Characterization of the distal tail fiber locus and determination of the receptor for phage AR1, which specifically infects Escherichia coli O157:H7

Abstract

Phage AR1 is similar to phage T4 in several essential genes but differs in host range. AR1 infects various isolates of Escherichia coli O157:H7 but does not infect K-12 strains that are commonly infected by T4. We report here the determinants that confer this infection specificity. In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction. The counterparts in AR1 may be similarly important and, therefore, were characterized. The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus. The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4. The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family. To identify the AR1-specific receptor, E. coli O157:H7 was mutated by Tn10 insertion and selected for an AR1-resistant phenotype. A mutant so obtained has an insertion occurring at ompC that encodes an outer membrane porin. To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an ompC disruption mutant (RM). When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored. Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule.

FIG. 1

Analysis of an E. coli O157:H7 mutant (TM) created by Tn10 transposition. (A) Southern blot analysis of bacterial chromosomal DNAs that had been digested with EcoRV and hybridized with an IS10 probe. (B) Schematic diagram of the sequenced IS10-containing fragment in TM. The EcoRV fragment of TM (indicated by an arrowhead in panel A) was inserted into EcoRV-cut pBluescript II SK(+) and sequenced. The junctions between the insert (open box) and the vector (thick line) are illustrated, and portions of the cloned sequence are shown beneath. The EcoRV restriction sites are underlined. The sequence in the middle portion illustrates where ompC (capital letters) was disrupted by the IS10 insertion (lowercase letters). (C) Northern blot analysis of RNAs isolated from the TM mutant. Total bacterial RNAs were extracted from different preparations of bacteria. The RNA (10 μg) was electrophoretically separated on an agarose gel, transferred to a nylon membrane, and hybridized with ompC (upper panel) and ompA (lower panel) probes, respectively. In this and the following experiments (Fig. 2), gene expression from the plasmids was induced with IPTG (1 mM). Wt, wild-type strain.

FIG. 2

Protein expression patterns of the wild-type and mutant E. coli strains. (A) Coomassie blue-stained profiles of the total proteins separated on an SDS–12% polyacrylamide gel. (B) Western blot analysis of OmpC expressed in the total bacterial lysates using mouse anti-OmpC antisera. (C) Coomassie blue-stained protein profiles of the outer membrane fractions. The identity of OmpA was confirmed by N-terminal microsequencing. Plasmids pTacOmp(O) and pTacOmp(K) encode OmpC of the O157:H7 origin and that of the K-12 origin, respectively. OmpC/F labels the band containing both OmpC and OmpF that are presumably not separated in these gel systems. Wt, wild-type strain.

FIG. 3

AR1 plaque formation on different preparations of bacteria. The AR1 phage stock was made on the wild-type O157:H7 strain and diluted in a 10-fold series. The phage solutions were then used to infect various preparations of bacteria and plated out on LB agar plates for PFU scoring. When transformed with plasmids, the bacteria were cultivated in the presence of IPTG (1 mM) prior to mixing with the phage. IPTG (1 mM) was also included in the plates used in the plaque assay. No plaque formation was observed with bacteria indicated in columns 2, 3, and 5 through 8. These experiments were repeated three times, with similar results. Wt, wild-type strain.

FIG. 4

AR1 binding assay. ³⁵S-labeled AR1 phages were incubated at 4°C for 2 h with different bacteria as indicated. Cells were collected by centrifugation and washed twice with cold PBS. Bacterium-bound AR1 was then quantitated by liquid scintillation counting. Counts derived from the wild-type strain and RM were set as reference points for 100 and 0%, respectively.