course content of microbial genetics (bio-515) (class :; time : 08:00-09:40; room :bio-2) week i....
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COURSE CONTENT OF MICROBIAL GENETICS(BIO-515)
(Class : ; Time : 08:00-09:40 ; Room : Bio-2 )
Week
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX
X.
XI.
XII.
XIII.
XIV.
XV.
Topic
Structure and Organization of the Genome:
Prokaryote and Eukaryote
DNA Replication and Recombination
Transcription and Translation
Post transcription and Post translation
----------------------Examination I ---------------------
Mutation and Reparation of DNA
Conjugation, Hfr, and Genetic Mapping
Transformation and Transduction
Transposition and DNA Rearrangement
Plasmid as Genetic Material
----------------------Examination II ---------------------
Regulation of Gene Expression: Paradigm of Operon I
Signal Transduction in Baqcteria
Phage genetics
Recombinant DNA Technology and Molecular Genetic Analysis GYT
Genetic of Fungi I
Genetic of Fungi II
----------------------Examination III ----------------------
Lecturer
ATL
ATL
ATL
ATL
ATL
ATW
ATW
ATW
ATW
ATW
ATW
GYT
GYT
GYT
GRH
GRH
GRH
Gene Transfer inBacteria:
Conjugation
1
2
An Overview
Three ways by which bacterial DNA can betransferred from cell to cell
3 ways to transfer DNA into bacteria
1. conjugation – plasmid-mediated transfer
2. transformation – absorb from environment
3. transduction – bacteriophage-mediated transfer
3
WORKING WITH MICROORGANISMS
binary fissionknown rate ~ °Cliquid mediumplating ...
serial dilutions ...
1 cell ~ 107 cellsvisible colonies
or undiluted ...
lawn
use both methods
WORKING WITH MICROORGANISMS
strainsprototrophs =
wild typegrow on minimal
medium
auxotrophs =mutantsdo not grow on
minimal mediumnutritioncarbon source
resistant mutants
Genetic information can be transferredbetween bacteria
In 1946, Lederberg and Tatum showed thattwo different strains of bacteria withdifferent growth requirements couldexchange genesLederberg and Tatum surmised that thebacterial cells must interact with each other -the process is now known as conjugation
BACTERIAL CONJUGATION
do bacteriahavegenetic exchange &recombination ?
Lederberg & Tatum,1946
Escherichia coli(E. coli)
single circular“chromosome”
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BACTERIAL CONJUGATION
experiment ... contactrequirement ?
2 strains, > 1 mutation
no colonies on A or B ...
no spontaneous back
or reversion mutations
some colonies (10-7) onmixed ...
prototrophs from
recombination
BACTERIAL CONJUGATION
experiment ...contact ? selective filter preventscell contact
no growth (prototrophs)on minimal medium
contact required forrecombination
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6
BACTERIAL CONJUGATION
fertility factor – F plasmidF+ donor & F– recipient strainsF+ x F– both F+
unidirectional rolling circle plasmid replicationF DNA transferred through a pore in the pilus
Fig. Physical and genetic map of transmissible plasmid pKM101
oriT: Origin of plasmidtransfer
tra Genes: encode manytransfer functions.
Many plasmids have transfer systemThat enable them to transfer DNABetween unrelated spesies:“Promiscuous Plasmids”
Plasmid:Self-transmissibleMobilizable
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Fig. Partial genetic and physical map of the self-transmissibleplasmid F (100 kb)
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Plasmid transfer by conjugation
Mechanism of DNA transfer byConjugation: Gram Negative Bacteria
tra genes: required for transferoriT: required for transfer (nick)
tra genes: 2 components-Dtr component~DNA transfer & Repto cluster in oriT
-Mpf component~Mating pair formationholding the mating cells
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The Dtr ComponentRelaxase:
~Specific DNA Endonuclease which make break/nick in the oriT
~to initiate the transfer prosess~Relaxase: Recyclizes the plasmid after transfer
break phosphodiester bond at the nick site by transfering
the bond from a deoxynucleotide to one of its own tyrosine~Relaxase protein still bound to the 5’ through its tyrosine
and transfer phosphate bond from its tyrosine back to 3’ hydroxyldeoxynucleotide in the DNA.
Relaxosome:~Proteins that are normally bound to the oriT~help relaxase bind to oriT
separate the strands at oriT to initiate transfercommunicate with coupling protein of Mpf when relaxase cut oriT
~One of the other protein of relaxosome: Helicasefunction: to separate DNA strands at the oriT
Primase:~are needed for chromosomal DNA replication to make mRNARNA primer to prime the synthesize of the DNA.
~To prime plasmid replication.
The oriT Sequence
~Site at which plasmid transfer is initiatedSite at which the DNA end rejoin to recyclizethe plasmid after transfer
~Spesific relaxase encoded by one of the tra geneswill cut at this sequence (oriT)
~Plasmid-encoded helicase will enter DNA onlyat this site/seq to separate strands.
~After transfer, the two ends at the DNA are heldtogether at oriT sequence, they can be religated
~plasmid to be transferred==plasmid musthave specific oriT.F plasmid oriT~~300 bp; AT-rich ; IR
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Mating Pair Formation~MPF system hold the donor & recipient cells during mating and formthe channel through which proteins and DNA are transferred.
~Involved in conjugal DNA transfer
The Pilus:~Tube-like structure that stick out of the cell surface10 nm or more in diameter
~Protein: PilinF plasmid encodes long, thin, flexible piluspKM101 encodes short, thick, rigid pilusRP4 encodes short, thick, rigid pilus
Determine efficiency of transfer
The Channel:MPF system encodes a channel/pore through whichDNA passes during conjugation.
Coupling Protein:~Part of the MPF system required for communication between Dtr & MPF~Provide the specificity for the transport process~is bound to the membrane channel
Mobilizable Plasmids
~Cannot transfer themselves but can be transferred byother plasmid: “Mobilizable Plasmid”
~Process by which they are transferred: “Mobilization”
~Contain oriT of self-transmissible plasmid, can be mobilizedby tra system of that plasmidMPF and Dtr systems can act trans on the cis acting oriT==Mobilization
~Applications in molecular genetics:Transposon mutagenesisPlasmid cloning vector
~M P minimal contain oriT of self-transmisible plasmid~Contain mob genes==mob region~~~mobilizationMechanisms of transfer (Fig.).
Fig. Mechanism of plasmid mobilization
Km 1tn p *
PstIX baISphISfiISalI
PstI
PstI
B glII
p U Tm in i-Tn5 Km 17.0 55 Kb
o ri R 6 K
A pR
PstIm o b RP4
EcoRI KpnI SfiI
(A)
Figure Map of pUTmini-Tn5Km1 (A), and transposon Mini-Tn5Km1 (B).
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Kan amycin R esistan ce G ene (Km 1)
IS (I) IS (O )
NotI
O end
EcoN ISspI
HindIIIXb aI PstI Sph I Sfi I
HindIIIXhoI
S fiI EcoR I Kpn IClaI
(B) I end
1835 bp
HincIIPstISphIHindIII
AccI
SmaIXmaIBamHISalI
EcoRISacIKpnI
EcoO109 2674AatII 2617
SspI 2501
XmnI 2294
ScaI 2177
PvuI 2066
AvaII 2059
FspI 1919
AvaII 1837BglI 1813
GsuI 1784Cfr101 1779
PpaI 1766
HgiEII 1387
PvuII 628
PvuI 276PvuII 306
NarI 235BglI 245FspI 256
(EcoRI/PvuII) 1HgiEII 181
NdeI 183
lacZ
lacI
AflIII 806
ORI
MmeI 996
MmeI 1180AlwNI 1217
Eco57I 1333
Ap pUC19
2686 bp
MCS
Figure . Map of plasmid pUC19
MCS
Fig. Map of plasmid pET-15b
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F factors can integrate intothe host chromosome
If an ‘F factor’ integrates it turns the hostchromosome into an “Hfr chromosome” and thecell into an Hfr cellHfr = “high frequency of recombination”Hfr cells can make pili and conjugate with F-
cellsWhen the F factor is transferred to F- cellsadjacent genes from the chromosome are alsotransferred
pili
Hfr
F+ orHfr
Conjugationtube
F-
F+
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BACTERIAL CONJUGATION
the F plasmid can integrate into the host chromosomegenerates a high frequency recombinant strain ... Hfr
15
BACTERIAL CONJUGATION
Hfr transfers part of thehost genome duringconjugation
Hfr x F– F– rarelyconverted to Hfr or F+
isolate & purify Hfr fromF+ for mapping
BACTERIAL CONJUGATION
Hfr x F– recombination of donor genes in hostterms: exogenote and endogenote
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BACTERIAL CONJUGATION
Wollman & Jacob, 1957 – gradient of transferselective marker – donor is strs & recipient is strr
origin of replication is transferred first
Hfr cell (cell with integrated F plasmid)
• transfers genomic markers, starting fromOrigin on F plasmid sequence
• Fertility factor is last element to be transferred-- therefore, recipients rarely become F+
• Allows mapping of chromosome in minutes ofconjugation required for transfer
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BACTERIAL CONJUGATION
interrupted-matingselective markers
donor is strs
recipient is strr
origin of replicationtransferred first
1st transferred markersmost frequent inexconjugants
BACTERIAL CONJUGATION
mapping in E. coli by interrupted-matingdistance measured in time (min)
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BACTERIAL CONJUGATION
bacterial chromosome is circularintegration of F factor is pseudo-randomintegration in either orientation
BACTERIAL CONJUGATION
F factor integrates by recombinationpairing regions of homology (hatched)episome = plasmid with free & integrated states
Bacterial Recombination Experiment
XHfr thr+ leu+ lac+gal+
streptomycin sensitive
_ _ _ _ _
F thr leu lac gal
streptomycin resistant
Grow on minimal mediumwith streptomycin
Chromosome Map:thr leu lac gal
8 8 1/2 15 25 min
0 minutes
(Experiment can beconducted with one mutantgene at a time or withmultiple types of media.)
_• F cells become threonine+ after 8 minutes of conjugation, become threonine andleucine+ at 8 1/2 minutes, become threonine, leucine and lactose+ after 15 minutes andbecome threonine, leucine, lactose and galactose+ after 25 minutes of conjugation.
Conjugation: Triparental Mating
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Conjugation in Gram PositiveBacteria
~Bacillus subtilisStreptomycesStaphylococcus
Contain self-trans-missible plasmids
StreptococcusEnterococcus
~E. faecalis excrete pheromone-like componentthat can stimulate mating.
~Pheromone: Small peptide that stimulate expression of tragenes and induce aggregation & mating
Mechanisms ofTransfer
~Genes encode pheromone are located on the chromosome of recipient.
cut from the signal peptide (Lipoprotein).~After signal sequence are cut from the lipoprotein, being transportedactive pheromone are produced by proteolysis of the C-terminal
~Pheromone is excreted from the cell.Sensing of pheromone==need protein specific located on thecell surface.
~Each of self-transmissible plasmid express a protein specificfor one type of pheromone.
TraC: protein binding pheromone in pAD1==pheromone CAD1~Pheromone inside the cell==induce exression of plasmid genes
involved in plasmid transferThis protein coat the cell surface=Cell contact
plasmid enter to the recipient~TraA: represses transcription of tra genes except TraC.
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Fig. Plasmid transfer in gram positive bacteria
Role of pheromone inplasmid transfer
~Recipient Cell. Pheromone genes are located on the chromosomePropheromone exported ==pheromone (Out side of the cell).
~The donor cell.TraA represses transcription of the other tra genes except TraC.TraC: encodes cell surface protein that can sense to pheromone
~Mating induction.Pheromone binds TraC on the cell surface a donor cell=enter the cell via oligopeptide permease system=Pheromone binds repressor TraA~~~releasing it from DNA
and derepressing the synthesis of TraE~~~~activatesthe expression of tra genes including those encoding theaggregation substance.
~Plasmid transfer.The donor cell contact with the recipient cell, and plasmidtransfer producing transconjugant
~Pheromone shutdown in transconjugant.Cell has become transconjugant, the inhibitor peptide iAD1 bindTraC and prevent autoinduction or pheromone stimulation ofmating with other donor cell.TraB=inhibitor protein function as preventing excretion of pheromone
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Regulation of the tra Genes(IncF Plasmid)
~TraJ is transcriptional activatorIts translation is normally blocked by finP and finO products
~FinP RNA===antisense RNA from promoter within andopposite orientation to the traJ gene.
~Complementary pairing FinP RNA and traJ transcript preventtraJ translation
~FinO stabilizes FinP antisense RNAPlasmid first enter the cell==No FinO and FinP RNAtraJ and other tra genes products are made==pilus appears on the cell and plasmid can be transferred
~After plasmid has become established==FinP and FinO proteinwill be synthesized, plasmid can no longer transfer
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Hfr cells can transfer host chromosomal genes
Chromosome Transfer in Bacteria
Genes are transferred in a fixed order, theoretically thewhole chromosome can be transferred
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