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

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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

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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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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

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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