Recombination bacteriophages and horizontal gene transfer
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Recombination, Bacteriophages, and Horizontal Gene Transfer. 2005. Bacterial Conjugation. transfer of DNA by direct cell to cell contact discovered 1946 by Lederberg and Tatum. F + x F – Mating. F + = donor contains F factor F – = recipient does not contain F factor

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Bacterial conjugation
Bacterial Conjugation

  • transfer of DNA by direct cell to cell contact

  • discovered 1946 by Lederberg and Tatum


F x f mating
F+x F– Mating

  • F+ = donor

    • contains F factor

  • F– = recipient

    • does not contain F factor

  • F factor replicated by rolling-circle mechanism and duplicate is transferred

  • recipients usually become F+

  • donor remains F+


F factor
F factor

  • The 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 recombination
Gene transfer and recombination

  • Genes are transferred in a linear manner

  • The F factor integrates into chromosomes at different points and its position determines the O site


F x f mating1
F x F– mating


Hfr conjugation
Hfr Conjugation

  • Hfr strain

    • donor having F factor integrated into its chromosome

  • both plasmid genes and chromosomal genes are transferred


Recombination bacteriophages and horizontal gene transfer
Hfr

  • Special class of F+ strains

  • This was discovered because this strain underwent recombination 1000x more frequently than F+ strains

  • In 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 mating
Interrupted mating

  • Wollman explained the cells that are different between F+ and Hfr. To facilitate the recovery, the Hfr was sensitive to antibiotics and the F+ wasn’t.

  • The cells were separated at intervals of 5 minutes is the F factor



Hfr x f mating
Hfr x F– mating

Figure 13.14b


Recombination
Recombination

  • Exogenote

  • Exogenote


Mating1
Mating

  • The two strains were mixed

  • There were incubated.

  • At intervals of 5 minutes, samples were taken of the F- cells

  • The 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 mating2
F+x F– mating

  • In 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 À sequence as well as genes for incompatibility and replication.


Recombination bacteriophages and horizontal gene transfer
F’

  • In 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 conjugation
F Conjugation

integrated F factor

  • F plasmid

    • formed by incorrect excision from chromosome

    • contains  1 genes from chromosome

  • F cell can transfer F plasmid to recipient

chromosomal gene

Figure 13.15a


Merozygotes
Merozygotes

  • When the F’ is then transferred to another bacterium

  • The 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 diploid

  • Merozygotes have been extremely beneficial in the study of gene regulation


Interrupted mating1
Interrupted mating

Figure 13.22a



Hfr mapping
Hfr mapping

  • used to map relative location of bacterial genes

  • based on observation that chromosome transfer occurs at constant rate

  • interrupted mating experiment

    • Hfr x F- mating interrupted at various intervals

    • order and timing of gene transfer determined



Recombinants
Recombinants

  • Map 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



Map distance
Map distance

  • The map distance is equal to the % recombination


Tra y
Tra Y

  • Characterization of the Escherichia coli F factor traY gene product and its binding sites

  • WC Nelson, BS Morton, EE Lahue and SW Matson Department of Biology, University of North Carolina, Chapel Hill 27599.


Tra genes
Tra Genes

  • Tra Y gene codes for the protein binds to the Ori T

  • Initiates the transfer of plasmid across the bridge between the two cells

  • Tra I Gene is a helicase responsible for the conjugation

  • strand-specific transesterification (relaxase)


Conjugative proteins
Conjugative Proteins

  • Key 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 transfer

  • Working in conjunction with the helicases they facilitate the transfer of ss RNA to the F- cell


Dna transformation
DNA Transformation

  • Uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form

  • Competent cell

    • capable of taking up DNA

  • May be important route of genetic exchange in nature


Recombination bacteriophages and horizontal gene transfer

Streptococcus pneumoniae

nuclease – nicks and degrades one

strand

DNA binding

protein

competence-specific

protein


Artificial transformation
Artificial transformation

  • Transformation done in laboratory with species that are not normally competent (E. coli)

  • Variety of techniques used to make cells temporarily competent

    • calcium chloride treatment

      • makes cells more permeable to DNA




Transformation mapping
Transformation mapping

  • used to establish gene linkage

  • expressed as frequency of cotransformation

  • if two genes close together, greater likelihood will be transferred on single DNA fragment


Bacteriophages

Bacteriophages

Microbial Genetics


Recombination bacteriophages and horizontal gene transfer
Diversification of Escherichia coli genomes: are bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • E. coli is a diverse species

  • 4.5 – 5.5 MB

  • E. coli strains are commensals of higher vertebrates, but some are pathogenic

  • There are 5subtypes of the diarrheagneic strainsd

  • The 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
E. Coli O 157 bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Sixteen sections of this pathogenic strain differ from the lab strain

  • These are subtype specific

  • Within sections of the DNA and these large segments

  • The G-C content varies from the lab strain

  • The 4.1 kb common backbone sequence mainly represent the DNA that RE. coli possesses from a common ancestor.


E coli
E. coli bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • There 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 transfer
Horizontal gene transfer bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Clearly this plays a central role in the diversity of E. coli

  • Among the 18 prophage remnants on O157 – 12 resemble lambda pahge

  • They all contain a variety of deletions and or insertions

  • Some of the phages are so similar that they contain a 20 kb segment tat is identical.


Recombinant phages
Recombinant phages bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • It is believed that the phages have undergone recombination and diversification

  • Recombination could occur with in a single cell

  • It could occur as the result of recombination


Virulence and strptococcus pyogenes
Virulence and bacteriophages the major contributors? Makoto Ohnishi – Trends in MicrobiologyStrptococcus 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 pyogenes
Streptococcus pyogenes bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • There are 15 prophages that have been identified in E. coli

  • These prophages belong to the group Siphoridae

  • All but one of these produce a toxin

  • In both strep and staph – the prophage is found at the site of recombination


Bacteriophages1
Bacteriophages bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Bacteriophages2
Bacteriophages bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Bacterial viruses

  • Obligate intracellular parasites

  • Inject themselves into a host bacterial cell

  • Take over the host machinery and utilize it for protein synthesis and replication


T 4 bacteriophage
T- 4 Bacteriophage bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Ds DNA virus

  • 168, 800 base pairs

  • Phage life cycles studied by Luria and Delbruck


Bacteriophage structure
Bacteriophage structure bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Bacteriophage structure con
Bacteriophage structure(con) bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Most bacteriophages have tails

  • The 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 bacterium

  • In the more complex phages the tail is surrounded by a contractile sheath for injection of the nucleic acids


Bacteriophage structure1
Bacteriophage structure bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Many bacteriophages have a base plate and tail fibers

  • Some have icosahedral capsids

  • M13 has a helical capsid


Bacteriophage structure con1
Bacteriophage structure(con) bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Most bacteriophages have tails

  • The 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 bacterium

  • In the more complex phages the tail is surrounded by a contractile sheath for injection of the nucleic acids


Bacteriophage structure2
Bacteriophage structure bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Many bacteriophages have a base plate and tail fibers

  • Some have icosahedral capsids

  • M13 has a helical capsid


Phix 174
PhiX 174 bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The 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 genes
PhiX174 – economy and overlapping genes bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The 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.


Phix174
PhiX174 bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Gene A – RF replication: viral strand synthesis

  • A* Turning off host DNA synthesis

  • B Formation of capsid

  • E Lysis of bacterium

  • F major coat protein

  • G Major spike protein


Genetics
Genetics bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Conversion of a parental single stranded DNA molecule to the viral + strand to a covalently closed double stranded molecule

  • This 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 encapsidation
Synthesis of + strands for encapsidation bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • There is not switch – It just occurs

  • During the period that the phage capsids( heads ) are being synthesized


Ss rna viruses
Ss RNA viruses bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • ssRNA phages

  • Tailess icosahedral

  • Single stranded linear molecule, having a great deal of intramolecular hydrogen bonding

  • Consists of 3600 nucleotides

  • Genes for attachment, coat protein and an RNA polymerase


Replication
Replication bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The RNA molecule serves a both a replication template and the mRNA


Filamentous phages
Filamentous phages bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Fd

  • Filamentous

  • Circular ss DNA

  • Lies in the middle of the filment

  • Infects through the pilus

  • Create a symbiotic relationship with the host


Recombination bacteriophages and horizontal gene transfer

M 13 bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Sequential steps m13 cloning vector joachim messier
Sequential steps bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology- M13Cloning vector – Joachim Messier

  • phage particles bind to F pilus

  • – only infects F+, Hfr, F' cells

  • single-stranded DNA genome enters cell

  • designated 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 particle

  • Important points for cloning vectors


M13 and cloning
M13 and cloning bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • M13 occurs in both single and double stranded forms

  • RF can be digested with restriction endonucleases

  • inserts can be cloned in – like plasmid

  • “+” strands from phage particles

  • – convenient source of single-stranded DNA


Recombination bacteriophages and horizontal gene transfer
M13 bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • – used for sequencing and site-directed mutagenesis

  • different sized DNA molecules packaged as phage particle

  • – (within reason)

  • – phage with inserts > 2 kb replicated slower

  • different sized DNA molecules

  • – produce different size phage particles


M13 phage
M13 Phage bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


General steps
General Steps bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


T even phages luria and delbruck
T even phages bacteriophages the major contributors? Makoto Ohnishi – Trends in MicrobiologyLuria and Delbruck

  • Four distinct periods in the release of phages from host cells

  • Latent period- follows the addition of phage( no release of virions)

  • Eclipse period – virions were detectable before infection and are now hidden or eclipsed

  • Rise or burst period – Host cells rapidly burst and release viruses

  • The 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
Steps in the life cycle bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Adsorption of the virus to the host

  • This is mediated by tail fibers or some analagous structure

  • When the tail fibers make contact, the base plate settles to the surface

  • This connection which is maintianed by electrostatic attraction and the ions Mg++ and Ca++


Attachment
Attachment bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • There is host specificity in the attachment and adsorption of the bacteriophage

  • There are receptors for the attachment. They vary from bacteria to bacteria

  • The receptors are on the bacteria for other purposes: the bacteriophages evolved to utilize them for their invasion


T even phages
T even phages bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The phage sheath shortens from 24 rings to 12 rings

  • The sheath becomes shorter and wider

  • This causes the central tube to push through the bacterial cell wall


Recombination bacteriophages and horizontal gene transfer
Gp5 bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

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


Penetration and other phages
Penetration and other Phages bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Penetration by other phages may differ

  • PRD1 phage attaches to a surface receptor by a spike on one of its capsid vertices


Conformational changes and prd1
Conformational Changes and PRD1 bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • As 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 P7

  • These phages have a major effect on the bacteriaceae


Early genes
Early Genes bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • E. coli RNA polymerase starts transcribing genes( phage genes) within minutes of entering the bacterial cell

  • The early m RNA direct the synthesis of proteins and enzymes that are needed for hostile tack over

  • Some early virus specific enzymes degrade host DNA to nucleotides wo that virus DNA synthesis can commence


Hydroxymethylcytosine
Hydroxymethylcytosine bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • HMC is needed for synthesis instead of cytosine

  • HMC must be glucosylated by the addition of glucose to protect from restriction enzymes


T4 and terminal redundancy
T4 and terminal redundancy bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The end has terminal redundancy

  • When multiple coies have been made enzymes join the copies by therse ends

  • When several untis are linked together this forms concatamers


Late mrna
Late mRNA bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Phage structural structural proteins

  • Proteins that help with pahge assembly

  • Proteins involved in cell lysis and release


Capsid
Capsid bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The base plate requires 12 protein products

  • The head or capsid requires 10 genes

  • The capside requires scaffolding proteins for assembly

  • DNA packaging a mysterious process

  • Many phages lyse their host cells at the end of the intracellular phase


Release
Release bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Interference with the synthesis of the bacterial cell wall

  • PhiX 174 produces a lytic enzyme that interfers with the urein precursos


Irreversible attachment
Irreversible attachment bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The attachment of the tail ribers to the bacterium is a weak attachment

  • The attachment of the bacterophage is also accompanied by a stronger interaction usually by the base plate


Sheath contraction
Sheath contraction bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The irreversible binding results in the sheath contraction


Injection
Injection bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • When 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 phages
Phage Multiplication Cycle – Lytic phages bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Lytic 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 period
Eclipse Period bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The bacteriophages may be seen inside or outside of the bacterial cells

  • The phages take over the cell’s machinery and phage specific mRNA’s are made

  • Early mRNA’s are generally needed for DNA replication

  • Later mRNA’s are required for the synthesis of phage proteins


Intracellular accumulation phase
Intracellular accumulation phase bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The bacteriophage sub units accumulate in the cytoplasm of the bacterial cell and are assembled


Lysis or release phase
Lysis or Release Phase bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • A lysis protein is released

  • The bacterial cell breaks open

  • The viruses escape to invade other bacterial cells


Plaque assay
Plaque assay bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Phage infection and lysis can easily be detected in bacterial cultures grown on agar plates

  • Typically bacterial cells are cultured in high concentrations on the surface of an agar plate

  • This produces a “ bacterial lawn”

  • Phage 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 assay1
Plaque assay bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Lambda and plaques
Lambda and Plaques bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The plaque produced by Lambda had a different appearance on the Petri Dish.

  • It is considered to be turbid rather than clear

  • The turbidiy is the result of the growth of phage immune lysogens in the plaque

  • The agar surface contains a ratio of about a phage /107 bacteria


Recombination bacteriophages and horizontal gene transfer
MOI bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Average number of phages /bacterium

  • After several lytic cycles the MOI gets higher due to the release of phage particles


Transduction
Transduction bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Transfer of bacterial genes by viruses

  • Virulent bacteriophages

    • reproduce using lytic life cycle

  • Temperate bacteriophages

    • reproduce using lysogenic life cycle


Generalized transduction
Generalized transduction bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transduction/gentran.html

  • http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/control/genrec/u4fg21a.html


Generalized transduction1
Generalized transduction bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • E. coli phage P21 or P22.

  • As a part of the lytic cycle, the phage cuts the bacterial DNA into fragments

  • This fragmentation prevents the expression of bacterial genes

  • Nucleotides can be used to make phage DNA

  • Occasionally these DNA fragments are about the same size as phage DNA

  • They become mistakenly packaged into phage capsids in place of phage DNA


Types of lysogenic cycle
Types of Lysogenic Cycle bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The most common type is the classic model of the Lambda phage

  • The DNA molecule is injected into a bacterium

  • In a short period of time, after a brief period of transcription, an integration factor and a repressor are synthesized

  • A 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 phage
The P1 temperate phage bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • There is not integration into the host

  • The phage becomes a plasmid

  • It exists as an independently replicating entity in the bacterial cell in the same way a plasmid exists.


Temperate
Temperate bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • A bacteriophage that can exist as a lytic or lysogenic phage is referred to as a temperate phage

  • A bacterium containing a full set of phage genes is a lysogen

  • The process of infecting a bacterial culture with a temperate phage is called lysogenization


Immunization
Immunization bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • A bacterial cell or lysogen cannot be reinfected by a phage of the same type

  • This is resistance to superinfection is called immunity

  • More than 90% of the bacteriophages are temperate

  • These are unable to produce bursts such as T4 and T7


Recombination bacteriophages and horizontal gene transfer

Lysogenic Phage bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Lambda phage
Lambda Phage bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Temperate phage

  • Alternate life cycle

  • Ds DNA – linear then circularizes when it enters the host

  • 48,502 base pairs

  • Molecular biology workhorse – because of its life cycle


Genes
Genes bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

Lambda genes

  • 46 genes have been identified

  • 14 are non esswential to the lytic cycle

  • Only 7 are nonessential to both the lytic and lysogenic cycles


Lambda gene map
Lambda Gene Map bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Genes are clustered according to function

  • There are four clusters

  • Head

  • Tail

  • Replication

  • Recombination genes

  • Regulatory genes act at specific site on the DNA


Restriction sites
Restriction sites bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Life cycle of phage
Life cycle of bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiologyλ Phage


Latency
Latency bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Lysogenic conversion can lead to virulence

  • Botulism, 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 lysogeny bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

cI yes

cII yes

cIII yes

Genes needed for maintenance of lysogeny

cI yes

cII no

cIII no

Control of lysogeny and lytic cycle


Lambda
Lambda bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • In 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 integration
Preferred site of integration bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • It 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 genes
Lambda Phage Genes bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • repression 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 genes1
Lambda Phage Genes bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • whether 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


Terminology
Terminology bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • LEGEND

  • 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.


Bacteriophages3
Bacteriophages bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Recombination bacteriophages and horizontal gene transfer

Specialized transduction bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Attachment site
Attachment site bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The 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 site
Recombination site bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The 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.


Integration
Integration bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Integration of bacteriophage lambda requires one phage-encoded protein - Int, which is the integrase - and one bacterial protein - IHF, which is IntegrationHostFactor.

  • 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 recombination
Enzymes and Recombination bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • There 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 recombination1
Enzymes and Recombination bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The 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 bacteriophages
Excision of bacteriophages bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Excision 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
Normal Excision bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Excision and lysis
Excision and lysis bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • The 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 transduction2
Generalized Transduction bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • Any part of bacterial genome can be transferred

  • Occurs during lytic cycle

  • During viral assembly, fragments of host DNA mistakenly packaged into phage head

    • generalized transducing particle


Generalized transduction3
Generalized transduction bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Specialized transduction
Specialized Transduction bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • also called restricted transduction

  • carried out only by temperate phages that have established lysogeny

  • only specific portion of bacterial genome is transferred

  • occurs when prophage is incorrectly excised


Specialized transduction1
Specialized transduction bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

Figure 13.20


Recombination bacteriophages and horizontal gene transfer

Figure 13.20 bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology


Generalized transduction mapping
Generalized Transduction Mapping bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • used to establish gene linkage

  • expressed as frequency of cotransduction

  • if two genes close together, greater likelihood will be carried on single DNA fragment in transducing particle


Recombination and genome mapping in viruses
Recombination and Genome Mapping in Viruses bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • viral genomes can also undergo recombination events

  • viral genomes can be mapped by determining recombination frequencies

  • physical maps of viral genomes can also be constructed using other techniques


Specialized transduction mapping
Specialized transduction mapping bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • provides distance of genes from viral genome integration sites

  • viral genome integration sites must first be mapped by conjugation mapping techniques


Recombination mapping
Recombination mapping bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • recombination frequency determined when cells infected simultaneously with two different viruses

Figure 13.24


Physical maps
Physical maps bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • heteroduplex maps

    • genomes of two different viruses denatured, mixed and allowed to anneal

      • regions that are not identical, do not reanneal

    • allows for localization of mutant alleles


Physical maps1
Physical maps… bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • restriction endonuclease mapping

    • compare DNA fragments from two different viral strains in terms of electrophoretic mobility

  • sequence mapping

    • determine nucleotide sequence of viral genome

    • identify coding regions, mutations, etc.


Lambda phage genes2
Lambda Phage Genes bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • repression 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 genes
Lamda Phage Genes bacteriophages the major contributors? Makoto Ohnishi – Trends in Microbiology

  • whether 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