1. Recombination, Bacteriophages, and Horizontal Gene Transfer
2005

2. Bacterial Conjugation
transfer of DNA by direct cell to cell contact
discovered 1946 by Lederberg and Tatum

3. F+ x F– Mating F+ = donor F– = recipient
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+

4. 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-".

5. 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

6. F x F– mating

7. Hfr Conjugation Hfr strain
donor having F factor integrated into its chromosome
both plasmid genes and chromosomal genes are transferred

8. 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

9. 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

10. Mating

11. Hfr x F– mating
Figure 13.14b

12. Recombination
Exogenote

13. 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

14. 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.

15. 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’

16. F Conjugation integrated F factor F plasmid chromosomal gene
formed by incorrect excision from chromosome
contains  1 genes from chromosome
F cell can transfer F plasmid to recipient
chromosomal gene
Figure 13.15a

17. 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

18. Interrupted mating Figure 13.22a
The linear transfer of genes is stopped by breaking the conjugation bridge to study the sequence of gene entry into the recipient cell.
Figure 13.22a

19. Example of results obtained by an interrupted mating experiment
Example of results obtained by an interrupted mating experiment. The gene order is lac-tsx-gal-trp.
Figure 13.22b

20. 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

21. Gene mapping

22. 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

23. Mapping results

24. Map distance
The map distance is equal to the % recombination

25. 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

26. 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)

27. 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

28. 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

29. The transforming DNA is in red and integration is at a homologous region of the genome.

30. 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.

31. Streptococcus pneumoniae
DNA binding
protein
nuclease – nicks and degrades one
strand
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.
competence-specific
protein

32. 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

33. Cloning vectors

34. pAmp

35. 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

36. Microbial Genetics
Bacteriophages

37. E. coli is a diverse species 4.5 – 5.5 MB
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

38. E. Coli O 157
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.

39. E. coli
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

40. Horizontal gene transfer
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.

41. Recombinant phages
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

42. 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

43. Streptococcus pyogenes
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

44. Bacteriophages

45. Bacteriophages 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

46. T- 4 Bacteriophage Ds DNA virus 168, 800 base pairs
Phage life cycles studied by Luria and Delbruck

47. Bacteriophage structure

48. Bacteriophage structure(con)
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

49. Bacteriophage structure
Many bacteriophages have a base plate and tail fibers
Some have icosahedral capsids
M13 has a helical capsid

50. Bacteriophage structure(con)
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

51. Bacteriophage structure
Many bacteriophages have a base plate and tail fibers
Some have icosahedral capsids
M13 has a helical capsid

52. PhiX 174
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.

53. PhiX174 – economy and overlapping genes
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.

54. PhiX174 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

55. Genetics
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

56. Synthesis of + strands for encapsidation
There is not switch – It just occurs
During the period that the phage capsids( heads ) are being synthesized

57. Ss RNA viruses 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

58. Replication
The RNA molecule serves a both a replication template and the mRNA

59. Filamentous phages Fd Filamentous Circular ss DNA
Lies in the middle of the filment
Infects through the pilus
Create a symbiotic relationship with the host

60. M 13

61. Sequential steps - M13 Cloning 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

62. M13 and cloning 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

63. M13 – 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

64. M13 Phage

65. General Steps

66. T even phages Luria 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

67. Steps in the life cycle 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++

68. Attachment
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

69. T even phages 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

70. Gp5
The baseplate contains the protein gp5 with lysozyme activity which made aid in the penetration of the host

71. Penetration and other Phages
Penetration by other phages may differ
PRD1 phage attaches to a surface receptor by a spike on one of its capsid vertices

72. Conformational Changes and PRD1
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

73. Early Genes
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

74. Hydroxymethylcytosine
HMC is needed for synthesis instead of cytosine
HMC must be glucosylated by the addition of glucose to protect from restriction enzymes

75. T4 and terminal redundancy
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

76. Late mRNA Phage structural structural proteins
Proteins that help with pahge assembly
Proteins involved in cell lysis and release

77. Capsid 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

78. Release Interference with the synthesis of the bacterial cell wall
PhiX 174 produces a lytic enzyme that interfers with the urein precursos

79. Irreversible attachment
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

80. Sheath contraction
The irreversible binding results in the sheath contraction

81. Injection
When the irreversible attachment has been made and the sheath contracts, the nucleic acid passes through the tail and enters the cytoplasm

82. Phage Multiplication Cycle – Lytic phages
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.

83. Eclipse Period
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

84. Intracellular accumulation phase
The bacteriophage sub units accumulate in the cytoplasm of the bacterial cell and are assembled

85. Lysis or Release Phase A lysis protein is released
The bacterial cell breaks open
The viruses escape to invade other bacterial cells

86. Plaque assay
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

87. Plaque assay

88. Lambda and Plaques
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

89. MOI Average number of phages /bacterium
After several lytic cycles the MOI gets higher due to the release of phage particles

90. Transduction Transfer of bacterial genes by viruses
Virulent bacteriophages
reproduce using lytic life cycle
Temperate bacteriophages
reproduce using lysogenic life cycle

91. Generalized transduction

92. Generalized transduction
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

94. Types of Lysogenic Cycle
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

95. The P1 temperate phage 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.

96. Temperate
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

97. Immunization
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

98. Lysogenic Phage

99. Lambda Phage 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

100. Genes 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

101. Lambda Gene Map Genes are clustered according to function
There are four clusters
Head
Tail
Replication
Recombination genes
Regulatory genes act at specific site on the DNA

102. Restriction sites

103. Life cycle of λ Phage

104. Latency Lysogenic conversion can lead to virulence
Botulism, cholera,and diptheria toxins are encoded by prophages that convert their host into a pathogenic bacterium

105. Control of lysogeny and lytic cycle
Genes needed to establish lysogeny
cI yes
cII yes
cIII yes
Genes needed for maintenance of lysogeny
cI yes
cII no
cIII no

106. Lambda
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.

107. Preferred site of integration
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)

108. Lambda Phage Genes 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

109. Lambda Phage Genes
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

111. Terminology
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.

113. Bacteriophages

115. Specialized transduction

116. Attachment site
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

117. Recombination site
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.

118. Integration
Integration 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.

119. Enzymes and Recombination
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 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.

120. Enzymes and Recombination
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.

121. Excision of bacteriophages
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.

122. Normal Excision

123. Excision and lysis 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

124. Generalized Transduction
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

125. Generalized transduction

126. Specialized Transduction
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

127. Specialized transduction
Figure 13.20

128. Figure 13.20

129. Generalized Transduction Mapping
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

130. Recombination and Genome Mapping in Viruses
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

131. Specialized transduction mapping
provides distance of genes from viral genome integration sites
viral genome integration sites must first be mapped by conjugation mapping techniques

132. Recombination mapping
recombination frequency determined when cells infected simultaneously with two different viruses
Figure 13.24

133. Physical maps 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

134. Physical maps… restriction endonuclease mapping sequence 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.

135. Lambda Phage Genes 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

136. Lamda Phage Genes
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