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

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 W

Wwas 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 plating ofP2 W

E. coli W ATCC9637 ? E. coli B B834E. coli C C-1a E. coli K-12 DH5

RR1

D1210

JM105

MM294MC1061

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 strainsPart 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 W host range

Part 1

A titer analysis was done

K-12 strains RR1 (rfbd+) E. Coli B B834

M94 (rfbd-) E. coli C C-1ATiter Comparison of P2, W phi hrb, and W phi

C-1A

B834

C-1A

B834

M94

RR1

C-1A

M94

RR1M94

RR1

B834

0.00001

0.00010

0.00100

0.01000

0.10000

1.00000

10.00000

P2 W phi hrb W phi

Phage

Lo

g (

Tit

ers

)

C-1A

B834

M94

RR1

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

NFV

GG

V F

P2

W

P2

W

P2

W

P2

W

WhrB

P2

W

P2

W

P2

W

WhrB

1 MSIKFRTVITTAGAAKLAAATAPGRRKVGITTMAVGDGGGKLPVPDAGQTGLIHEVWRHALNKISQDKRNSNYIIAELVIPPEVGGFWMRELGLYDDAGT 100 || ||:|||||||| ||||||||| ||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 1 MSTKFKTVITTAGAVKLAAATAPGGRKVNITTMAVGDGGGKLPVPDAGQTGLIHEVWRHALNKISQDKRNSNYIIAELVIPPEVGGFWMRELGLYDDAGT 100 . . . . . . . . . .

101 LIAVANMAESYKPALAEGSGRWQTCRMVIIVSSVASVELTIDTTTVMATQDYVDDKIAEHEQSRRHPDASLTAKGFTQLSSATNSTSETLAATPKAVKAA 200 ||||||||||||| ||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||.|||||||||||||||||||101 LIAVANMAESYKPTLAEGSGRSQTCRMVIIVSSVASVELTIDTTTVMATQDYVDDKIAEHEQSRRHPDASLTAKGFTQLSNATNSTSETLAATPKAVKAA 200 . . . . . . . . . .

201 YDLANGKYTAQDATTARKGLVQLSSATNSTSETLAATPKAVKTVMDETNKKAPLNSPALTGTPTTPTARQGTNNTQIANTAFVMAAIAALVDSSPDALNT 300 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||201 YDLANGKYTAQDATTARKGLVQLSSATNSTSETLAATPKAVKTVMDETNKKAPLNSPALTGTPTTPTARQGTNNTQIANTAFVMAAIAALVDSSPDALNT 300 . .* * . . . * . . . . .

301 LNELAAALGNDPNFATTMTNALAGKQPKDATLTALAGLATAADRFPYFTGNDVASLATLTKVGRDILAKSTVAAVIEYLGLQETVNRAGNAVQKNGDTLS 400 ||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||| ||301 LNELAAALGNDPNFATTMTNALASKQPKDATLTALAGLATAADRFPYFTGNDVASLATLTKVGRDILAKSTVAAVIEYLGLQETVNRARNAVQKNGDILS 400 . . . . . . . . . .

401 GGLTFENDSILAWIRNTDWAKIGFKNDADGDTDSYMWFETGDNGNEYFKWRSRQSTTTKDLMTLKWDALNILVNAVINGCFGVGTTNALGGSSIVLGDND 500 ||:|||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||401 GGITFENDSILAWIRNTDWAKIGFKNDADGDADSYMWFETGDNGNEYFKWRSRQSTTTKDLMTLKWDALNILVNAVINGCLGVGTTNALGGSSIVLGDND 500 * . . . . . . . . . . 501 TGFKQNGDGILDVYANSQRVFRFQNGVAIAFKNIQAGDSKKFSLSSSNTSTKNITFNLWGASTRPVVAELGDEAGWHFYSQRNTDNSVIFAVNGQMQPSN 600 ||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||| ||||||||||.||||||||||||||||||||||||||501 TGFKQNGDGILDVYANSQRVFRFQNGVAIAFKNIQVGDSKKFSLSSSNTSTKNITFNLWGASNRPVVAELGDESGWHFYSQRNTDNSVIFAVNGQMQPSN 600 . . . . . . . . . . 601 WGNFDSRYVKDVRLGTRVVQLMARGGRYEKAGHTITGLRIIGEVDGDDEAIFRPIQKYINGTWYNVAQV 669 |||||||||||||||||| |||||||||||||||||||||||||||||||| |||||||||||| ||||601 WGNFDSRYVKDVRLGTRVDQLMARGGRYEKAGHTITGLRIIGEVDGDDEAIVRPIQKYINGTWYIVAQV 669 . . . . . .

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 Whost range mutantand insert it into the pCR BLUNT 2 TOPO vector and compare its growth to the wild type W

pCR BLUNT 2 TOPO vector

Procedure

H gene

PCR with jkh1 and jkh2 primers

2 KB

TOPO Cleaning-

A 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

Hyper Ladder 1

1 2 3 4 5

Supercoiled ladder

6 7 8 5 5 66 7 7

Hyper

Ladder 1

5.5 KB

2 KB

3.5 KB

Quick Check

Cut With EcoR1Plasmid

Marker RescueTOPO Vector with H gene insert

Rapid TransformationE. Coli C-1A

Infected with wild type W phi

Recombination occurs resulting in W phi receiving mutant H gene

Lytic Cycle Initiates

Newly Encoded Tail SequenceThe phage that underwent recombinationshould grow on E. coli B.

E. Coli B

W

Part 3

Analyzing the rfbD gene

The E. coli K-12 rfbD gene• A comparison of the genotypes of the E. coli K-12 strains that fail to

plate W reveals that they all carry a common mutation, rfbD1.

• 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.

• The rfbD1 mutant has been shown to have altered LPS.

rfb

D1

rfb

D+

Klena & Schnaitman (1994)J. Bacteriol. 176:4006

HYPOTHESIS: The rfbD1 mutation blocksadsorption of W because of the altered LPS

Experimental design: Construct a pair of otherwise identical E. coli K12 strains that are rfbD and rfbD1 and test plating of W.

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+ rfbD1

A G G G A A T

A G G G G A A T

5’ 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• P1 transduction

– P1 transduction is advantageous because it packages from random double-stranded 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 2 3 4 5836 colonies 648 colonies 118 colonies 0 colonies 0 colonies

Indistinguishable Phage growth

24 tet resistant colonies were test for growth on W. Controls:

M94 is rfbD-

RR1 is rfbD+

W

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 w phage is able to bind.

Acknowledgements• 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

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