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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$e mutants (dierent phenotypes
• )ecombination
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Mechanisms o Genetic
'6change• Transormation - uptake o ree /"A
molecules released rom one bacterium by
another bacterium• Con8ugation 2 direct transer o /"A rom
donor cell to a recipient cell
• Transduction 2 bacterial genes are carriedrom a donor cell to recipient cell by a bacteriophage
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Bacterial )ecombination
• ederberg and Tatum, 5E?F
• 'scherichia coli, E. coli, strain 5%
2 +train A 2 thr+, leu+, thi+, met-, bio-
2 +train B 2 thr-, leu-, thi-, met+, bio+
• Gre# separately, then mi6ed together and
plated on minimal media• 3rototrophs appeared at rate 5 cell;5&H cells
plated! Controls
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Bacterial )ecombination
• Genetic recombination occurred 2
replacement o one or more genes present in
one strain #ith those rom a geneticallydistinct strain
• Do# did it happen
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Cells "eed Contact 7or
)ecombination
• Bernard /a$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&&× 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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Dr Bacteria And Chromosome Mapping
• In any gi$en Dr strain, certain genes are ====================== recombined thanothers
• ================== pattern o transer • Interrupted mating techni@ue 2ollman and acob
• Chromosome o Dr bacterium #as transerredlinearly and order and distance bet#een genes
could be determined• Ma8or dierence bet#een Dr strains 2 point o
the origin and =======================
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Dr Bacteria And Chromosome Mapping
• 'ntire E. coli chromosome #as mapped by
this techni@ue
• =============== long
• ?&&& =============== se@uences
• Appro6imately ============= genes
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Dr Bacteria And Chromosome Mapping
• Conclusions<
2 E.coli chromosome is circular
2 7 actor integrates into chromosome at dierent points
2 Its position determines the origin o transer (-
site or original point o transer 2 Genes ad8acent to are transerred irst
2 hole chromosome is ne$er transerred
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Dr Bacteria And Chromosome Mapping
• 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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Bacterial )ecombination
)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