Recombination, Bacteriophages, and Horizontal Gene Transfer2005

Bacterial Conjugationtransfer of DNA by direct cell to cell contactdiscovered 1946 by Lederberg and Tatum

F+ x F MatingF+ = donorcontains F factorF = recipientdoes not contain F factorF factor replicated by rolling-circle mechanism and duplicate is transferredrecipients usually become F+donor remains F+

F factorThe F factor can exist in three different states: F+ refers to a factor in an autonomous, extrachromosomal state containing only the genetic information described above. The "Hfr" (which refers to "high frequency recombination") state describes the situation when the factor has integrated itself into the chromosome presumably due to its various insertion sequences. The F' or (F prime) state refers to the factor when it exists as an extrachromosomal element, but with the additional requirement that it contain some section of chromosomal DNA covalently attached to it. A strain containing no F factor is said to be "F-".

Gene transfer and recombinationGenes are transferred in a linear mannerThe F factor integrates into chromosomes at different points and its position determines the O site

F x F mating

Hfr ConjugationHfr straindonor having F factor integrated into its chromosomeboth plasmid genes and chromosomal genes are transferred

HfrSpecial class of F+ strainsThis was discovered because this strain underwent recombination 1000x more frequently than F+ strainsIn certain Hfr strains certain stains are more likely to recombine than others.The nonrandom pattern of gene transfer was shown to vary from Hfr strain to Hfr strain

Interrupted matingWollman explained the cells that are different between F+ and Hfr. To facilitate the recovery, the Hfr was sensitive to antibiotics and the F+ wasnt.The cells were separated at intervals of 5 minutes is the F factor

Mating

Hfr x F matingFigure 13.14b

RecombinationExogenoteExogenote

MatingThe two strains were mixedThere were incubated. At intervals of 5 minutes, samples were taken of the F- cellsThe cells were centrifuged so that they would know which genes were transferred.The distance between genes was measured by the time that it took for the genes to be transferred.During the first five minutes, the strains were mixed there was no recombination

F+ x F matingIn its extrachromosomal state the factor has a molecular weight of approximately 62 kb and encodes at least 20 tra genes. It also contains three copies of IS3, one copy of IS2, and one copy of a sequence as well as genes for incompatibility and replication.

FIn 1959 during his experiments with the Hfr strains of E. coli Adelberg discovered that the F factor could lose its integrated status and revert to its F+ status. When this occurred, the F factor carries along several adjacent bacterial genes. When you have the F factor + bacterial genes the condition is known as the F

F ConjugationF plasmidformed by incorrect excision from chromosomecontains 1 genes from chromosomeF cell can transfer F plasmid to recipientFigure 13.15aintegrated F factorchromosomal gene

MerozygotesWhen the F is then transferred to another bacteriumThe bacterium may contain genomic copies of a gene as well as an additional copy of the gene in the F.As a result the situation is a partial diploidMerozygotes have been extremely beneficial in the study of gene regulation

Interrupted matingFigure 13.22a

Figure 13.22b

Hfr mappingused to map relative location of bacterial genesbased on observation that chromosome transfer occurs at constant rateinterrupted mating experimentHfr x F- mating interrupted at various intervalsorder and timing of gene transfer determined

Gene mapping

RecombinantsMap distance can be determined by replating the resulting colonies on agar For example

leu+ exconjugants by plating them on medium containing no leucine but containing methionine and arginine

Mapping results

Map distanceThe map distance is equal to the % recombination

Tra YCharacterization of the Escherichia coli F factor traY gene product and its binding sitesWC Nelson, BS Morton, EE Lahue and SW Matson Department of Biology, University of North Carolina, Chapel Hill 27599.

Tra GenesTra Y gene codes for the protein binds to the Ori TInitiates the transfer of plasmid across the bridge between the two cells

Tra I Gene is a helicase responsible for the conjugationstrand-specific transesterification (relaxase)

Conjugative ProteinsKey players are the proteins that initiate the physical transfer of ssDNA, the conjugative initiator proteins They nick the DNA and open it to begin the transferWorking in conjunction with the helicases they facilitate the transfer of ss RNA to the F- cell

DNA TransformationUptake of naked DNA molecule from the environment and incorporation into recipient in a heritable formCompetent cellcapable of taking up DNAMay be important route of genetic exchange in nature

DNA bindingproteinnuclease nicks and degrades onestrandcompetence-specificproteinStreptococcus pneumoniae

Artificial transformationTransformation done in laboratory with species that are not normally competent (E. coli)Variety of techniques used to make cells temporarily competentcalcium chloride treatmentmakes cells more permeable to DNA

Cloning vectors

pAmp

Transformation mappingused to establish gene linkageexpressed as frequency of cotransformationif two genes close together, greater likelihood will be transferred on single DNA fragment

Microbial GeneticsBacteriophages

Diversification of Escherichia coli genomes: are bacteriophages the major contributors? Makoto Ohnishi Trends in MicrobiologyE. coli is a diverse species4.5 5.5 MBE. coli strains are commensals of higher vertebrates, but some are pathogenicThere are 5subtypes of the diarrheagneic strainsdThe pathogenicity of the strains has been traced to a subtype that retains a large segment of virulence factors or pathogenicity islands

E. Coli O 157 Sixteen sections of this pathogenic strain differ from the lab strainThese are subtype specificWithin sections of the DNA and these large segmentsThe G-C content varies from the lab strainThe 4.1 kb common backbone sequence mainly represent the DNA that RE. coli possesses from a common ancestor.

E. coliThere are 98 copies of IS elements within this section as well as genes enxoding hemolysins, proteases, and other virulence factors.More interesting O 157 also contains 18 remnants of prophages

Horizontal gene transferClearly this plays a central role in the diversity of E. coliAmong the 18 prophage remnants on O157 12 resemble lambda pahgeThey all contain a variety of deletions and or insertionsSome of the phages are so similar that they contain a 20 kb segment tat is identical.

Recombinant phagesIt is believed that the phages have undergone recombination and diversificationRecombination could occur with in a single cellIt could occur as the result of recombination

Virulence and Strptococcus pyogenes Streptococcal pyrogenic exotoxins(SPE) contribute to the diverse symptoms of a streptococcal infection. These antigens compare to Staphylococcal antigens of the same type. The A + C genes coding for these toxins were horizontally transferred from strain to strain by a lysogenic bacteriophage. In addition the genes contributed by the phages produce hyaluronidase, mitogenic factor, and leukocyte( WBC) toxins

Streptococcus pyogenesThere are 15 prophages that have been identified in E. coliThese prophages belong to the group SiphoridaeAll but one of these produce a toxinIn both strep and staph the prophage is found at the site of recombination

Bacteriophages

BacteriophagesBacterial virusesObligate intracellular parasitesInject themselves into a host bacterial cellTake over the host machinery and utilize it for protein synthesis and replication

T- 4 BacteriophageDs DNA virus168, 800 base pairsPhage life cycles studied by Luria and Delbruck

Bacteriophage structure

Bacteriophage structure(con)Most bacteriophages have tailsThe size of the tail varies.It is a tube through which the nucleic acid is injected as a result of attachment of the bacteriophage to the host bacteriumIn the more complex phages the tail is surrounded by a contractile sheath for injection of the nucleic acids

Bacteriophage structureMany bacteriophages have a base plate and tail fibersSome have icosahedral capsidsM13 has a helical capsid

Bacteriophage structure(con)Most bacteriophages have tailsThe size of the tail varies.It is a tube through which the nucleic acid is injected as a result of attachment of the bacteriophage to the host bacteriumIn the more complex phages the tail is surrounded by a contractile sheath for injection of the nucleic acids

Bacteriophage structureMany bacteriophages have a base plate and tail fibersSome have icosahedral capsidsM13 has a helical capsid

PhiX 174The spherical phage (PhiX174, G4, S13) are broadly similar to the filamentous phage. The capsid is icosahedral not helical and is not enveloped (these phage lyse the host cell). Their genome consists of a circular ssDNA molecule. A well-known examples is PhiX174, which was the first genome to be sequenced - by Fred Sanger's group in 1976. Its genome of 5386 bp coded for 11 genes, including several examples of overlapping genes.

PhiX174 economy and overlapping genesThe coding frames for 7 proteins overlap: A* is a truncated form of A; B is coded within A in a different reading frame; K is encoded in a third reading frame at the end of A which extends into and overlaps with that of C; E is coded within D in a different reading frame. These were the first examples of overlapping genes. Other relatives of PhiX174 are G4 and S13.

PhiX174Gene A RF replication: viral strand synthesisA* Turning off host DNA synthesisB Formation of capsidE Lysis of bacteriumF major coat proteinG Major spike protein

GeneticsConversion of a parental single stranded DNA molecule to the viral + strand to a covalently closed double stranded moleculeThis is called the Replicative form( RFI)Synthesis of many copies of RFI. The strand is transcribed. Gene A product is made and the process continuew

Synthesis of + strands for encapsidationThere is not switch It just occursDuring the period that the phage capsids( heads ) are being synthesized

Ss RNA virusesssRNA phagesTailess icosahedralSingle stranded linear molecule, having a great deal of intramolecular hydrogen bondingConsists of 3600 nucleotidesGenes for attachment, coat protein and an RNA polymerase

ReplicationThe RNA molecule serves a both a replication template and the mRNA

Filamentous phagesFdFilamentousCircular ss DNALies in the middle of the filmentInfects through the pilusCreate a symbiotic relationship with the host

M 13

Sequential steps- M13Cloning vector Joachim Messier phage particles bind to F pilus only infects F+, Hfr, F' cellssingle-stranded DNA genome enters celldesignated as + strand+ strand repaired double-stranded replicative form (RF)RF contains + and strands strand is template for mRNA synthesis for production of new + strands by rolling circle replication+ strands are packaged in phage coat protein exit cell as phage particleImportant points for cloning vectors

M13 and cloningM13 occurs in both single and double stranded formsRF can be digested with restriction endonucleasesinserts can be cloned in like plasmid+ strands from phage particles convenient source of single-stranded DNA

M13 used for sequencing and site-directed mutagenesisdifferent sized DNA molecules packaged as phage particle (within reason) phage with inserts > 2 kb replicated slowerdifferent sized DNA molecules produce different size phage particles

M13 Phage

General Steps

T even phagesLuria and DelbruckFour distinct periods in the release of phages from host cellsLatent period- follows the addition of phage( no release of virions)Eclipse period virions were detectable before infection and are now hidden or eclipsedRise or burst period Host cells rapidly burst and release virusesThe total number of phages released can be determined by the burst size the number of viruses produced per infected cell

Steps in the life cycle

Adsorption of the virus to the hostThis is mediated by tail fibers or some analagous structureWhen the tail fibers make contact, the base plate settles to the surfaceThis connection which is maintianed by electrostatic attraction and the ions Mg++ and Ca++

AttachmentThere is host specificity in the attachment and adsorption of the bacteriophageThere are receptors for the attachment. They vary from bacteria to bacteriaThe receptors are on the bacteria for other purposes: the bacteriophages evolved to utilize them for their invasion

T even phagesThe phage sheath shortens from 24 rings to 12 ringsThe sheath becomes shorter and widerThis causes the central tube to push through the bacterial cell wall

Gp5The baseplate contains the protein gp5 with lysozyme activity which made aid in the penetration of the host

Penetration and other PhagesPenetration by other phages may differPRD1 phage attaches to a surface receptor by a spike on one of its capsid vertices

Conformational Changes and PRD1As a result of the binding a tubular structure is formed that allows the virus to penetrate the Penetration of the membrane tube is made by the membrane enzyme P7These phages have a major effect on the bacteriaceae

Early GenesE. coli RNA polymerase starts transcribing genes( phage genes) within minutes of entering the bacterial cellThe early m RNA direct the synthesis of proteins and enzymes that are needed for hostile tack overSome early virus specific enzymes degrade host DNA to nucleotides wo that virus DNA synthesis can commence

HydroxymethylcytosineHMC is needed for synthesis instead of cytosineHMC must be glucosylated by the addition of glucose to protect from restriction enzymes

T4 and terminal redundancyThe end has terminal redundancyWhen multiple coies have been made enzymes join the copies by therse endsWhen several untis are linked together this forms concatamers

Late mRNAPhage structural structural proteinsProteins that help with pahge assemblyProteins involved in cell lysis and release

CapsidThe base plate requires 12 protein productsThe head or capsid requires 10 genesThe capside requires scaffolding proteins for assemblyDNA packaging a mysterious processMany phages lyse their host cells at the end of the intracellular phase

ReleaseInterference with the synthesis of the bacterial cell wallPhiX 174 produces a lytic enzyme that interfers with the urein precursos

Irreversible attachmentThe attachment of the tail ribers to the bacterium is a weak attachmentThe attachment of the bacterophage is also accompanied by a stronger interaction usually by the base plate

Sheath contractionThe irreversible binding results in the sheath contraction

InjectionWhen the irreversible attachment has been made and the sheath contracts, the nucleic acid passes through the tail and enters the cytoplasm

Phage Multiplication Cycle Lytic phagesLytic phages or virulent phages enter the bacterial cell, complete protein synthesis, nucleic acid replication, and then cause lysis of the bacterial cell when the assembly of the particles has been completed.

Eclipse PeriodThe bacteriophages may be seen inside or outside of the bacterial cellsThe phages take over the cells machinery and phage specific mRNAs are madeEarly mRNAs are generally needed for DNA replicationLater mRNAs are required for the synthesis of phage proteins

Intracellular accumulation phaseThe bacteriophage sub units accumulate in the cytoplasm of the bacterial cell and are assembled

Lysis or Release PhaseA lysis protein is releasedThe bacterial cell breaks openThe viruses escape to invade other bacterial cells

Plaque assayPhage infection and lysis can easily be detected in bacterial cultures grown on agar platesTypically bacterial cells are cultured in high concentrations on the surface of an agar plateThis produces a bacterial lawnPhage infection and lysis can be seen as a clear area on the plate. As phage are released they invade neighboring cells and produce a clear area

Plaque assay

Lambda and PlaquesThe plaque produced by Lambda had a different appearance on the Petri Dish.It is considered to be turbid rather than clearThe turbidiy is the result of the growth of phage immune lysogens in the plaqueThe agar surface contains a ratio of about a phage /107 bacteria

MOIAverage number of phages /bacteriumAfter several lytic cycles the MOI gets higher due to the release of phage particles

TransductionTransfer of bacterial genes by virusesVirulent bacteriophagesreproduce using lytic life cycleTemperate bacteriophagesreproduce using lysogenic life cycle

Generalized transductionhttp://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transduction/gentran.htmlhttp://www.cat.cc.md.us/courses/bio141/lecguide/unit1/control/genrec/u4fg21a.html

Generalized transductionE. coli phage P21 or P22.As a part of the lytic cycle, the phage cuts the bacterial DNA into fragmentsThis fragmentation prevents the expression of bacterial genesNucleotides can be used to make phage DNAOccasionally these DNA fragments are about the same size as phage DNAThey become mistakenly packaged into phage capsids in place of phage DNA

Types of Lysogenic CycleThe most common type is the classic model of the Lambda phageThe DNA molecule is injected into a bacteriumIn a short period of time, after a brief period of transcription, an integration factor and a repressor are synthesizedA phage DNA molecule typically a replica of the injected molecules is inserted into the DNA As the bacterium continue to grow and multiply and the phage genes replicate as part of the bacterial chromosome

The P1 temperate phageThere is not integration into the hostThe phage becomes a plasmidIt exists as an independently replicating entity in the bacterial cell in the same way a plasmid exists.

TemperateA bacteriophage that can exist as a lytic or lysogenic phage is referred to as a temperate phageA bacterium containing a full set of phage genes is a lysogenThe process of infecting a bacterial culture with a temperate phage is called lysogenization

ImmunizationA bacterial cell or lysogen cannot be reinfected by a phage of the same typeThis is resistance to superinfection is called immunityMore than 90% of the bacteriophages are temperateThese are unable to produce bursts such as T4 and T7

Lysogenic Phage

Lambda PhageTemperate phageAlternate life cycleDs DNA linear then circularizes when it enters the host48,502 base pairsMolecular biology workhorse because of its life cycle

GenesLambda genes46 genes have been identified14 are non esswential to the lytic cycleOnly 7 are nonessential to both the lytic and lysogenic cycles

Lambda Gene MapGenes are clustered according to functionThere are four clustersHeadTail ReplicationRecombination genesRegulatory genes act at specific site on the DNA

Restriction sites

Life cycle of Phage

LatencyLysogenic conversion can lead to virulenceBotulism, cholera,and diptheria toxins are encoded by prophages that convert their host into a pathogenic bacterium

Control of lysogeny and lytic cycle

Genes needed to establish lysogenycI yescII yescIII yesGenes needed for maintenance of lysogenycI yescII nocIII no

LambdaIn order for the lambda prophage to exist in a host E.coli cell, it must integrate into the host chromosome which it does by means of a site-specific recombination reaction.

Preferred site of integrationIt is inserted into the E. coli chromosome between the gal operon and the biotin operon.The site of attachment is specific just for the Lambda phage ( att)

Lambda Phage Genesrepression of all lytic functions cI: the lambda repressor, when present and active, will repress lytic functions. The counterpart of cI is cro, a repressor of cI. The initial "decision" that lambda makes is based on the outcome of a battle over cI synthesis lambda DNA is injected and circularized initially, cII is made cII is a positive regulator of cI synthesis. If cII is around long enough, then cI will be made, and cI will repress cro and other lytic functions if cII is degraded quickly, cro will build up, and cI will not be synthesized

Lambda Phage Geneswhether cII is degraded or not depends on the health of the host. A healthy cell will degrade cII quickly, and in effect signal to lambda that a lytic cycle would be good. An unhealthy cell will not degrade cII, which is like telling lambda that the cell is too sick to make viral progeny, so lysogeny is the better idea

TerminologyLEGEND att: an E.coli seqence for the "attachment" or integration of lambda's circular chromosome. oriC: E.coli's origin of Chromosome replication (given here for orientation only) gal: E.coli's gene for galactose utilization pe:prophage ends (site of integration) cos: joined sticky ends of vegetative DNA; sometimes called ve ("vegetative ends") int: gene for the enzyme integrase c: gene for lambda repressor to maintain lysogeny Q: another gene concerned with lysogeny h: the last of the many capsomer genes.

Bacteriophages

Specialized transduction

Attachment siteThe E.coli chromosome contains one site at which lambda integrates. The site, located between the gal and bio operons, is called the attachment site and is designated attB since it is the attachment site on the bacterial chromosome. The site is only 30 bp in size and contains a conserved central 15 bp region where the recombination reaction will take place. The structure of the recombination site was determined originally by genetic analyses and is usually represented as BOB', where B and B' represent the bacterial DNA on either side of the conserved central element

Recombination siteThe bacteriophage recombination site - attP - is more complex. It contains the identical central 15 bp region as attB. The overall structure can be represented as POP'. However, the flanking sequences on either side of attP are very important since they contain the binding sites for a number of other proteins which are required for the recombination reaction. The P arm is 150 bp in length and the P' arm is 90 bp in length.

IntegrationIntegration of bacteriophage lambda requires one phage-encoded protein - Int, which is the integrase - and one bacterial protein - IHF, which is Integration Host Factor. Both of these proteins bind to sites on the P and P' arms of attP to form a complex in which the central conserved 15 bp elements of attP and attB are properly aligned. The integrase enzyme carries out all of the steps of the recombination reaction, which includes a short 7 bp branch migration.

Enzymes and RecombinationThere are two major groups of enzymes that carry out site-specific recombination reactions; one group - known as the tyrosine recombinase family - consists of over 140 proteins. These proteins are 300-400 amino acids in size, they contain two conserved structural domains, and they carry out recombination reactions using a common mechanism involving a the formation of a covalent bond with an active site tyrosine residue.

Enzymes and RecombinationThe strand exchange reaction involves staggered cuts that are 6 to 8 bp apart within the recognition sequence. All of the strand cleavage and re-joining reactions proceed through a series of transesterification reactions like those mediated by type I topoisomerases.

Excision of bacteriophagesExcision of bacteriophage lambda requires two phage-encoded proteins: Int (again!) and Xis, which is an excisionase. It also requires several bacterial proteins. In addition to IHF, a protein called Fis is required. All of these proteins bind to sites on the P and P' arms of attL and attR forming a complex in which the central conserved 15 bp elements of attL and attR are properly aligned to promote excision of the prophage.

Normal Excision

Excision and lysisThe reverse of integration happens upon induction UV light is a good inducer induction actually involves RecA. RecA is activated by ssDNA. Activated RecA interacts with cI, and causes cI to proteolyze itself. Without cI, lytic functions are derepressed, and the lytic cycle begins. induction also results in the synthesis of both Int and Xis only int is required for integration, but both are required for excision normal excision (which usually occurs) produces the cicular viral genome, and lysis continues

Generalized TransductionAny part of bacterial genome can be transferredOccurs during lytic cycleDuring viral assembly, fragments of host DNA mistakenly packaged into phage headgeneralized transducing particle

Generalized transduction

Specialized Transductionalso called restricted transductioncarried out only by temperate phages that have established lysogenyonly specific portion of bacterial genome is transferredoccurs when prophage is incorrectly excised

Specialized transductionFigure 13.20

Figure 13.20

Generalized Transduction Mappingused to establish gene linkageexpressed as frequency of cotransductionif two genes close together, greater likelihood will be carried on single DNA fragment in transducing particle

Recombination and Genome Mapping in Virusesviral genomes can also undergo recombination eventsviral genomes can be mapped by determining recombination frequenciesphysical maps of viral genomes can also be constructed using other techniques

Specialized transduction mappingprovides distance of genes from viral genome integration sitesviral genome integration sites must first be mapped by conjugation mapping techniques

Recombination mappingrecombination frequency determined when cells infected simultaneously with two different virusesFigure 13.24

Physical mapsheteroduplex mapsgenomes of two different viruses denatured, mixed and allowed to annealregions that are not identical, do not reannealallows for localization of mutant alleles

Physical mapsrestriction endonuclease mappingcompare DNA fragments from two different viral strains in terms of electrophoretic mobilitysequence mappingdetermine nucleotide sequence of viral genomeidentify coding regions, mutations, etc.

Lambda Phage Genesrepression of all lytic functions cI: the lambda repressor, when present and active, will repress lytic functions. The counterpart of cI is cro, a repressor of cI. The initial "decision" that lambda makes is based on the outcome of a battle over cI synthesis lambda DNA is injected and circularized initially, cII is made cII is a positive regulator of cI synthesis. If cII is around long enough, then cI will be made, and cI will repress cro and other lytic functions if cII is degraded quickly, cro will build up, and cI will not be synthesized

Lamda Phage Geneswhether cII is degraded or not depends on the health of the host. A healthy cell will degrade cII quickly, and in effect signal to lambda that a lytic cycle would be good. An unhealthy cell will not degrade cII, which is like telling lambda that the cell is too sick to make viral progeny, so lysogeny is the better idea

The linear transfer of genes is stopped by breaking the conjugation bridge to study the sequence of gene entry into the recipient cell.Example of results obtained by an interrupted mating experiment. The gene order is lac-tsx-gal-trp.The transforming DNA is in red and integration is at a homologous region of the genome.Transformation with a plasmid is often induced artificially in the laboratory. The transforming DNA is in red and integration is at a homologous region of the genome.Mechanism of transformation in S pneumoniae. A long double stranded DNA binds cell surface with the aid of DNA-binding proteins and is nicked by nuclease. One strand is degraded by nuclease. The undegraded strand associates with a competence-specific protein. The single strand enters the cell and is integrated into the host chromosome at a homologous site in the host DNA.