Effect of mutations in the outer membrane components on bacteriophage T4 adsorption to Escherichia coli

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1 Effect of mutations in the outer membrane components on bacteriophage T4 adsorption to Escherichia coli ALICE WANG AND KEVIN LIN Department of Microbiology and Immunology, UBC Esherichia coli strains with mutations that affect the occurrence of outer membrane proteins and the structure of the lipolysaccharide (LS) in the outer membrane were tested to determine whether particular structures were essential for infection by coliphaget4. The infectivity was poor in the strains lacking OmpC. It was also poor in strains lacking the phosphate group on second heptose but containing -EEN on the first heptose on LS, strains lacking the branching heptose on the second heptose on LS, lacking the 3 glucose residues that are linked to the second heptose on LS and strains containing all the sugar residues on LS. Average infectivity was seen with strains lacking the terminal β-glc on LS and with strains lacking OmpF. Efficient infectivity was seen for strains lacking only OmpA, strains lacking all of the phosphate groups and EEN on LS as well as strains lacking a branching galactose and the terminal β-glc on LS. These results suggest that some components are essential but overall efficiency depends on several protein and lipopolysaccharide features. Bacteriophage T4 adsorbs to the cell wall of its host cell, Escherichia coli. Various literatures have indicated that the adsorption sites are lipopolysaccharide (LS) and outer membrane protein C (OmpC) on the outer membrane of the bacteria. revious experiments have shown that the presence of different polysaccharides that resemble the structure of LS inhibit phage binding (1,3,4,,6), which indicates T4 probably do bind to LS on bacterial cells. However, the precise LS residues used for T4 binds remains undetermined. To study T4 binding determinants, the production of T4 phage on E. coli strains with mutations causing variation in the LS structure was examined. Other tests used strains with mutations in specific outer membrane proteins in order to investigate whether the presence of particular proteins was essential for T4 infectivity. MATERIALS AND METHODS The efficiency of E. coli mutants for T4 infection was assayed via overlay method. Small broth tube (13x100mm) containing 3 ml of molten soft overlay agar was placed into a 50 0 C water bath. T4 stock was diluted to a concentration that, when plated, will give plaques on a plate. 4 x 10 8 cells of different E. coli mutants (~0.1 ml of a saturated culture) were added to each tube as host cells, along with 0.1 ml or 0.2 ml of diluted phage, pending on the dilution. Tubes were mixed by phage style mixing, and then rapidly transferred to a plate containing bottom agar. lates were incubated for hours at 37 0 C and plaques were counted after incubation. The strains of E.coli were provided by S. Farmer 9University of British Columbia, British Columbia). They were from the collection of C. Whitfield (University of Guelph, Ontario). The strain characteristics described in Table I and II have were studied and reported by Whitfield and others (2, 5) RESULTS The structure of the LS and genotypes of the tested strains are described in Tables I and II. From Fig. 1, it was shown that strains with all of one of the following characteristics are infected poorly by phage. Lacking OmpC (strain -158, ) Lacking the phosphate group on second heptose but contain -EEN on the first heptose on LS. Lacking the branching heptose on the second heptose on LS. Lacking the 3 glucose residues that are linked to the second heptose on LS. Containing all the sugar residues (the parental strain) on LS. Mutants with the following characteristics show average infection levels. Lacking the terminal β-glc on LS. Lacking OmpF 47

2 Table 1 Structure of LS and genotype of E. coli mutants Name 1 Name 2 Structure C149 Sex: f-, proc-24, aroa-357, his-53, pure-41, ilv-277, met- 65, lacy-29, xyl-14, rpsl-97, cyca-1, cycb-2?, tsx-63, lambda- C150 Wild-type Sex F-, procc-24, ompf-254, his-53, pure-41, ilv-277, met-65, lacy-29, xyl-14, rpsl-97, cyca-1, cycb-2?, tsx- 63, lambda- OmpF- strain, protein 1a- SexF-, proc-24, ompa-256, his-53, pure-41, ilv-277, met- 65, lacy-29, xyl-14, rpsl-97, cyca-1, cycb-2?, txs-63, lambda- rotein 3a- (ompa) SexF-, proc-24, aroa-357, his-53, ompc-264, pure-41, ilv-277, met-65, lacy-29, xyl-14, rpsl-97, cyca-1, cycb- 2?, tsx-63, lambda- OmpC- (protein 1b-) SexF-, proc-24, ompa-252, his-53, ompc-262, pure-41, ilv-277, met-65, lacy-29, xyl-14, rpsl-97, cyca-1, cycb?, tsx-63, lambda- rotein 1b- (ompc) 3a- (ompa) SexF-, proc-24, ompf-254, ompa-256, his-53, pure-41, ilv-277, met-65, lacy-29, xyl-14, rpsl-97, cyca-1, cycb- 2?, tsx-63, lambda- rotein 1a- (ompf) and protein 3a ompa (reduced) SexF, proc-24, ompf-254, his-53, ompc-263, pure-41, nmpa1, ilv-277, met-65, lacy-29, xyl-14, rpsl-97, cyca- 1, cycb-2?, tsx-63, lambda- rotein 1a- (ompf) and lb- (ompc), protein E present (phoe) C842 F470 Hep Hep-Hep-Glc-Glc-βGlc attaches to O-antigen in wild type EEN 48

3 C843 Cwg296 Hep-Hep-Glc-Glc-βGlc attaches to O-antigen in wild type C844 Cwg297 Hep-Hep-Glc-Glc-βGlc attaches to O-antigen in wild type EEN C845 Cwg303 Hep-Hep EEN C846 Cwg308 Hep Hep-Hep-Glc EEN C847 Cwg309 Hep Hep-Hep-Glc-Glc EEN C848 Cwg310 Hep Hep-Hep-Glc-Glc EEN C849 Cwg311 Hep Hep-Hep-Glc-Glc EEN C850 Cwg312 Hep Hep-Hep-Glc-Glc-βGlc attaches to O-antigen in wild type EEN 49

4 Table 2 - Structures of surface proteins and LS Tested Number Structure 1 OmpF -, OmpA -, OmpC - 2 -Hep-Hep-Glc-Glc 3 -Hep-Hep-Glc-Glc-βGlc 4 5 Hep -Hep-Hep-Glc-Glc-βGlc Hep -Hep-Hep-Glc-Glc-βGlc 6 Hep -Hep-Hep-Glc 7 Hep -Hep-Hep-Glc-Glc 8 -Hep-Hep-Glc-Glc 9 Hep -Hep-Hep-Glc-Glc 50

5 10 Hep -Hep-Hep-Glc-Glc 11 -Hep-Hep-Glc-Glc-βGlc 12 Hep -Hep-Hep-Glc-Glc-βGlc Mutants with following characteristics are efficiently infected by phage: Lacking OmpA only. Lacking all of the phosphate groups and EEN on LS. Lacking a branching galactose and the terminal β-glc on LS. The permissive strains showed some large differences in infectivity between the different tests. The poorly infective strains showed low levels of infectivity in all of the tests. DISCUSSION The results showed that E. coli mutants lacking OmpC are unable to bind to T4, indicating the OmpC is essential for phage adsorption (Fig.1). It also appeared that the phosphate group and EEN on LS interfere with phage infection, as mutants lacking these two molecules tended to be very efficiently infected. The two glucose molecules following the two heptose molecules on LS seemed to facilitate phage infection and might be part of the T4 binding site since mutants lacking these two molecules poorly infected by phage. The deletion of OmpA appears to increase infection by phage and this effect may be due to changes in membrane structure caused by the removal of OmpA. The change in membrane structure may make the phage attachment site more exposed and in this way help in phage adsorption. To fully investigate T4 adsorption sites, more mutants with different LS structures will need to be tested for their binding efficiency toward T4. Table 2 provides the specific LS structures that are to be tested, and the results obtained will help in determining the structure of T4 adsorption site. The interaction between OmpC and T4 can be clarified with two-hybrid system. REFERENCES 1. Coombs, D. H. a. A., F. (1994) in Molecular Biology of Bacteriophage T4 (Karam, J. D., ed), pp , American Society of Microbiology, Washington, DC. 2. Heinrichs, D., J. Yethons,. Amor and C.Whitfield (1998) J. Biol. Chem. 273, Sutherland, I. W. (1977) Surface Carbohydrates of the rokaryote Cells, 1 st Ed., Academic ress Inc., London 4. Watanabe, T. (1976) Canadian Journal of Microbiology 22, Yethon, J., D. Heinrichs, M.Monteiro, M.erry and C. Whitfield, J. Biol. Chem. 273, Yu, F. a. M., S. (1982) J. Bacteriol. 151,

6 Fig. 1- Efficiency of infection observed for different strains of Escherichia coli. Figures A, B, C and D represent tests done on different days. A. C850 C849 C848 C847 C846 C845 C844 C843 C842 C150 C149 B Concentration of Free hage (10^8 pfu/ml) 60 B. C850 C849 C848 C847 C846 C845 C844 C843 C842 C150 C149 B Concentration of Free hage (10^8 pfu/ml) 52

7 C. C850 C849 C847 C845 C844 C842 B Concentration of Free hage (10^8 pfu/ml) D. c850 c849 c848 c847 c846 c845 c844 c843 c842 c160 c159 c158 c157 c156 c150 c149 b Concentration of Free hage (10^8 pfu/ml)