The bacteriophage HK97 gp15 moron element encodes a novel superinfection exclusion protein

Abstract

A phage moron is a DNA element inserted between a pair of genes in one phage genome that are adjacent in other related phage genomes. Phage morons are commonly found within phage genomes, and in a number of cases, they have been shown to mediate phenotypic changes in the bacterial host. The temperate phage HK97 encodes a moron element, gp15, within its tail morphogenesis region that is absent in most closely related phages. We show that gp15 is actively expressed from the HK97 prophage and is responsible for providing the host cell with resistance to infection by phages HK97 and HK75, independent of repressor immunity. To identify the target(s) of this gp15-mediated resistance, we created a hybrid of HK97 and the related phage HK022. This hybrid phage revealed that the tail tube or tape measure proteins likely mediate the susceptibility of HK97 to inhibition by gp15. The N terminus of gp15 is predicted with high probability to contain a single membrane-spanning helix by several transmembrane prediction programs. Consistent with this putative membrane localization, gp15 acts to prevent the entry of phage DNA into the cytoplasm, acting in a manner reminiscent of those of several previously characterized superinfection exclusion proteins. The N terminus of gp15 and its phage homologues bear sequence similarity to YebO proteins, a family of proteins of unknown function found ubiquitously in enterobacteria. The divergence of their C termini suggests that phages have co-opted this bacterial protein and subverted its activity to their advantage.

Fig 1

Expression of HK97 gp15 inhibits phage infection. (A) Plating of serial dilutions of phages HK97, λ, HK022, and HK75 on cells alone (594 cells) or transformed with plasmids expressing the moron elements from HK97 (pEx15; gp15) and HK022 (pEx20; gp20) reveals the inhibition of HK97 and HK75 by gp15. The introduction of a nonsense mutation in pEx15 abrogates this inhibition (pEx15am). (B) Western blot analysis probing for the FLAG epitope fused to the C terminus of gp15 and gp20 reveals that the proteins are expressed in E. coli and that the detectable expression of gp15 is correlated with the inhibition phenotype. (C) Expression of gp15 from an HK97 lysogen provides resistance to HK97 and HK75 that is independent of the repressor protein.

Fig 2

Genome map of the morphogenetic regions of phages HK97 and HK022. Gene coding regions are indicated by boxes, and gene names are noted above the boxes. Shading of the boxes indicates the percent identity between the two phage homologues, with dark gray (■) indicating >95%, medium gray 75 to 95%, and white (□) <35%. The morons, gp15 and gp20, are boxed. The sites of recombination in the HK97/022 hybrid phage are shown with arrows.

Fig 3

Sequence comparison of gp15, gp20, and the YebO family of bacterial proteins. The upper group of sequences is encoded by phages, and the lower group consists of the YebO family of proteins encoded within bacterial genomes. The percent identity of each sequence to gp15 is shown in the column marked “% ID.” Unnamed protein sequences are designated by their GI numbers. Three highly conserved hydrophobic positions and two conserved Arg residues are indicated by arrows, and the N-terminal putative membrane helix is indicated above the sequences.

Fig 4

Potassium efflux induced by phages HK97 and λ as measured by a potassium-selective electrode. (A) Phage genome injection into wild-type E. coli by HK97 (♢) leads to efflux of potassium out of the cell and into the surrounding medium. This effect is abrogated in cells lacking the HK97 receptor (lamB) on their surface (+). The expression of pEx15-FLAG is able to block the entry of the HK97 (▲) genome but not the λ (×) genome. This effect is alleviated when pEx15-FLAG carries a nonsense mutation in gp15 (●). (B) The resistance to phage DNA injection is provided by gp15 expressed from an HK97 prophage. The presence of a wild-type HK97 lysogen in E. coli blocks the injection of the HK97 (▲) genome but not λ (×). The introduction of a nonsense mutation into gp15 relieves the inhibition for HK97 DNA injection (●).

Fig 5

Gp15 does not affect the lytic growth of HK97. (A) Mitomycin induction of a wild-type HK97 prophage (●), a prophage with pEx15-FLAG (○), and a prophage bearing a nonsense mutation in gp15 where lysis of the culture was followed by monitoring the OD₆₀₀. (B) Samples taken at various time points following mitomycin induction in panel A were lysed with chloroform. The resulting phage lysates were top-plated on E. coli and plaques enumerated, revealing no difference in the number of phage particles produced by the wild-type lysogen (□) compared to a lysogen with pEx15-FLAG (■) and a lysogen with a nonsense mutation in gp15 .