Transcript 4th Annual VCU-BBSI Closing Symposium

An Analysis of The Host
Range Determinants of a P2
Related Phage W phi
James Kokorelis
Introduction
•Bacteriophages are important in microbial evolution and
pathogenesis as they facilitate the transfer of genetic
information between hosts
•Bacteriophages are specific to certain host bacteria
- require different surface structures to allow
adsorption
l requires the maltose receptor
• T4 adsorbs to the TonB (iron) receptor
• Many phages (P1, P2, Mu) recognize LPS
• filamentous phages adsorb to F pili

Introduction to Wf
Wf was isolated from E. coli strain W. It is similar in
morphology to temperate bacteriophage P2, and genome
sequence analysis reveals extensive homology to P2.
However, a striking difference is seen in the plating of these
phages on different E. coli strains.
E coli
strain
E. coli W
E. coli B
E. coli C
E. coli K-12
ATCC9637
B834
C-1a
DH5a
RR1
M94
803
D1210
JM105
MM294
MC1061
plating of
P2 W f
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Research Aims
My research is focused on three related aims:
Part 1- Quantifying the infection efficiency
of P2, Whi, and W phi host range mutant on
several E. coli strains
Part 2- confirming the role of C-terminal
amino acids in the phage tail fiber gene
Part 3 - confirming the role of E. coli gene
rfbD in phage WF host range
Part 1
A titer analysis was done
K-12 strains RR1 (rfbd+)
M94 (rfbd-)
E. Coli B B834
E. coli C C-1A
Titer Comparison of P2, W phi hrb, and W phi
10.00000
RR1
C-1A
C-1A
C-1A
1.00000
B834
0.10000
M94
RR1
Log (Titers)
M94
RR1
B834
C-1A
B834
0.01000
M94
RR1
0.00100
0.00010
B834
M94
0.00001
P2
W phi hrb
Phage
W phi
Summary of Part 1
• It is clear the W phi has a significantly limited
host range when compared to P2 and the Wphi
host range mutant.
• Wphi infects the RR1 strain which is rfbd+ but
not M94 which is rfbd• Wphi host range mutant is able to infect all of the
same strains as P2
Therefore, to analyzing these trends lets begin at
the tail fibers, our second research aim.
Comparison of Tail Fiber Gene Sequences
P2
Wf
P2
Wf
P2
Wf
P2
Wf
1 MSIKFRTVITTAGAAKLAAATAPGRRKVGITTMAVGDGGGKLPVPDAGQTGLIHEVWRHALNKISQDKRNSNYIIAELVIPPEVGGFWMRELGLYDDAGT 100
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1 MSTKFKTVITTAGAVKLAAATAPGGRKVNITTMAVGDGGGKLPVPDAGQTGLIHEVWRHALNKISQDKRNSNYIIAELVIPPEVGGFWMRELGLYDDAGT 100
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101 LIAVANMAESYKPALAEGSGRWQTCRMVIIVSSVASVELTIDTTTVMATQDYVDDKIAEHEQSRRHPDASLTAKGFTQLSSATNSTSETLAATPKAVKAA 200
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101 LIAVANMAESYKPTLAEGSGRSQTCRMVIIVSSVASVELTIDTTTVMATQDYVDDKIAEHEQSRRHPDASLTAKGFTQLSNATNSTSETLAATPKAVKAA 200
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201 YDLANGKYTAQDATTARKGLVQLSSATNSTSETLAATPKAVKTVMDETNKKAPLNSPALTGTPTTPTARQGTNNTQIANTAFVMAAIAALVDSSPDALNT 300
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201 YDLANGKYTAQDATTARKGLVQLSSATNSTSETLAATPKAVKTVMDETNKKAPLNSPALTGTPTTPTARQGTNNTQIANTAFVMAAIAALVDSSPDALNT 300
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301 LNELAAALGNDPNFATTMTNALAGKQPKDATLTALAGLATAADRFPYFTGNDVASLATLTKVGRDILAKSTVAAVIEYLGLQETVNRAGNAVQKNGDTLS 400
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301 LNELAAALGNDPNFATTMTNALASKQPKDATLTALAGLATAADRFPYFTGNDVASLATLTKVGRDILAKSTVAAVIEYLGLQETVNRARNAVQKNGDILS 400
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401 GGLTFENDSILAWIRNTDWAKIGFKNDADGDTDSYMWFETGDNGNEYFKWRSRQSTTTKDLMTLKWDALNILVNAVINGCFGVGTTNALGGSSIVLGDND
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Wf 401 GGITFENDSILAWIRNTDWAKIGFKNDADGDADSYMWFETGDNGNEYFKWRSRQSTTTKDLMTLKWDALNILVNAVINGCLGVGTTNALGGSSIVLGDND
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WfhrB
GG
P2 501 TGFKQNGDGILDVYANSQRVFRFQNGVAIAFKNIQAGDSKKFSLSSSNTSTKNITFNLWGASTRPVVAELGDEAGWHFYSQRNTDNSVIFAVNGQMQPSN
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Wf 501 TGFKQNGDGILDVYANSQRVFRFQNGVAIAFKNIQVGDSKKFSLSSSNTSTKNITFNLWGASNRPVVAELGDESGWHFYSQRNTDNSVIFAVNGQMQPSN
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P2
601 WGNFDSRYVKDVRLGTRVVQLMARGGRYEKAGHTITGLRIIGEVDGDDEAIFRPIQKYINGTWYNVAQV 669
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Wf 601 WGNFDSRYVKDVRLGTRVDQLMARGGRYEKAGHTITGLRIIGEVDGDDEAIVRPIQKYINGTWYIVAQV 669
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WfhrB
VV
F
N
P2
500
500
600
600
Part 2.
• The H gene in the phage DNA encodes
the tail fiber sequence
• The overall idea is to take the H gene from
the WF host range mutant and insert it
into the pCR BLUNT 2 TOPO vector and
compare its growth to the wild type WF.
pCR BLUNT 2 TOPO vector
Procedure
Hyper Ladder 1
PCR
with jkh1
and jkh2
primers H gene
2 KB
TOPO CleaningA mixture of salt,
magnesium chloride,
TOPO vector and H gene
were incubated for 10 min
at room temperature
Chemically
transform plasmid
into E. coli DH5
alpha cells.
Grow on Kan plates
1
2
3
Supercoiled
ladder
5 6
4
Hyper
7
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5
6
7
5
6
7
Ladder 1
3.5 KB
5.5 KB
2 KB
Quick Check
Plasmid
Cut With EcoR1
Marker Rescue
TOPO Vector with H
gene insert
E. Coli C-1A
Rapid Transformation
Infected
with wild
type W phi
Lytic Cycle
Initiates
Recombination occurs resulting in
W phi receiving mutant H gene
Newly Encoded Tail Sequence
The phage that underwent recombination
should grow on E. coli B.
WF
E. Coli B
Part 3
Analyzing the rfbD gene
The E. coli K-12 rfbD gene
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The rfbD gene product catalyzes the final step in dTDP-rhamnose
biosynthesis. This is the synthesis pathway for an element of bacterial
LPS as well as O antigen.
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The rfbD1 mutant has been shown to have altered LPS.
HYPOTHESIS: The rfbD1 mutation blocks
adsorption of Wf because of the altered LPS
rfbD+
A comparison of the genotypes of the E. coli K-12 strains that fail to
plate Wf reveals that they all carry a common mutation, rfbD1.
rfbD1
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Klena & Schnaitman (1994)
J. Bacteriol. 176:4006
Experimental design: Construct a pair
of otherwise identical E. coli K12 strains
that are rfbD+ and rfbD1 and test plating
of Wf.
First need to identify the rfbD1 mutation.
The wild type strain used is CAG12099(P1)
The mutant strain is M94
Sequence analysis of rfbD1 and rfbD+
rfbD+
AGGGGAAT
5’
rfbD1
AGGGAAT
3’
299aa
285 aa
ccccttgcactcaacaagctcaacgcagtaccaacaacagcctatcctacaccagctcgtcgtccacataact…
P L A L N K L N A V P T T A Y P T P A R R P H N …
P
L H S T S S T Q Y Q Q Q P I L H Q L V V H I T …
rfbD+
rfbD1
Creating Isogenic Strains
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P1 transduction
– P1 transduction is advantageous because it packages from random doublestranded breaks on the chromosome that are generated during phage lysis.
Plating
• Transduction was done via linkage – tet
markers close together on the chromosome.
• Therefore the colonies are plated on
tetracycline plates to see if they will grow.
There should not be any growth on the tubes 4
and 5 because they are the controls and either
lack cells or P1.
1
836 colonies
2
648 colonies
3
4
118 colonies
0 colonies
5
0 colonies
Indistinguishable Phage growth
24 tet resistant colonies were test for growth on WF.
Controls:
WF
M94 is rfbDRR1 is rfbD+
Where to Go From Here
• heteroduplex analysis
– DNA formed by strands from different
sources
– will have loops and bubbles in regions where
the two DNAs differ.
– electrophoretic mobility in MDE gel is less
than that of homoduplex,
• can be detected by an extra slow moving band.
Synopsis
• Titers
– It is clear the W phi has a significantly limited host range
when compared to P2 and the Wphi host range mutant.
• Tail Fiber
– The complementation will demonstrate that the presence
of the altered sequence on a plasmid is sufficient to
confer the change in specificity thus demonstrating that
these alteration contribute to the modifying of the host
range.
• rfbD
– The transferring of the rfbD+ gene purportedly alters the
LPS that was encoded from the rfbD1 gene thus the wf
phage is able to bind.
Acknowledgements
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This work was supported by grant EEC0234104 from the
NSF/NIH Bioinformatics and Bioengineering Summer Institute
program.
People in the VCU/MCV Lab:
– Dr. Gail E. Christie
– Sandra Tallent
– Michael Harwich
– Nicholas Olivarez
– Megan Feltcher
Home mentor at James Madison University (JMU):
– Dr. Louise Temple
References:
Jensen EC, Schrader HS, Rieland B, Thompson TL, Lee KW, Nickerson KW, Kokjohn
TA. Prevalence of broad-host-range lytic bacteriophages of sphaerotilus natans,
escherichia coli, and pseudomonas aeruginosa. Applied and Environmental
Microbiology 1998 Feb;64(2):575-580.
Wang, J., M. Hofnung, and A. Charbit. 2000. The C-terminal portion of the tail fiber
protein of bacteriophage lambda is responsible for binding to LamB, its receptor at
the surface of Escherichia coli K-12. J. Bacteriol. 182:508-512. [PubMed].
Yao Z, Valvano MA. Genetic analysis of the O-specific lipopolysaccharide biosynthesis
region (rfb) of escherichia coli K-12 W3110: Identification of genes that confer group
6 specificity to shigella flexneri serotypes Y and 4a. Journal of Bacteriology 1994
Jul;176(13):4133-4143.
Questions