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An Analysis of The Host Range Determinants of a P2 Related Phage W phi

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

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An Analysis of The Host Range Determinants of a P2 Related Phage W phi

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  1. An Analysis of The Host Range Determinants of a P2 Related Phage W phi James Kokorelis

  2. 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

  3. 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

  4. 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 plating of • P2 W f • E. coli W ATCC9637 ? + • E. coli B B834 + - • E. coli C C-1a + + • E. coli K-12 DH5a + - RR1 + + M94 + - 803 + - • D1210 + + • JM105 + + • MM294 + - • MC1061 + +

  5. 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

  6. Part 1 A titer analysis was done

  7. K-12 strains RR1 (rfbd+)E. Coli B B834 M94 (rfbd-) E. coli CC-1A

  8. 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.

  9. P2 Wf P2 Wf P2 Wf P2 Wf P2 Wf WfhrB P2 Wf P2 Wf WfhrB Comparison of Tail Fiber Gene Sequences 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 . . . . . . G G V V F F N

  10. 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 mutantand insert it into the pCR BLUNT 2 TOPO vector and compare its growth to the wild type WF.

  11. pCR BLUNT 2 TOPO vector

  12. Hgene Procedure Hyper Ladder 1 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 Grow on Kan plates Chemically transform plasmid into E. coli DH5 alpha cells.

  13. Supercoiled ladder Hyper Ladder 1 7 1 2 3 5 6 8 4 5 6 7 5 6 7 3.5 KB 5.5 KB 2 KB Quick Check Cut With EcoR1 Plasmid

  14. 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

  15. Newly Encoded Tail Sequence The phage that underwent recombination should grow on E. coli B. WF E. Coli B

  16. Part 3 Analyzing the rfbD gene

  17. The E. coli K-12 rfbD gene • 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. • 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. rfbD1 rfbD+ HYPOTHESIS: The rfbD1 mutation blocks adsorption of Wf because of the altered LPS Klena & Schnaitman (1994) J. Bacteriol. 176:4006

  18. 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

  19. 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 Sequence analysis of rfbD1 and rfbD+ rfbD+ rfbD1 A G G G G A AT A G G G A AT 5’ 3’ 299aa 285 aa

  20. 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.

  21. 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. 4 1 2 3 5 836 colonies 648 colonies 118 colonies 0 colonies 0 colonies

  22. Indistinguishable Phage growth 24 tet resistant colonies were test for growth on WF. Controls: M94 is rfbD- RR1 is rfbD+ WF

  23. 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.

  24. 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.

  25. 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.

  26. Questions ?

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