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BIOL2P98D3Spring2014
PrinciplesofMicrobiology
Dr.CarolynnEPietrangeliPhD
Lecture5June30,2014
CHSC2P98PrinciplesofMicrobiology
SpringD3June16July182014
INSTRUCTOR: Dr.CarolynnE.Pietrangeli,PhD
CONTACT: email: cpietrangeli@brocku.ca
Office:MCF212
OFFICEHOURS: Monday/Friday12PM1PM
Andbyappointment email
request
FORMAT: Lectures,3hourstwiceweekly
Monday/Friday9AM12PM
AS216
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WheretoNow?
LetstryonsomeGenes
Seewhathappenswhentheymutateand
Transfer(donate?)them
OrderofBusiness
Thebacterialgenome
Thebacterialchromosomeandreplication
Plasmids Plasmids
Transposableelements
Mutation
Assayingmutation
Bacterialgenetransfer
Recombination
Transformation
Transduction
Assayingbacterialgenetransfer
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BeforeWeVisitLetsLearntheLanguage
Genome entirecomplementofgenes
presentinacell(orvirus),carryingallthe
heritabletraits
Chromosome principalgeneticelement
carryinggenesessentialforlifeprocesses,
includingproliferation
Additional genetic elements Additionalgeneticelements
Plasmids
Transposableelements
Originof
replication
lac operon
(lactose
degradation)
100/0
90
10
HfrH
P804
HfrC
t
Not1restriction
sites,inkbp
4,639,679bp
EscherichiacoliK12
80
70
30
20
KL14
Figure4.8
trp operon
(tryptophan
biosynthesis)
his operon
(histidine
biosynthesis)
60
50
40
Hfr44
2012 Pearson Education, Inc.
E.coli
chromosome
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Plasmids:Extrachromosomal ProkaryoticDNA
Replication
functions
mer
sul
str
cat
IS1
94.3/0kbp
SmallcircularorlineardsDNA
molecules;oftensupercoiled
tra
IS1
IS10
IS10
75kbp 25kbp
50kbp
Figure4.10
Figure 4.9
tet
oriT
Tn10
MostplasmidsarecirculardsDNA
Replicateindependentlyofthebacterialchromosome
Mayintegrateintothebacterialchromosome=episome
Severaltypesmaycoexistinasinglebacterium;mustbefromdifferent
Incompatibility(Inc)Groups
Fplasmids(conjugative)maybetransferredbetweenbacteria
Figure4.9
ExamplesofBacterialPlasmids
CN=Copynumber
http://www.gs.washington.edu/academics/courses/manoil/41109/lecture/lecFeb23.pdf
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ReplicationoftheBacterialChromosome
ReplicationoftheBacterialChromosome
Theta()
replication
http://www.youtube.com/watch?v=HBwyNrkYnp0
1.DNAreplicationinprokaryotes
http://www.youtube.com/watch?v=w7TjoiubgF4
2. Claymation DNAreplication
http://www.youtube.com/watch?v=qSmTtk54JAs
3.DNAreplicationinEcoli
replication
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PlasmidReplicationStrategies
Theta()replication
Rolling circle replication Rollingcirclereplication
http://www.youtube.com/watch?v=YydvqhNOcVA
RollingCircleReplicationofDNA
TransposableElements
Jumpinggenes
DNAsegmentsthatmovefromonesitetoanotheron
thesameoradifferentDNAmolecule
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Conservative
Replicative
TypesofTransposableElements(TEs)
Insertionsequences
Composite transposons (Tn) Compositetransposons (Tn)
Replicative transposons:
PhageMu viral
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InsertionSequences
ThePriceofAdmission
PropertiesofInsertionSequences(Is)
Short:7002500bp
Genesrestrictedtotransposition:
transposase + regulatory sequences transposase +regulatorysequences
IS911:1250bp
orfB isTransposase gene
Transposase =enzymerequiredtoachievetransposition
IScarryONLYthegenesrequiredfortransposition
Compositetransposons (Tn)carry:
Insertionsequences
Additionalgenes antibioticresistance
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ReplicativeTransposon PhageMu
[MutatorPhage]
Randomintegrationinto
b t i l h bacterialchromosome
Insertionproduces
mutationinhostDNA
Replicationby
transposition
i h transposon acquireshost
DNAsequences
MorganGJetalJ.Mol.Biol.(2002)317,337359
FunctionsofTransposableElements(TE)/
MobileGeneticElements(MGE)
Generatemutations
IncreaseordecreasetheamountofDNAinthe
genome
Promotegenomerearrangement
Regulategeneexpression
Induce chromosome breakage and rearrangement Inducechromosomebreakageandrearrangement
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TheMolecularToolkit:HowWeUseTransposons
Geneticmapping TEinsertions
Behave as point mutations mostly producing null alleles Behaveaspointmutations,mostlyproducingnullalleles
Areusefulindeletionmapping determiningthegene
order whennearbutnotwithinageneofinterest
Candeterminethelocationofgeneswithinoroutsideof
operons (polarityofinsertionintooperons)
Mutationtransferselectedforbyantibioticresistance y
Tracklossoftransposon bylossofantibioticresistance
WheretoNow?...
BacterialGenomePlasticity Mutation
Definingmutations
Macrolesions Macrolesions
Microlesions
Natureornurture howdomutationsoccur?
Spontaneousmutations
Inducedmutations
l f l d Geneticanalysisofmutants selectionand
screening
Howdoweexploitbacterialmutations?
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TheGroundRules
Mutationsarestochasticevents
Onceamutation occurs,unlessarevertant
b l h f d subsequentlyoccurs,theprogeny ofamutated
cellwillcarrytheparentmutation
Threelevelsofevaluationofmutations
DNAbp alteration
Aminoacidcodon change g
Functionalprotein/tRNA/rRNA phenotypicchange
BasicsofMutationandMutants
Wildtypebacterialstrain strainisolatedfromnature=
parentalstrain
Mutation Heritable changeinDNAsequencethatcan
lead to a change in phenotype leadtoachangeinphenotype
Mutantbacterialstrain strainthatdiffersfromparental
straininanyportionofthegenomenucleotidesequence
Revertant bacterialstrain straininwhichthe
parental/wildtypephenotypeisrestored
WildtypeMutant
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DefiningMutationsandMutants
Perspective:DetectionofMutantClones
Selectablemutations Geneticresearchtools
Mutantacquiresgrowthadvantageunder
controlledconditions
Detectedbycontrolledassay
Nonselectable mutations
ChangesinDNAfromparentthatprovideneither
anadvantageordisadvantagetotheorganism
Detectedbyscreening
Macrolesional Mutations
Position with respect to centromere Positionwithrespecttocentromere
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DNA
MUTATION
NormalDNA
replication
Microlesional Mutations
DNA
mRNA
Asparagine
codon
Stop
codon
Tyrosine
codon
Tyrosine
codon
Transcription
Figure10.3
Protein
Missense
mutation
Nonsense
mutation
Silent
mutation
Wildtype
Faulty
protein
Incomplete
protein
Normal
protein
Normal
protein
Translation
2012 Pearson Education, Inc.
TypesofForwardMutations
Forwardmutation:WildtypeMutant
ProteinFrameofReference
Type of Mutation DNA Level Protein Level TypeofMutation DNALevel ProteinLevel
Silent Change innucleotidecoding
sequence
Nochangeinaminoacid
(aa)
Missense Singlebasesubstitution Exchange ofaa
Nonsense Singlebasesubstitutionormore
resultsinSTOP codon (CAG
UAG)
Chaintermination
Frameshift Insertion OR deletion of 1 2 bp Mutant or non functional Frameshift InsertionORdeletionof12bp
into/out ofgenecodingregion
Readingframeshift
Mutant ornonfunctional
protein
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Stopcodons
DNA:
TAG/TAA/TGA
RNA:
UAG/UAA/UGA
SubstitutionPointMutation:
THECATISINTHEBAG
THEHATISINTHEBAG
Howwouldyouclassifythismutation?
UAG/UAA/UGA
UHOHSTOP
Noproblem!
Sameaa;same
protein
Wrongprotein
Wrongfunction
CODON noprotein
Missensebutclose!
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Frameshift Mutations
CausedbyIndels (InsertionsandDeletions)
Reading
frame
mRNA DNA
Normal
Codons
Insertion
Transcription
offoflight
greenstrands
THE BIG CAT ATE THE FAT RAT Wild type
protein
Deletion
2012 Pearson Education, Inc.
THEBIGCATATETHEFATRAT
THEBIGCATTETHEFATRAT.
Wildtype
Mutant
ReversionofMutation
Samesitereversion
Secondsitereversion/Suppressormutation
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MutationOrigins
Spontaneous arisingwithoutexernal
intervention
Induced resultingfromexternalintervention
Exposuretotoxins,radiation
Laboratorytooltoidentifygeneticallyengineered
organisms
Sitedirectedmutagenesis
InducedandConditionalMutations
Inducedmutations Mutagen externalpressurethatcausesaheritable
alterationinDNA
Chemicals
Radiation ioninzing (Xray)ornonionizing(UV)
Viralincorporation
Conditionalmutation expressedonlyunderfixedenvironmentalconditions
Permissivevs.Restrictiveconditions
Biochemicalmutants Auxotrophs
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MutationRates
DNAreplicationerrors:10
6
to10
7
/kb
DNA virus error rates: 10
3
to10
4
/kb DNAviruserrorrates:10 to10 /kb
RNAgenomemutationrateis1000foldhigher
thanDNAgenomemutationrate
DNARepair
Directreversal basecanbecorrectedwithoutreferenceto
complementarystrand
Singlestrandrepair utilizescomplementarystrand
Doublestrandrepair highlyerrorprone
SOSregulatoryrepair largescaledamage;highlyerrorprone;
responsibleforacquisitionofantibioticresistanceinsomebacteria
Clancy,S.(2008)DNAdamage&repair:mechanismsformaintainingDNAintegrity.NatureEducation1
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InducedandConditionalMutagenesis
Inducedmutations Mutagen externalpressure
thatcausesaheritablealterationinDNA
Chemicals
Radiation ioninzing (Xray)ornonionizing(UV)
Viralincorporation
Conditionalmutation expressedonlyunderfixed
environmentalconditions
Permissivevs.Restrictiveconditions
Biochemicalmutants Auxotrophs
ExamplesofChemicalMutagens
Nucleotidebaseanalogs:resemblenucleotides
Alkylating agents e.g.nitrosoguanidine
Hydroxylating agents
Intercalatingagents induceframeshift
mutations e.g.acridines
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Prescott
Table14.1
DoMutationsOccurRANDOMLYorin
RESPONSEtoEnvironmentalPressure?
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UtilizingVirusestoDetectBACTERIAL Mutants
Bacteriophage (Phage) virusthatinfectsbacteria
Lytic phage T4 bacteria lysed following viral Lytic phage T4 bacterialysed followingviral
replication
Temperatephage Phage viralgenome
integratesintohostDNAandreplicateswithit
Prophage phagegenomeinsertedand
integratedintothecircularbacterialDNA
chromosome
Replication
Lytic cycle
Lysogenic cycle
HowdoWeDetectMutantsina
BacterialPopulation?
Useaselectablemarker antibioticresistance;phage
resistance;nutritionalalteration auxotroph
Screen forchangesincolonymorphology
Performreplicaplating
T1phage
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NatureorNurture:HowdoMutationsOccur?
Spontaneousmutation
TheLuriaandDelbruckFluctuationTest1943
NobelPrize1969
Observation:
PhageT1killsmostbacteria(Ton
s
)
Rarebacteriasurvive(Ton
r
)
PossibleExplanationsforTon
r
phenotype
Randommutation:Ton
s
Ton
r
Inducedadaptation:Mutationinduced byphysiologic
responsetophage
ReplicaPlating
E.coliTon
r
A t h Auxotroph
Temperature
sensisitive
Griffthsetal.,Fig.1522,7thed.
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Master plate; growth
DetectingNutritionalAuxotrophs
throughReplicaPlating
Masterplate;growth
oncompletemedium
Velveteen;
sterilized
Plastic
hoop
Wooden
block
Velveteen;
withimprint
ofall
colonies
Completemedium Allcoloniesgrow
Minimalmedium Mutantsdonotgrow
Pressplateonto
velveteen
Transferimprint
ofcoloniesto
freshmedia
Incubate
Figure10.2 2012 Pearson Education, Inc.
Auxotroph bacteriathatcarrya
mutationresultingininabilityto
synthesizeanessentialcompound
TheLuriaandDelbruckFluctuationTest
System:growbacteriaoverseveralgenerationsin
presenceofphage
Readout:numberofTon
r
bacterialcolonies
Hypothesistesting:
Sourceofmutation Expectedresult
NumberofTon
r
colonies
Spontaneous Highly variable
Mutation occursbeforecontactwith
phage
Physiologic
Mutationis inducedbycontactwith
phage
Uniform
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20x0.2mL
1x10mL
Early
culture
Late
cultures
FluctuationTest
LargeEarlyCultureResults
FluctuationTest
SmallLateCultureResults
Ifmutation
occurredin
RESPONSEto
stimulus,the
variancewould
be
verylow
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MutagenesisatWorkforUs:
TheAmesTest
InterpretingtheResultoftheAmesTest
Figure10.8
2012 Pearson Education, Inc.
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Mutation:IfitsWRONG,whydoit?
Mutationgeneratesdiversity
Mutation is a major means of achieving Mutationisamajormeansofachieving
Laboratories
Schedule:AsperpostingonSakai
LaboratoryDemonstrators
ChristeneCarpenterClelandMCF210
Extension5788ccarpenterclela@brocku.ca
MarkLukewich MCF213
Extension3398mlukewich@brocku.ca
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Evaluation
Laboratory 35% SeeLaboratoryManual
MidtermExamination 20% FridayJuly49:00AM10:30AM
Discussion Forum will follow DiscussionForumwillfollow
FinalExamination 20% 2hourduration
DiscussionForum 25% 5exercises/5markseach
SeeSakaiforInstructions
20%content
5%participation p p
ThereareNOmakeuporsupplemental examinationsinBIOL2P98
ThisappliestoLectureExaminations
(MidtermANDFinalExamination)ANDtothe
LaboratoryExamination
BIOL2P98SPLaboratorySchedule
LaboratorySchedule
Weekof LaboratoryExerciseTitle
Jun 16 Lab 1 Lab Safety Techniques & Enumeration
LabsareinIH309.
Jun16 Lab1 LabSafety,Techniques&Enumeration
Jun23 Lab2 BacterialGrowth&Identification
Jun30 CanadaDayweek NOLABS Assignment2onSakai
Jul07 Lab3 TheControlofMicrobialGrowth
Jul14 LabExam Basedonalllabmaterial
Labsections IH309
Section5 Monday 2:00pm 5:00pm
Section3 Tuesday 10:00am 1:00pm
Section1 Tuesday 2:00pm 5:00pm
Section2 Wednesday 2:00pm 5:00pm
ReadallAppendices&Lab1onSakai
CreateaflowchartforLab1
Bringalabcoat&safetyglasses/goggles,Lab1,
flowchartandPhotographicAtlas
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DiscussionForums
OccureachFridayduringlastlecturehour(11AM12PM)
Eachweek,weevaluateacaserelevanttolecture/laboratory
content in Microbiology in assigned Small Groups contentinMicrobiologyinassignedSmallGroups
Eachcasewillbeaccompaniedbysetsofquestionstobediscussed
andworkedonbyeachGroup
EachGroupsubmitsaSINGLEelectronicReport(nopaper
submission)respondingtothecasequestionssetforthatGroup
ELECTRONICReport(nopapersubmission)dueby5PMonthe
T d f th f ll i k ( C O tli ) Tuesdayofthefollowingweek(seeCourseOutline)
TheGroupwillreportthespecificcontributionofeachmember
EachmemberoftheGroupearnstheSAMEGRADEfortheReport
DiscussionForumInstructionspostedonSakai includesGroup
listings
DiscussionForumParticipation
20%ofthetotalgradeearnedforthe
Discussion Forums (5% of the final grade in DiscussionForums(5%ofthefinalgradein
BIOL2P98)isderivedfromparticipationin
DiscussionForumexercises
Attendancewillberecordedbysigninsheet
atthebeginningofeachDiscussionForum
Di i G ill b k d t d ib DiscussionGroupswillbeaskedtodescribe
thespecificcontributionofeachmemberto
eachGroupReport
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Resources
RequiredTextbook
BrockBiologyofMicroorganisms.FourteenthEdition.MadiganMT,Martinko
JM,BenderKS,BuckleyDH,StahlDA.PearsonBoston2012
Providesabasicreviewofconceptstobediscussedduringthecourse
DoesNOTsubstituteforcourselecturesorDiscussionForumcontent
Textbookchapterswillnotbeassigned;studentswillmatchthelecture
contenttorelevantchaptersinthetextbook
TextbookcontentwillbesupplementedbyreadingsmadeavailableonSakai.
Postingsofadditionalreadingswillbeannouncedduringlecturesandon
Sakai
RecommendedTextbookforLaboratory
APhotographicAtlasfortheMicrobiologyLaboratory.Leboffe MJ,PierceBE
4thEdition.MortonPublishing2011
GeneralBackgroundMicrobiology
http://www.mhhe.com/biosci/cellmicro/talaro/links.mhtml
AcademicIntegrity
AReflectionofPersonalIntegrity
Manythingsmaybestolenfromusbyother
peopleoverthecourseofourlives
Integrityisonethingwecanstealonlyfrom
ourselves
Integrity
Respecting it Respecting yourself Respectingit=Respectingyourself
Preserveit
Beproudofupholdingit