CHAPTER 2 Genetics of Bacteria and Their Viruses by dr nik

7/21/2019 CHAPTER 2 Genetics of Bacteria and Their Viruses by dr nik

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GENETICS OF

BACTERIA & THEIR VIRUSES II

bacterial conjugation

bacterial transor!ation

Bacterial trans"uction


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Prokaryote Basics The largest and most obvious division of living organisms is

into prokaryotes vs. eukaryotes.

Eukaryotes are defined as having their genetic materialenclosed in a membrane-bound nucleus, separate from the

cytoplasm. In addition, eukaryotes have other membrane-bound organelles such as mitochondria, lysosomes, andendoplasmic reticulum. almost all multicellular organisms areeukaryotes.

In contrast, the genome of prokaryotes is not in a separatecompartment it is located in the cytoplasm !althoughsometimes confined to a particular region called a "nucleoid#$.Prokaryotes contain no membrane-bound organelles% their

only membrane is the membrane that separates the cell from

the outside &orld. 'early all prokaryotes are unicellular.


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Three (omains of )ife


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Prokaryote vs. Eukaryote *enetics Prokaryotes are haploid, and they contain a single circular

chromosome. In addition, prokaryotes often contain smallcircular ('+ molecules called "plasmids#, that confer useful

properties such as drug resistance. nly circular ('+molecules in prokaryotes can replicate.

In contrast, eukaryotes are often diploid, and eukaryotes havelinear chromosomes, usually more than .

In eukaryotes, transcription of genes in '+ occurs in the

nucleus, and translation of that '+ into protein occurs in thecytoplasm. The t&o processes are separated from each other.

In prokaryotes, translation is coupled to transcriptiontranslation of the ne& '+ molecule starts beforetranscription is finished.


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Bacterial /ulture

0urprisingly, many, perhaps even most, of thebacteria on Earth cannot be gro&n in the laboratorytoday.

Bacteria need a set of specific nutrients, the correctamount of o1ygen, and a proper temperature to gro&.The common gut bacteriumEscherichia coli!E.coli$ gro&s easily on partially digested e1tracts madefrom yeast and animal products, at 23 degrees in anormal atmosphere. These simple gro&th conditionshave made E. coli a favorite lab organism, &hich isused as a model for other bacteria.


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4ore /ulture

Bacteria are generally gro&n in either of 5 &ayson solid media as individual colonies, or in li6uidculture.

The nutrient broth for li6uid culture allo&s rapidgro&th up to a ma1imum density. )i6uid cultureis easy and cheap.

0olid media use the same nutrient broth as li6uidculture, solidifying it &ith agar. +gar apolysaccharide derived from sea&eed that mostbacteria can7t digest.

The purpose of gro&th on solid media is to isolateindividual bacterial cells, then gro& each cell upinto a colony. This is the standard &ay to create a

pure culture of bacteria. +ll cells of a colony areclosely related to the original cell that started thecolony, &ith only a small amount of geneticvariation possible.

0olid media are also used to count the number ofbacteria that &ere in a culture tube.


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Bacterial 4utants

4utants in bacteria are mostly biochemical in nature, because &e can7t generally see the cells.

The most important mutants are au1otrophs. +n au1otroph needs some nutrient that the &ildtype strain !prototroph$ can make for itself. 8or e1ample, a trp- au1otroph can7t make its o&ntryptophan !an amino acid$. To gro& trp- bacteria, you need to add tryptophan to the gro&th

medium. Prototrophs are trp9% they don7t need any tryptophan supplied since they make theiro&n.

/hemoau1otrophs are mutants that can7t use some nutrient !usually a sugar$ that prototrophscan use as food. 8or e1ample, lac- mutants can7t gro& on lactose !milk sugar$, but lac9

prototrophs can gro& on lactose.

esistance mutants confer resistance to some environmental to1in drugs, heavy metals,bacteriophages, etc. 8or instance, +mp causes bacteria to be resistant to ampicillin, acommon antibiotic related to penicillin.

+u1otrophs and chemoau1otrophs are usually recessive% drug resistance mutants are usuallydominant.


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#OR$ING #ITH %ICROORGANIS%S

strains

rototro's()il" t*e gro) on !ini!al

!e"iu! au+otro's(

!utants

"o not gro) on!ini!al !e"iu!

nutrition

carbon source

resistant!utants


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eplica Plating

+ common &ay to find bacterial mutants is replica plating, &hich meansmaking t&o identical copies of the colonies on a petri plate underdifferent conditions.

8or instance, if you &ere looking for trp- au1otrophs, one plate &ould

contain added tryptophan and the other plate &ould not have anytryptophan in it.

Bacteria are first spread on the permissive plate, the plate that allo&s bothmutants and &ild type to gro&, the plate containing tryptophan in thiscase. They are allo&ed to gro& for a &hile, then a copy of the plate ismade by pressing a piece of velvet onto the surface of the plate, thenmoving it to a fresh plate &ith the restrictive condition !no tryptophan$.The velvet transfers some cells from each colony to an identical positionon the restrictive plate.

/olonies that gro& on the permissive plate but not the restrictive plate are!probably$ trp- au1otrophs, because they can only gro& if tryptophan issupplied.


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eplica Plating, pt. 5


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Bacterial Para-se1ual Processes

Eukaryotes have the processes of meiosis to reducediploids to haploids, and fertili:ation to return the cells tothe diploid state. Bacterial se1ual processes are not so

regular. ;o&ever, they serve the same aim to mi1 thegenes from t&o different organisms together.

The three bacterial para-se1ual processes


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#OR$ING #ITH %ICROORGANIS%S


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2

Plasmids

4any ('+ se6uences in bacteria are mobile and can betransferred bet&een individuals and among species.

Plasmids are circular ('+ molecules that replicateindependently of the bacterial chromosome

Plasmids often carry antibiotic resistance genes

Plasmids are used in genetic engineering as gene transfervectors


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T'e U,Tube E+eri!ent

e+eri!ent --- contact .

sensiti/e to 0Nase.

selecti/e ilter re/entscell contact

no gro)t' 1rototro's2

on !ini!al !e"iu! contact re3uire" or

reco!bination


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Transformation

It is very important for recombinant ('+ &ork. Theessence of recombinant ('+ technology is to remove('+ from cells, manipulate it in the test tube, then put it

back into living cells. In most cases this is done bytransformation.

In the case of E. coli, cells are made "competent# to betransformed by treatment &ith calcium ions and heatshock. E. coli cells in this condition readily pick up ('+from their surroundings and incorporate it into theirgenomes.


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TRANSFOR%ATION IN BACTERIA

con/ersion o one genot*e to anot'er b* uta4e o

e+ogenous 0NA

transor!ation rincile5 "e!onstrate" t'at 0NA)as resonsible or in'erite" "ierences in

ol*sacc'ari"e c'aracter o S. pneumoniae


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e+tracte" 0NA 1in an e+eri!ent2 brea4s at ran"o!

co,transor!ationo 6 tig'tl* lin4e" "onor genes is

!ore li4el* t'an 6 "istant "onor genes

Co-transformationis the simultaneous transformation

of two or more genes.

cells !ust be !a"e co!etentto enable 0NA entr*


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0NA !ust enter an"reco!bine into t'e 'ost


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/on=ugation

/on=ugation is the closest analogue inbacteria to eukaryotic se1.

The ability to con=ugate is conferred by the8 plasmid. + plasmid is a small circle of('+ that replicates independently of thechromosome. Bacterial cells that containan 8 plasmid are called "89#. Bacteria

that don7t have an 8 plasmid are called"8-#.

89 cells gro& special tubes called "se1pilli# from their bodies. >hen an 89 cellbumps into an 8- cell, the se1 pilli holdthem together, and a copy of the 8 plasmidis transferred from the 89 to the 8-. 'o&both cells are 89.

>hy aren7t all E. coli 89, if it spreads likethat? Because the 8 plasmid can bespontaneously lost.

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5

8 factor and /on=ugation

8 !fertility$ factor is a con=ugative plasmid transferred fromcell to cell by con=ugation

8 factor is an episome F genetic element that can insert intochromosome or replicate as circular plasmid

The 8 plasmid is a lo&-copy-number plasmid GAA kb in

length, and is present in


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Intracellular Events in /on=ugation

The piece of chromosome that enters the 8- form the ;fr islinear. It is called the "e1ogenote#.

The 8- cell7s o&n chromosome is circular. It is called the

"endogenote#. nly circular ('+ replicates in bacteria, so genes on the

e1ogenote must be transferred to the endogenote for the 8-to propagate them.

This is done by recombination 5 crossovers bet&een

homologous regions of the e1ogenote and the endogenote.In the absence of recombination, con=ugation isineffective the e1ogenote enters the 8-, but all the geneson it are lost as the bacterial cell reproduces.


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52

8 factor and /on=ugation /on=ugation is a process in &hich ('+ is

transferred from bacterial donor, 89 cell to a

recipient, 8- cell by direct contact.

The transfer is mediated by a tube-like

structure called a pilus, formed bet&een the

cells, through &hich the plasmid ('+ passes.

nce in contact, con=ugation, ('+ transfer is

unidirectional. The lagging strand template

peels a&ayH and is transferred to the

recipient.

The leading strand template is replicated in the

donor &hile the lagging strand template is

replicated in the recipientH so that both cells

&ind up &ith the plasmid.


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5

;fr 8 factor can integrate into chromosome via

genetic e1change bet&een plasmid

elements present in 8 and homologous copylocated any&here in bacterial chromosome

/ells &ith the 8 plasmid integrated into the

bacterial chromosome are kno&n as ;fr

cells

>hen an ;fr cell undergoes con=ugation,the process of transfer of the 8 factor is

initiated in the same manner as in an 89cell

;o&ever, because the 8 factor is part of the

bacterial chromosome, transfer from an ;fr

cell also includes ('+ from thechromosome

;fr F high fre6uency of recombination


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5C

;fr and /on=ugation Transfer begins &ithin anintegrated 8 factor and proceedsin one direction

+ part of 8 is the first ('+transferred, chromosomal genesare transferred ne1t, and theremaining part of 8 is the last

The con=ugating cells usuallybreak apart long before the entirebacterial chromosome istransferred, and the finalsegment of 8 is almost nevertransferred

The recipient cell remains 8-


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8-prime !87$

The process of making an ;fr from an 89 involves a crossover bet&een the 8plasmid and the chromosome. This process is reversible an ;fr can revert tobeing 89 &hen the 8 plasmid ('+ incorporated into the ;fr chromosome hasa crossover and loops out of the chromosome forming an 8 plasmid onceagain.

0ometimes the looping-out and crossing-over process doesn7t happen at theproper place. >hen this happens, a piece of the bacterial chromosome canbecome incorporated into the 8 plasmid. This is called an 87 !8-prime$plasmid.

87 plasmids can be transferred by con=ugation. /on=ugation &ith an 87 !or aregular 8 plasmid$ is much faster and more efficient than &ith an ;fr, becauseonly a very small piece of ('+ is transferred.

+ cell containing an 87 is "merodiploid# part diploid and part haploid. It isdiploid for the bacterial gene carried by the 87 !one copy on the 87 and theother on the chromosome$, and haploid for all other genes.


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TRANS0UCTION IN BACTERIA

alternati/e lie c*cles o te!eratebacterio'age


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generali7e" trans"uction8 ran"o! incororation

l*tic c*cle9 non,integrate" 'age


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'age

integration


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seciali7e" trans"uction8 non, ran"o! incororation

l*sogenic c*cle9 integrate" 'age

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trans"uction: 'age ac3uire 'ost genes an"transer t'e! to ot'er bacterial cells

generali7e" trans"uction: transers any'ost geneDan" occurs )'en 'age ran"o!l* ac4age'ost

0NA

seciali7e" trans"uction: ault* searation oro'age 1'age incororate" into 'ost geno!e2D

ne) 'age contains a"jacent 'ost genes onl*


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SU%%AR: RECO%BINATION IN BACTERIA

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CHAPTER 2 Genetics of Bacteria and Their Viruses by dr nik