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BIOL2P98D3Spring2014
PrinciplesofMicrobiology
Dr.CarolynnEPietrangeliPhD
Lecture6July7,2014
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
7/6/2014
2
FinalExaminationBIOL2P98
Date:Saturday,July19
Time:20:00(8:00PM)22:00(10:00PM)
Location:BobDavisGymnasium Walker
Complex
MultipleChoice
/ Cumulative 2/3fromMidtermtoEndof
Course
DiscussionForum#5July18
AnoralQuestionandAnswerSession
EachGroupwillreceiveReviewQuestions
basedontheentireSemesterscontentby
WednesdayJuly16
EachGroupwillconferontheForumtoarrive
atanswerstotheReviewQuestions
Each member of each Group will have an EachmemberofeachGroupwillhavean
opportunitytorespond
MembersoftheGroupwillearnthesame
grade
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Mutation ASummary
Stopcodons
DNA:
TAG/TAA/TGA
RNA:
UAG/UAA/UGA
SubstitutionPointMutation:
THECATISINTHEBAG
THEHATISINTHEBAG
Howwouldyouclassifythismutation?
UAG/UAA/UGA
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AssayingforMutations
Selectablemutation Drugresistance
C f l d t t t t i d Confersclearadvantageonmutantstrainunder
definedenvironmentalconditions
AssayedbySELECTION
Nonselectablemutation Colour change
withgrowthonagar(nonutritional
t) component)
Confersnoadvantageordisadvantagetosurvival
AssayedbySCREENING
Agar:Kan
Colonies:WT
Agar:Kan+
Colonies:WT
Agar:Kan+
Colonies:
Mutant
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ReversionMutations
AgarPlatingResult
BacterialColonyColour
WildType(WT) Blue
Mutant White
Revertant Blue
Possible
mechanism SameAlternate
it it site site
TrueSuppressor
reversionmutation
DoMutationsOccurRANDOMLYorin
RESPONSEtoEnvironmentalPressure?
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UtilizingVirusesI:
ToDetectBACTERIAL 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
Bacterial
colonies
Virallawn
NOTE
CORRECTION
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ReplicaPlating
E.coliTon
r
A t h Auxotroph
Temperature
sensisitive
Griffthsetal.,Fig.1522,7thed.
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
Auxotroph bacteriathatcarrya
mutationresultingininabilityto
synthesizeanessentialcompound
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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
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
Experiment1
Sample1
SmallLateCultureResults
Culture
12345
Experiment1
IfmutationoccurredinRESPONSEtophage,
thevariancewouldbeverylow
Sample2
Culture
12345
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MutagenesisatWorkforUs:
TheAmesTest
Testingcompoundsin
drugdevelopment
Doesmutagenesis=carcinogensis?
InterpretingtheResultoftheAmesTest
Figure10.6
2012 Pearson Education, Inc.
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Mutation:IfitsWRONG,whydoit?
Mutationgeneratesdiversity
Mutationisamajormeansofachieving
SoHowdoBacteriaTRANSFERGeneticMaterial?
Conjugation physicalcontactbetweenlive
bacteriaresultinginexchangeofgenetic
material
Transformation bacterialuptakeofDNA p
fragmentsandincorporationintothegenome
Transduction transferofgeneticmaterial
fromonebacteriumtoanotherbya
bacteriophage
Generalized
Specialized
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DonorDNA
Recipient
Nick
SSBprotein
Endonuclease
nicksDNA
Bindingof
SSBprotein
Recombination Physical
exchangeofDNAbetween
Genetic
Recombination
Recipient
DNA
RecA
protein
Strand
invasion
Development
ofcrossstrand
exchange
geneticelements
Homologousrecombination
Physicalexchangeof
homologousDNAfromtwo
differentsources
Crossing
over
Figure10.11
Patches Splices
Resolution
atsites
Resolution
atsites
2012 Pearson Education, Inc.
SSB=singlestrandbindingprotein
RecA =proteinforDNAmaintenanceandrepair
(RAD51inhumans)
DNAfrom
Trp

cells
UsingSelectiveMediatoDetect
RareGeneticRecombinants
Figure10.12
Agarlacking
tryptophan
Agarlacking
tryptophan
Trp

cells Trp

cells
Nogrowth
Recombinants
formcolonies
2012 Pearson Education, Inc.
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2StrainsofPneumococcus:IIIS(Smooth)Strain/IIR(Rough)Strain
Transformation
Griffith1928
Live
Scells
Heatkilled
Scells
Live
Rcells
LiveRcells
heatkilled
Scells
Live
Scells
Figure10.12 2012 Pearson Education, Inc.
Transformingprinciple:elementthatchangesIIRStrain(nonlethal)
toIIISStrain(lethal)
Transformation:GenetictransferprocessbywhichDNAis
incorporatedintoarecipientcellandbringsaboutgeneticchange
WhatIS theTransformingPrinciple?
ProteinorDeoxyribonucleicAcid?
Protein
DNA
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TheTransformingPrincipleisDNA
Avery,MacLeodandMcCarty1944
HersheyandChase1952
I.AveryMacLeodandMcCarty1944
II.Hershey
andChase
1952
II.Hershey
andChase
1952

32
Plabeled
DNAenters
hostcellsand
directssynthesisof
newphage
Bacterial
chromosome
TransformingDNA
fromdonorcell
DNAbindingprotein
Competencespecific,singlestrand
DNAbindingprotein
Recipientcell
Nuclease
BindingDNA
Transformation
Transfection =
Freenucleotides
Nuclease
RecAprotein
UptakeofssDNA
Transfection =
Transformation
withaspecial
twist:
VIRAL DNAis
Figure10.13
Transformed
recipientcell
RecAmediated
homologousrecombination
2012 Pearson Education, Inc.
the
transforming
agent
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Transformation ReadyorNot?
Transformationcompetent:Cellscapableof
t ki DNA d b i t f d takingupDNAandbeingtransformed
Naturalcompetence
Inducedcompetence/Artificialcompetence
Heatshockaftercoldincubationwithdivalent
cations (calcium chloride) cations (calciumchloride)
Electroporation
UtilizingVirusesII:
ToTransferBACTERIALGeneticMaterial
Bacteriophage (Phage) virusthatinfectsbacteria
Lytic (virulent) phage T4 bacteria lysed Lytic (virulent)phage T4 bacterialysed
followingviralreplication
Temperatephage Phage viralgenome
integratesintohostDNAandreplicateswithit
Prophage phagegenomeinsertedand
integratedintothecircularbacterialDNA
chromosome
Replication
Lytic cycle
Lysogenic cycle
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Bacteriophage Lysogenic Cyclevs.Lytic Cycle
http://www.desktopclass.com/education/fafsc/virusesfscbiologychapter5part3.html
Phage
Donor
PhageDNA
HostDNA
Lyticcycle
Normallytic
events
Transduction
Generalizedtransduction
Lowefficiency
Transducing
particle(contains
donorcellDNA)
Donor
cell
Normal
phage
Transduced
recipientcell
Recipient
cell
Transduction
Recipientinfected
bytransducing
particle
Homologous
recombination
Figure10.14
2012 Pearson Education, Inc.
TransferofDNAfromonecelltoanotherbyabacteriophage
Generalizedtransduction anyportionofhostDNA
Specializedtransduction specificregionofhostDNA
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Normalevent: Rareevent:
Galactose
genesof
hostDNA
Host
chromosome
Lysogenized
cell
PhageDNA
Induction
SpecializedTransduction
Startingpoint:Lysogenic phage
Viral DNA excision host genes
PhageDNA
circularizes
and detaches
fromhostDNA
A portion of
hostDNA is
exchangedfor
phageDNA
Detached
DNA
replicates
ViralDNAexcision hostgenes
detachalongwithviralgenes
Transducing efficiency high
Temperatephageonly
Figure10.15
Normal
phage
Defectivephage
thatcantransduce
galactosegenes
Phagereplication
iscompleted.
Cell lyses
2012 Pearson Education, Inc.
Conjugation
RecipientCell
MatingPairattached
viapilus
DonorCell
F+
F
Plasmidencoded mechanism of genetic
Figure10.17 2012 Pearson Education, Inc.
Plasmid encodedmechanismofgenetic
transferthatrequirescellcellcontact
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Tn1000
IS3
IS3
tra
region
99.2kbp/0
Requirementsfor
Conjugation
F(fertility)plasmid
Sexpilus
DNAreplicationby
rollingcircle
oriV =originofreplication
oriT = origin of transfer
Fplasmid
F+=nonintegrated
IS2
75kbp 25kbp
oriT =originoftransfer
IS=Insertionsequence
Tn =Transposon
tra =transfer
Figure10.16
oriV
oriT
50kbp
2012 Pearson Education, Inc.
Bacterial
chromosome
F plasmid
Pilus
F

cell
(donor)
F

cell
(recipient)
Pilusretracts
Cell pairsstabilized.
Fplasmid nicked inonestrand
Retainedstrand
Donor
Conjugation
F+=cell inwhichtheF
plasmidisnotintegrated
into the host chromosome
Transferof onestrand fromF

cell to F

cell.
Fplasmid simultaneouslyreplicatedinF

cell
Synthesisof complementary strand
beginsinrecipient cell
Primer
Cell walls
DNA polymerase
DNA polymerase
Unwindingprotein
(Tral)
Plasmidencoded
membraneproteins
Specific outer
membraneprotein
ofrecipient
Donated strand
intothehostchromosome
Pilus forms
Pilus retracts
Matingpairstabilizes
Fplasmid:nickinonestrand
Transferof1FplasmidstrandintoF
ReplicationofFplasmidinF+cell
Complementary strand synthesis in F
Figure10.18
F

cell F

cell
Completionof DNAtransfer
and synthesis. Cellsseparate
Primer
Recipient
2012 Pearson Education, Inc.
ComplementarystrandsynthesisinF
plasmidrecipientcell
CompletionofDNAtransfer
Separationofbacterialcells
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Ability to synthesize F pilus
FPlasmidassociatedAlterations
inRecipientBacterium
AbilitytosynthesizeFpilus
DNAmobilizationforcelltransfer
Lossofabilityactasarecipientinconjugation
Acquisitionofabilitytoactasdonorinconjugation
FPlasmidsandFPlasmidHosts
F+ BacterialcellthatcontainsanonintegratedF
plasmid plasmid
F plasmid PreviouslyintegratedFplasmidthathas
excisedandcapturedsomechromosomalgenes
Hfr (highfrequencyofrecombination)bacterial
strain Bacterialstrainwithepisomal Fplasmid
(integrated) (integrated)
Hfr recombinant Hfr strainthathasacquirednew
traitsthroughhomologousrecombinationwith
otherbacterialstrains
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Hfr StrainsandChromosomeMobilization
Result:Highrateofgeneticrecombination
betweengenesondonorchromosomeand
thoseonrecipientchromosome
Fplasmidisanepisome:
Canintegrateinto
hostchromosome
Chromosome IntegratedF
plasmid
Hfrcell
(donor)
F

cell
(recipient)
Fplasmidnickedinonestrand
Hfr DonorRecipientPair
TransferofFfollowedby
chromosomalDNA
Figure10.21
F

cell Hfrcell
Synthesisofsecondstrandin
recipient
2012 Pearson Education, Inc.
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Fplasmid
oriT
tra
rep
High frequency of crossovers
Chromosome
IS3
pro lac
Recombination
Highfrequencyofcrossovers
promotes useofHfr strainsas
genemappingtools
Figure10.19
IS3
IS3 IS3
pro
rep oriT tra lac
2012 Pearson Education, Inc.
TransfertoF

recipient
IS3
oriT
rep
pro
tra
oriT =origin
oftransfer
Insertionsequence
proline
l i
Transfer
Insertionsequence
Integrated
Fplasmid
Chromosome
leu
thr
lac
IS3
leucine
threonine
Figure10.20
arg
his
trp
gal
2012 Pearson Education, Inc.
arginine
histidine
tryptophan
galactose
lactose
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Hfr3
Hfr1
Hfr4
F
F
F
chr
Hfr2
F
Hfr1
CDE XYZAB
GeneCdonatedfirst;
clockwiseorder
Figure10.22
Hfr3
Hfr4
Hfr2
LKJ BAZYX ONM
XYZAB UVW
GFE BAZYX JIH
GeneLdonatedfirst;
counterclockwiseorder
GeneXdonatedfirst;
clockwiseorder
GeneGdonatedfirst;
counterclockwiseorder
2012 Pearson Education, Inc.
GeneMapping:TheInterruptedMatingPairMethod
http://www.bio.miami.edu/dana/250/25008_7.html
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Hfrdonor:Thr

Leu

Lac

Str
s
F

recipient:Thr

Leu

Lac

Str
r

Conjugation,followedby
platingontoagarmedia
Mappingby
Recombination
Frequency
Fartherapart2genes
are,thehigherthe
recombination
frequency:HigherRF=
greatermapdistance
Thr

Leu

Str
r
recombinants
Lac

Str
r
recombinants
Selectionfor
recombinants
SelectingRecombinants:
Agarminimalmediumwith
streptomycinandglucose;
selectsformarkersThr

Leu

;
doesnotselectforLac
Agarminimalmediumwith
streptomycin,lactose,threonine,
leucine;selectsformarkerLac

;
doesnotselectforThrorLeu
ProofofConjugation
SummaryofTransformation,
Transduction,Conjugation
SeeFigure10.10inourTextbook
ReaddiscussioninTextbookthoroughly
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Thr

Leu

Gal

Trp

Thr

Leu

8min 25 33 0
60
50
40
n
a
n
t
s
r
i
a
Gal

Trp

30
20
0
N
u
m
b
e
r

o
f

r
e
c
o
m
b
i
n
p
e
r

1
0
0

H
f
r

b
a
c
t
e
r
Time,aftermixingparentalcultures(min)
p
10
10 20 30 40 50 60 70
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
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Genetic
Recombination