biol 3301 - genetics ch9a - genetic analysis and mapping in bacteria

8/18/2019 BIOL 3301 - Genetics Ch9A - Genetic Analysis and Mapping in Bacteria

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Genetic Analysis and Mapping In

Bacteria And Bacteriophages• Bacteria, bacteriophages - prokaryotes

• Circular single chromosome

• They are haploid (no masking!

• "e# generation is produced e$ery %& minutes!

• 'asy to gro# in '")M*+ "*MB')+!

• Indi$idual members o these large populations areG'"'TICA. I/'"TICA (or $ery nearly so!


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Bacteria

• Genetic inormation o most bacteria is stored in asingle main chromosome #ith a e# thousand

genes and a $ariable number o 0mini-chromosomes1- plasmids and episomes 2 3lasmid 2 autonomously replicating, circular /"A, 4 to

5&&s genes• +ome bacteria can contain up to 55 dierent plasmids

• +ome can contain only one plasmid (lo# copy and high copynumber

2 'pisomes - same as plasmid but can be integrated into bacterial chromosome


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3lasmids

• '6trachromosomal heredity unit

• '6ist autonomously in bacterial cytoplasm

• /ouble-stranded closed circle molecule o /"A 2 7 (ertility actors (or plasmids

2 ) (resistance actors 2 e!g! antibiotic resistance

2 Col (colicinogenic actors

• 'pisomes 2 plasmids that can be in integratedcondition! "ot all plasmids can integrate


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3lasmids

• ) plasmids

2 )esistance transer actor 2 essential to transer

o the plasmid $ia con8ugation

2 r-determinants 2 genes conerring resistance to

antibiotics


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3lasmids

• ColE1 2 encodes one or more proteins that

are highly to6ic to bacterial strains, colicins

• Colicinogenic bacteria contain a plasmid

gene encoding an immunity protein

protecting the host

• "ot transmittable by con8ugation


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Bacterial Mutants

2 Color and morphology o a colony

2 Block ability to utili9e speciic energy source 2

can:t utili9e lactose, galactose, arabinose, etc 2 *nable to synthesi9e an essential metabolite

(prototrophs;au6otrophs

2 )esistance to drugs and antibiotics


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CHANGES in BACTERIA

(PHENOTYPE)• 5! Colony morphology <

• a! ==========================

• b! +mooth→ rough ( loss o capsule• %! Biochemical acti$ity

• Change in ===================

• 4! >irulence

• Change in ability =====================

• ?! /rug resistance


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Bacteria• Gro# in li@uid culture or on solid agar

• Minimal medium 2 organic carbon source(glucose, lactose, $ariety o inorganic salts

• Capable o gro#ing on minimal media 2 prototroph! Able to synthesi9e all essential organiccompounds! ild type

• "ot capable o li$ing on minimal media 2au6otroph, lose the ability to synthesi9e one or

more organic components - mutant


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Gene nomenclature• *sually %-4 letters #ith or 2 symbol

• 3rototrophy;au6otrophy 2 met+ can synthesi9e methionine, a #ild-type prototroph

2 met- re@uires methionine, a mutant au6otroph• 'nergy e6traction

2 gal +< can utili9e galactose, #ild-type

2 gal 2 < can not utili9e galactose, a mutant

• /rug resistance 2 str R


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Bacterial Gro#th

• Characteristic gro#th pattern<

2 ag phase 2 small amount o bacterial cells

2 og phase 2 cell start di$iding by =======

2 +tationary phase 2 nutrients become limiting

2 /eath

Ma6imum cell density achie$ed usually duringo$ernight incubation


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Bacterial Gro#th

• To @uantiy number o cells in li@uid culturesmall amount o culture is transerred to a

3etri dish #ith solid medium• 'ach bacterial cell #ill di$ide and produce

a $isible colony

• Count colonies, multiply by dilutions (idone 2 determine number o bacterial cellsin li@uid culture


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+electi$e systems

• Allo#s the desired mutant to reproduce

- antibiotic resistance

2 minimal medium supplemented #ith speciic

nutrient

• )e$ertant< re$erse change rom mutant to

#ild-type 2 similar selection regimens


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Do# To Map Genes In Daploids

• Da$e circular chromosome

• Daploid

• /o not ha$e =================

• Da$e mutants (dierent phenotypes

• )ecombination


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Mechanisms o Genetic

'6change• Transormation - uptake o ree /"A

molecules released rom one bacterium by

another bacterium• Con8ugation 2 direct transer o /"A rom

donor cell to a recipient cell

• Transduction 2 bacterial genes are carriedrom a donor cell to recipient cell by a bacteriophage


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Bacterial )ecombination

• ederberg and Tatum, 5E?F

• 'scherichia coli, E. coli, strain 5%

2 +train A 2 thr+, leu+, thi+, met-, bio-

2 +train B 2 thr-, leu-, thi-, met+, bio+

• Gre# separately, then mi6ed together and

plated on minimal media• 3rototrophs appeared at rate 5 cell;5&H cells

plated! Controls


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• Genetic recombination occurred 2

replacement o one or more genes present in

one strain #ith those rom a geneticallydistinct strain

• Do# did it happen


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Cells "eed Contact 7or

)ecombination

• Bernard /a$is, e6periment #ith *-tube

• /ierent strains o bacteria are in$ol$ed in aunidirectional transer o genetic material 2

bacterial se6

•7 cells 2 donors o genetic material

• 7- cells 2 recipients o genetic material


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7 and 7- Bacteria•

Cell contact is essential or chromosomal transer • 3hysical interaction occur through a con8ugation tube 7 pilus (or se6 pilus, pili

• 7 cells contain 7 actor ( plasmid, one o many types!

• 7 actor (ertility actor rom E. coli is the most studied plasmid! 2 7 actor carries genes encoding the se6 pilus or physical

transer o genetic material!

2 7 actor 2 circular, double-stranded /"A molecule about5&&,&&& bp, J%& genes (plasmid

• Ater con8ugation 7- al#ays become 7


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Con8ugation< /"A transer

• Transer is originated at a speciic site _______ 2 theorigin o traner

• 7 actor also ha$e ________ 2origin o replicationK and

oriS 2 secondary origin o replicationK do not take part intranser

• /uring con8ugation one strand at oriT is cut by an en9yme,one end is transerred ===================

• 7 actor replicates during transer by rolling-circle

replication• /uring con8ugation, only ============ o the donor

chromosome is synthesi9ed in the donor cell and thetranserred strand is replicated in the recipient cell


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Con8ugation

• 7 actor

2 7 actor can e6ist in autonomous state, replicate

independently (7 cell 2 r integrated state, inserted into bacterial

chromosome 2 episome 7:

2 hen 7 cell con8ugates #ith 7- cell, only 7actor is transerred


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Dr Bacteria And Chromosome Mapping

• 5EL&, Ca$alli-+or9a used a mutagen (nitrogen

mustard on E. coli 5%

• "e# mutant #ith recombination rate 5&-? (5&&&times more

• Dr mutation 2 high re@uency o recombination

•7

-

6 7B

----------- recipient becomes 7• Dr 6 7- ---------- recipient remains 7-


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Dr cells

• A cell that carries 7 actor integrated in bacterialchromosome is called Dr cell

• 7 actor integrates into bacterial chromosome bysite-speciic recombination, mediated by shortse@uences that are present in multiple copies in

both chromosomes 2 integration in multiple sites

• In integrated state, 7 actor mediates transer o/"A rom Dr cell to a recipient 7- cell 2 partialtranserK 7 actor is not transerred ully


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Con8ugation< /"A transer in Dr

cells• Transer is initiated #ithin the integrated 7 actor

• 3art o 7 actor is transerred prior to transer o

chromosomal /"A in Dr 6 7-• The rest o the 7 actor is transerred ater the

chromosomal genes

•Thereore 7- cell does not recei$e ull 7 actor($ery rarely the #hole chromosome is transerred


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• In any gi$en Dr strain, certain genes are ====================== recombined thanothers

• ================== pattern o transer • Interrupted mating techni@ue 2ollman and acob

• Chromosome o Dr bacterium #as transerredlinearly and order and distance bet#een genes

could be determined• Ma8or dierence bet#een Dr strains 2 point o

the origin and =======================


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• 'ntire E. coli chromosome #as mapped by

this techni@ue

• =============== long

• ?&&& =============== se@uences

• Appro6imately ============= genes


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• Conclusions<

2 E.coli chromosome is circular

2 7 actor integrates into chromosome at dierent points

2 Its position determines the origin o transer (-

site or original point o transer 2 Genes ad8acent to are transerred irst

2 hole chromosome is ne$er transerred


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• Dr cells 2 7 actor integrated into

chromosome

• 7 - 7 actor is present in cytoplasm

• 7N - 7 actor in Dr strain re$erts to

cytoplasmic condition but carries se$eral

ad8acent chromosomal genes

• Mero9ygote 2 partial diploid


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Transormation

• Another #ay o introducing /"A into cell,

not through con8ugation<

2 'ntry o oreign /"A into a recipient cell

2 )eplacement by the donor /"A o its

homologous region on recipient chromosome


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Transormation

• Competent bacterial cells take up /"A

• 'ntry occurs through a limited number o receptorsites on the surace o bacterial cells, re@uires

energy• "ucleases digest /"A molecule lea$ing only one

strand

• /"A aligns #ith complementary chromosomalregion

• This segment o /"A replaces the chromosomal/"A ( e6cision and degradation


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Transormation

• Can occur #ith 8ust taking up the plasmid

• Bacterial cell #ill ha$e

======================= i plasmidcarries dierent genes


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)ec 3roteins• Mutants #ith almost no recombination 2 recA

(5&&& times less

• ther recB, recC and recD 2recombination lo#er5&& times

• Compared #ild type and mutant bacteria

• 7ound )ecA protein

• +ingle-strand displacement as a mechanism o

recombination 2 )ecA acilitates the process


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Bacterial )ecombination 2 )ec

3roteins• )ecA protein 2 single strand recombination

• )ecBC/ protein 2polypeptide, en9yme,

important or double-stranded /"ArecombinationK un#inds the heli6


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/"A )ecombination


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Transfer Of Genetic Material In

Bacteria• Three methods o transer<

• A! Common properties<

• 5! *nidirectional - donor cell→ recipient cell

• %! 7ragment o /"A transerred• B! Conjuation

• a! ccurs amongst bacteria #ith se6 pili

• b! Bridge ormed bet#een cells by pilus

• c! 7ragment o /"A (plasmid transerred rom one bacterialcell (donor to another bacterial cell (recipient

• C! Transfor"ation• >iable bacteria absorb Onaked1 ragments o /"A released rom dead

bacteria

• /! C! Trans#uction

• Bacterial $irus (bacteriophage transers /"A ragments rom one bacterial cell to another


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Gene Transer In Bacteria

• *nidirectional

• )ecombination occur bet#een a ragment o one

chromosome (rom a donor cell and a complete

chromosome (in a recipient cell

• )ecipient cell become partial diploid (merodiploid

• Crosso$ers must occur in pairs and must insert a segment

o the donor chromosome into recipient chromosome

• dd number o crosso$ers #ill destroy the integrity o the

recipient chromosome - non$iable

biol 3301 - genetics ch9a - genetic analysis and mapping in bacteria

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