Bacteriophage P22 SieA-mediated superinfection exclusion

Abstract

Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. Salmonella phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the c2 (repressor), gtrABC, sieA, and sieB genes. Here we report that the P22-encoded SieA protein is necessary and sufficient for exclusion by the SieA system and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants that overcome the SieA block were isolated, and they have amino acid changes in the C-terminal regions of the gene 16 and 20 encoded ejection proteins. Three different single-amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in phage target specificity. Our data strongly suggest a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.IMPORTANCEThe ongoing evolutionary battle between bacteria and the viruses that infect them is a critical feature of bacterial ecology on Earth. Viruses can kill bacteria by infecting them. However, when their chromosomes are integrated into a bacterial genome as a prophage, viruses can also protect the host bacterium by expressing genes whose products defend against infection by other viruses. This defense property is called "superinfection exclusion." A significant fraction of bacteria harbor prophages that encode such protective systems, and there are many different molecular strategies by which superinfection exclusion is mediated. This report is the first to describe the mechanism by which bacteriophage P22 SieA superinfection exclusion protein protects its host bacterium from infection by other P22-like phages. The P22 prophage-encoded inner membrane SieA protein prevents infection by blocking transport of superinfecting phage DNA across the inner membrane during injection.

Keywords: P22; SieA; bacteriophage; ejection proteins; superinfection exclusion.

Fig 1

SieA superinfection exclusion. Soft agar containing sieA⁺ or sieA^– S. enterica lawn cells (indicated with relevant genotype on the left) was allowed to solidify on L broth plates, and 10-μL spots of parental “wild-type” (WT) phage (P22 UC-0937), as well as isogenic double-mutant (P22 UC-0972) and isogenic triple-mutant (P22 UC-0979) phages that overcome sieA exclusion, were applied (indicated on the right). See text and Table 1 for descriptions and complete genotypes of these phages and lawn cells. The plates were incubated overnight at 37°C. Phage stocks were about 10¹⁰ PFU/mL, and they were diluted by the factors shown above. Ten microliters of undiluted P22 (UC-0937) phage stock also showed no plaques on sieA⁺ host (UB-2520).

Fig 2

Potassium ion release after P22 infection. S. enterica strains growing in log phase were infected with P22 at 37°C, and K⁺ measurements (expressed as percent released relative to total K⁺ released after boiling for 10 min) were performed as described (57). (A) Potassium ion release by infectious P22 virions. The upper key gives the infections that were analyzed. The host with no sieA gene was UB-0002 and with a sieA⁺ gene was UB-2520. The infecting phages were as follows: WT P22 (UC-0937), triple SieA escape mutant P22 16 G532W, 16 P546S, and 20 G350D (UC-0979). (B) Lack of potassium ion release by P22 particles lacking E-proteins. The host with no sieA gene (UB-0002) was infected as indicated in the lower key with “WT” P22 (UC-0937) or by virion-like particles whose genomes carry a deletion of one of the E-protein genes and which do not make plaques. The latter defective particles were produced by induction of isogenic 7^–D-1, 16^–D-1, and 20^–D-1 prophages in strains UB-2289, UB-2288, and UB-2285, respectively. The virions and virion-like particles were CsCl gradient purified, and relative particle concentrations were measured by quantitating the amount of coat protein in Coomassie brilliant blue-stained SDS-PAGE gels.

Fig 3

P22 SieA is an inner membrane protein. (A) P22 SieA protein membrane topology. Membrane topology of P22 SieA protein was predicted by a transmembrane hidden Markov model (TMHMM) (61). (B) Cell fractionation. The inner and outer membrane fractions of S. enterica strains carrying a P22 sieA gene (UB-2520) or a C-terminally FLAG-tagged P22 sieA gene (UB-2668) were isolated as described in Materials and Methods. Samples derived from equal numbers of cells of whole-cell lysate and inner membrane and outer membrane fraction proteins were separated by 15% SDS-PAGE. The SDS-PAGE immunoblot was probed with an anti-FLAG monoclonal antibody (Materials and Methods). Precision Plus Protein Kaleidoscope (Bio-Rad) molecular weight markers are indicated on the left. Although some additional bands reacted with the anti-FLAG antibodies, the only difference between the tagged and untagged samples identifies the band that runs at the expected size of SieA protein (about 19 kDa) as the FLAG-tagged SieA protein band.

Fig 4

Phage P22 with foreign ejection protein genes. The phage P22 genome is diagrammed above with the location of the sieA genes indicated. Red arrows indicate regions expressed from the prophage, while green and blue arrows are regions expressed during early and late lytic growth, respectively. Below, replacement E-protein genes from phages Sf6 (strains UB-2349, UB-2416, UB-2417, UB-2418, and UB-2669), L (UB-2464), HK620 (UB-2537), or CUS-3 (UB-2614) are shown in different colors. P22 gene names are indicated on the P22 genes, and percent identities to their P22 homologs are indicated on the foreign genes. The viability and SieA resistance phenotypes are summarized on the right (see also Table 4).

Fig 5

Neighbor-joining tree of representative SieA proteins. An unrooted Clustal neighbor-joining tree (71) of SieA proteins chosen to display the extent of their diversity is shown. The tree shows selected bootstrap values out of 1,000 trials above the branches and fractional distances below the branches. Gray boxes indicate the three sequence classes of SieA protein homologs. A bar representing a fractional distance of 0.05 is shown in the upper left. Table S2 gives locus_tags for the SieA sequences.