacterialcellsofseveralgenera:Bacillus
andClostridium(rods),Sporosarcina(cocci),andothers.
•Thesestructuresareextraordinarilyresistanttoenvironmentalstressessuch
asheat,ultravioletradiation,gammaradiation,chemicaldisinfectants,and
desiccation.
•Becauseoftheirresistanceandthefactthatseveralspeciesofendospore-
formingbacteriaaredangerouspathogens,endosporesareofgreatpractical
importanceinfood,industrial,andmedicalmicrobiology.
•Therearemanyreportsofsporesremainingviableover10,000years,and
revivalofsporesmillionsofyearsoldhasbeenclaimed.Thereisonereportof
viablesporesofBacillusmarismortuiinsaltcrystalsapproximately250million
yearsold.

3. EndosporeStructure
•Anendosporehasfollowinglayers:
–Exosporium
–Sporecoat
–Outermembrane
–Cortex
–Sporecellwallorcorewallorgermcellwall
–Innermembrane
–Core

4. Exosporium:
•Itistheoutermostlayermadeupofproteinthatenclosessporecoat.
•Insomebacterialspore,exosporiumismadeupofpolysaccharideandlipid.
Sporecoat:
•Itisthickdoublelayeredcoveringthatenclosesoutermembrane.
•Sporecoatconsistsofsporespecificprotein,mainlycontainscysteineand
hydrophobicaminoacids.Duetopresenceoftheseaminoacids,sporeare
resistanttoadverseenvironmentalcondition.
Outermembrane:
•Theprecisefunctionoftheoutermembranethatliesunderthesporecoatsis
notclear,althoughthismembraneisanessentialstructureinsporeformation.
Cortex:
•Cortexmadeupoflooselyarrangedpeptidoglycanlayer.
•Innerlayer:comprisesabout20%ofpeptidoglycan,itistightlyarranged
•Outerlayer:itislooselyarranged,itcanbehydrolysedduringspore
germination.itcomprisesalalnine(55%),tetra-peptide(15%)andmuramic
lactum(30%).
Sporecellwall/corewall,germcellwall:
•Presentinsidethecortexandsurroundstheprotoplastorcore,madeup
ofpeptidoglycan.

5. Innermembrane:
•Theinnersporemembraneisastrongpermeabilitybarrierthatplaysamajorrolein
sporeresistancetomanychemicals,inparticularthosethatcoulddamagethespore
DNA
Core:
•Itistheinnermostpartofspore,alsoknownassporeprotoplast.
•Coreconsistsofcytoplasm,nucleoid,ribosomesandothercellularmaterials
surroundedbyinnerorcytoplasmicmembrane.
•Corecontains(10-25%)waterso,thecytoplasmisgellike.
•Itcontainshighamountofcalciumanddipicolinicacidascalciumdipicolinate(CaDPA)
(10-15%bydryweight).
•Corealsocontainshighpercentageofsmallacidsolubleprotein(SASP).
•SASPissynthesizedduringsporulationanditbindstoDNAincoreandprotectitfrom
potentialdamagecausedbyUVradiation,desiccationanddrying.
•Inaddition,SASPsalsoprovidesnutritionandenergyforsporegermination.
•CorealsocontainsomeDNArepairenzymes.DNAisrepairedduringgerminationand
outgrowthafterthecorehasbecomeactiveonceagain.
Insummary,endosporeheatresistanceprobablyisduetoseveralfactors:calcium-
dipicolinateandacid-solubleproteinstabilizationofDNA,protoplastdehydration,the
sporecoat,DNArepair,thegreaterstabilityofcellproteinsinbacteriaadaptedtogrowth
athightemperatures,andothers.

6. Sporogenesis/ sporulation
•Sporeformation,sporogenesisorsporulation,normallycommenceswhengrowth
ceasesduetolackofnutrients.
•Itisacomplexprocessandmaybedividedintosevenstages.
Stage0
•Normalconditionsofavegetativecell
StageI:Axialfilamentformationstage
•Inthisstagebacterialchromosomebecomethreadlikeknownasaxialfilament
•Axialfilamentsattachedtocytoplasmicmembranebymesosome
•Elongationofcelltakeplaces
•PHBAisthereservedfoodmaterialinBacillussppisutilizedinsporulation.
StageII:foresporeformation
•Asymmetriccelldivisionoccurs
•CellmembraneformsseptumnearoneendwhichenclosesasmallportionofDNA
formingforespore
StageIII:engulfmentofforespore
•Mothercellmembranegrowaroundtheforesporeengulfingit.
•Foresporenowhastwomembranelayer

7. Stage IV: synthesis of exosporium
•Chromosome of mother cell disintegrates
•Exosporiumsynthesis occurs
•Foresporestarts Forming primodialcortex between two
membrane.
•Dehydration of cell
Stage V: synthesis of dipicoloniccacid
•Production of SASPs and dipicolinicacid occurs
•Incorporation of calcium ions with dipicolonicacid occur
forming calcium dipicolonate
•Further dehydration of cytoplasm
•Formation of coat layer
Stage VI: maturation
•Maturation of endospore
Stage VII: release of endospore
•Lyticenzymes destroy the sporangium releasing the spore

9. Endospore germination
•Germinationistriggeredbyenvironmentalstimulithatarethentransducedintoa
seriesofinterrelateddegradationevents.Thisultimatelyresultsinthelossof
typicaldormantendosporeproperties.
•Thetransformationofdormantsporesintoactivevegetativecellsoccursinthree
stages:(1)activation,(2)germination,and(3)outgrowth.
Activation:
•Thereareanumberofdifferenttypesofagentsthattriggersporegermination,
andsomeofthese,suchasCaDPAandcationicsurfactantslikedodecylamine,and
hightemperature,probablyareimportantonlyinthelaboratory.
•However,innature,itislikelythatthepresenceofspecificnutrientsiswhat
triggerssporegermination.
•Theavailableevidenceisconsistentwithnutrientgerminantsbindingina
stereospecificmannertospore-specificproteincomplexes,termedgerminant
receptors(GRs),inthesporemembrane.
•Oncetriggered,thegerminationprocesscontinuesintheabsenceofnutrients,a
findingthatindicatesthatgerminationiscontrolledbythesequentialactivationof
preexistingenzymesanddoesnotrequireproteinsynthesis.

10. Germination:
•Aftergerminantreceptoractivation,theendospore’scoreisrehydratedandcations
fromtheendosporecore,includingthelargedepotofCaDPAcomplex,arereleased.
Evennow,littleisknownabouttheidentitiesandfunctionsoftheproteinsthat
mediatethesefluxes.
•CaDPAreleasecompletesstageIofgerminationandalsotriggersentryintostageII,
whencortexlyticenzymes(CLEs)degradethepeptidoglycancortex.
•AnothermajorprocessthatoccursduringIIstageofgerminationisthebreakdown
ofendospore-specificproteins(e.g.SASPs)andstructures(e.g.cortex).
•Themetabolitesreleasedduringthesedegradationprocessesarebelievedtoserve
asrawmaterialsforthedevelopingcell.
•Someendosporescanalsousereleasedaminoacidsasquorumsensingsignalsto
synchronizeentryintovegetativegrowth.
Outgrowth:
•Cellstaketheirusualrod-shapeandgetreadytodivide.
•SinceendosporesaredevoidofmRNA,outgrowthisdependentondenovo
transcriptionandtranslation.mRNAsynthesisbeginsinthefirstminutesof
germinationandprecedesproteinsynthesisbyseveralminutes.
•DNAsynthesisdoesnotstartuntil30minutesaftergerminationonsetwhen
chromosomereplicationbegins.

12. Questions
•Explainsporogenesisindetail.Whatarethe
eventsinvolveinendosporegermination?
•Explainsporogenesisgivingonlydiagram.
•Writeshortnoteonendosporegermination.
•Discussstructureofendospore.Whatarethe
componentresponsibleforendospore
tolerancetoharshenvironmentconditions.