- Research article
- Open Access
Genomic analysis of bacteriophage ε34 of Salmonella entericaserovar Anatum (15+)
BMC Microbiologyvolume 8, Article number: 227 (2008)
The presence of prophages has been an important variable in genetic exchange and divergence in most bacteria. This study reports the determination of the genomic sequence of Salmonella phage ε34, a temperate bacteriophage that was important in the early study of prophages that modify their hosts' cell surface and is of a type (P22-like) that is common in Salmonella genomes.
The sequence shows that ε34 is a mosaically related member of the P22 branch of the lambdoid phages. Its sequence is compared with the known P22-like phages and several related but previously unanalyzed prophage sequences in reported bacterial genome sequences.
These comparisons indicate that there has been little if any genetic exchange within the procapsid assembly gene cluster with P22-like E. coli/Shigella phages that are have orthologous but divergent genes in this region. Presumably this observation reflects the fact that virion assembly proteins interact intimately and divergent proteins can no longer interact. On the other hand, non-assembly genes in the "ant moron" appear to be in a state of rapid flux, and regulatory genes outside the assembly gene cluster have clearly enjoyed numerous and recent horizontal exchanges with phages outside the P22-like group. The present analysis also shows that ε34 harbors a gtrABC gene cluster which should encode the enzymatic machinery to chemically modify the host O antigen polysaccharide, thus explaining its ability to alter its host's serotype. A comprehensive comparative analysis of the known phage gtrABC gene clusters shows that they are highly mobile, having been exchanged even between phage types, and that most "bacterial" gtrABC genes lie in prophages that vary from being largely intact to highly degraded. Clearly, temperate phages are very major contributors to the O-antigen serotype of their Salmonella hosts.
Recent studies of tailed-phages have shown their enormous numbers and wide overall diversity [1–3], but relatively few studies have had as a long-range goal an attempt to analyze the range of diversity in tailed-phages that infect a particular bacterial species or cluster of species (see, for example, [4, 5] and references therein for the phages of Mycobacterium). We are interested in the diversity of dsDNA tailed-bacteriophages that infect Salmonella; such analyses of enteric bacteria enjoy the major benefit of the huge amount of previous genetic and biochemical characterization of the proteins encoded by the Escherichia coli and Salmonella enterica "model system" phages. Many members of the Caudovirales (tailed-phages) specific for Salmonella species have been reported, but no careful comparison of these many phages has been done with a goal of understanding the detailed nature of the diversity of these viruses. In 1950, Boyd recognized that S. enterica serovar Typhimurium carried "symbiotic bacteriophages", which we now call prophages . Genomic sequencing has shown that essentially all Salmonellae carry prophages [7–13]. Some of these prophages are fully functional and some are clearly defective and no longer competent to program a complete phage lytic cycle.
Temperate phages often modify the host cell surface lipopolysaccharide upon lysogenization in a process called "lysogenic conversion." Salmonella phage ε34, the subject of this report, is a temperate phage that was isolated in the 1950s  that was historically important in the proof of this phenomenon [14–20]. Previous studies have provided an early genetic map for ε34, and identified the conversion genes as well as the virion O-antigen polysaccharide receptor-recognizing tailspike encoding gene [19–25], and ε34 will only adsorb to and thus infect its Salmonella enterica serovar Anatum host cell if the latter carries an ε15 prophage [15, 18]. This type of modification of surface polysaccharides has been implicated in bacterial virulence . We report here the genome sequence of phage ε34 and compare it to its relatively close P22-like relatives.
Results and discussion
1. Analysis of the ε34genome sequence
Double-stranded DNA from ε34 carrying the clear plaque mutation(s) c99 was sequenced. The alterations in the clear mutant c99 strain were determined to affect genes 46 and 48 (see below). The sequencing runs assembled into a single circular sequence, and its annotated genome sequence is arbitrarily opened at the 5'-end of the small terminase subunit gene according to convention with this type of phage. The genome is 43016 bp long and is 47.26% G+C which is somewhat lower than the host bacterium 53% . The ε34 genome is predicted to contain 71 protein-coding genes (shown diagrammatically in figure S1 in the Additional file 1) and genes for at least two antisense RNAs, Sar and Q antisense RNA. A complete list and description of the genes is presented in Table S1 as Additional file 1. Comparison of proteins predicted to be encoded by these 71 genes with the extant database (BLASTP and PSI-BLAST ) along with the gene order and orientation show clearly that ε34 is a P22-like member of the larger "lambdoid" phage group. Its morphogenetic genes show a clear one-to-one relationship with those of phage P22, while its early regions show a typical mosaic relationship with other lambdoid phages. Of the 71 predicted genes, only seven (genes 15, 40, 44, 45, 59, 60, and 63) do not have a phage- or prophage-borne match in the current database; of these, gene 15 matches numerous bacterial genes of unknown function. Space limitations preclude citation of all the molecular and genetic studies that have lead to the current understanding of the genes in the lambdoid phages (see refs. [29, 30]).
2. The ε34genes
a. Early left operon
The ε34 early left operon contains 21 open reading frames, all of which have homology to genes in currently known lambdoid phages. The first gene in this operon, gene 43, encodes a homolog of the highly studied λ transcriptional antitermination N protein that has high similarity in its C-terminal region (AAs 28–120) to the C-terminal regions of orthologues in other lambdoid phages, however its N-terminal 27 AAs are at best only 62% identical to those proteins. Since the C-terminal part is thought to be involved in association with host RNA polymerase and the BoxB-nut RNA site [29, 30], it seems likely that the ε34 protein has a novel BoxB specificity. Immediately transcriptionally downstream (left) of gene 43, genes 31 through 42 are somewhat similar to and syntenic with the parallel region of P22-like phage ST64T. These include homologues of phage λ cIII (gene 39) and kil (gene 38) genes, which control establishment of lysogeny and inhibition of host septation, respectively. Genes 33 through 36 encode the following proteins that are likely to be involved in catalyzing homologous recombination: a P22 type anti-RecBCD protein Abc2, a possible endonuclease, a bacterial type single-strand DNA binding protein, and a P22-type Erf ("essential recombination function") protein. The putative gene 34-encoded nuclease is unusual in this context, and gene 36 is called a homologue of the well studied phage P22 Erf protein only because their C-terminal 56 amino acids are 86% identical. Parallel Erf "domain swaps" have been discussed previously [31–33], and these are consistent with the known structure of the protein . Downstream gene, 31, appears to be an in-frame fusion of the N-terminus of a gene that is similar to eaE found in this location in the P22 genome and a putative HNH homing endonuclease. Interestingly, the latter is homologous to endonucleases from phage infecting Gram-positive bacteria including Lactobacillus phages A2 (NP_680539.1) and ΦAT3 (YP_025078.1). Downstream genes 29 and 30 encode proteins that are identical to P22 EaF and Orf45, respectively, while genes 25-28 specify proteins that are closely related to those of ε15 genes 48 through 45, respectively (e.g., ε34 gp28 and ε15 gp45 are 96% identical over their 277 amino acids). Finally, genes 23 and 24 are oriented like their P22 homologues and encode putative integrase and excisionase proteins that are discussed in more detail below.
b. Early right operon
The ε34 early right operon contains sixteen genes, all but two of which are homologous to known lambdoid phage early right operon genes. The first two genes in this operon are similar to lambdoid cro and cII genes, respectively, and the next four genes, 49 through 53, are very similar to the phage ES18 DNA replication gene region; of these, ε34 gp50 is likely the origin binding protein because of its λ gpO homology and gp51 is a putative helicase . Genes 54 through 61, represent a typically mosaic lambdoid "nin region" that includes homologues of λ and P22 ninA, B, D, E, F and Z genes. Only the short gene 59 has no known homologues and gene 60 only has two closely related (but unannotated) homologues in Sodalis glossinidius prophages . The last gene in the operon (62) encodes a protein that is 98% identical to the phage λ gene Q antitermination protein, and the putative Q protein target (qut) in ε34 is identical to that of phage λ, indicating that their target specificities are the same.
c. Late operon
The lambdoid phage late operons are turned on by Q antitermination and encode the genes for virion assembly and cell lysis. The ε34 virion is indistinguishable from the P22 virion by negative stain electron microscopy , and most of the morphogenetic genes of ε34 are closely related to those of P22. The only major differences are in ε34 genes 6, 12, 13 and 19. The P22 gene 6 homologue is partly deleted relative to ε34 (see below). Homologues of genes 12 and 13 encode proteins in P22 that are released with the DNA during injection, and 19 encodes the tailspike (discussion of the latter below). SDS polyacrylamide electrophoresis gels of the proteins of the ε34 virions are nearly identical to those of P22 except for a small size difference in the ejection protein gp13, the tailspike protein and, the presence of a strong ~15 kDa band that is not present in P22 virions [21, 22]. N-terminal sequence analysis of the latter band excised from such gels gave a sequence of NH2-ANPNF (performed as described in ), indicating that it is encoded by gene 71, and that its N-terminal methionine is removed. This protein is 97% identical to the Dec protein of phage L that has been shown to make virions more stable to magnesium ion chelation .
By homology with other lambdoid phages that integrate into tRNA genes, the putative integration attachment site (attP) of ε34 is found just downstream of the int (23) gene within a 43 bp sequence that is identical to part of the tRNA argU gene. This strongly suggests that ε34 integrates into, and in the process replaces, the 3'-portion of the argU gene in the Salmonella chromosome. The closest known relative to ε34 integrase is that of the E. coli K12 defective prophage DLP12 which is also integrated into argU . Although these two integrases are only about 80% identical in sequence, most of the differences are outside the following very similar regions: amino acids 1–80, 145–260 and 300–360. These similar regions include or overlap the target specificity generating "arm binding" N-terminal (AAs ~1–60) and "core binding" (AAs ~75–175) domains of λ integrase , so identical target specificity of the ε34 and DLP12 integrases is reasonable. We note that several Salmonella P22-like phages that integrate into the tRNA thrW gene, P22, ST104 and ST64T, have integrases that are about 30% different from ε34.
e. Lysogenic conversion
Phage ε34 has at least six genes (16, 20, 21, 22, 44, and 45) that should be, by their position and experimental work on homologues in other phages, expressed from the prophage. Two of these, 44 and 45, appear to be in the same operon and downstream of the prophage repressor gene (46) gene (like rexA and rexB in λ), but they have no matches in the current sequence database. Genes 16 is a homologue of the P22 mnt maintenance repressor gene discussed below in the context of the "ant moron". Genes 21 and 22 have similarity to genes known to be involved in surface polysaccharide modification, and ε34 is known to perform such modifications [18, 19, 39]. Genes 22 and 21 proteins are very similar to phage-encoded bactoprenol-linked glucose flippases that translocate glucosyated undecaprenyl phosphate from the cytoplasmic face to the periplasmic face of the inner membrane and bactoprenol glucosyl transferases that catalyze transfer of the glucose from UDP-glucose to the above prenyl intermediate, respectively. Genes in these two families are designated gtrA and gtrB [26, 40], respectively. In addition, highly sequence variable but similarly sized genes encoding integral membrane proteins, called gtrC here, are found immediately transcriptionally downstream of gtrA and gtrB (in the position of ε34 gene 20) in similar operons in numerous other phages, prophages, and bacterial chromosomes (see below). In the best studied cases, Shigella flexneri temperate phages SfII, SfV and SfX, their similar operons are known to be responsible for enzymatic modification of the O-antigen polysaccharide to give "serotype conversion" of the bacterial host by the prophage. These downstream variable genes have in some cases been shown to be serotype-specific glucosyl transferases that add various glucosyl residues to the O-antigen backbone repeat. The first two genes, gtrA and gtrB, are highly conserved and not thought to be serotype specific, while gtrC genes are serotype specific . Thus, the ε34 genes 20–22, which appear to constitute an operon since the three genes are arranged with very little or no space between them, are almost certainly responsible for the conversion of Salmonella from serotype 3,10,15 to serotype 3,15,34 by the ε34 prophage (figure 1A) [14–20].
The ε34 gene 20 protein is moderately similar (30–40% identity) to proteins encoded by genes that lie in the gtrC position of several otherwise very similar operons in S. enterica genomes (e.g., Typhimurium LT2 (locus_tag STM0557) and serovar Typhi CT18 (locus_tag STY0605). Further analysis of the currently available (March 2008) database results in fifty-one "gtrC" genes, most of which lie in apparently intact operons with adjacent gtrA and gtrB genes in bacterial genomes. These putative gtrC genes fall into nine different sequence types whose encoded proteins are at best 40% identical (figure 1B). Some of these types have very little sequence similarity (e.g., ε34 gene 20 protein and phage ST64T GtrC). GtrC type proteins GtrII, GtrV and GtrX, encoded by Shigella phages (above), have been shown to have nine transmembrane helices [41–43], and all of the nine types of GtrC protein in the Salmonella operons are very strongly predicted to have between nine and eleven transmembrane helices (by TopPred II analysis ). The ε34 GtrC protein, which is predicted to have nine or ten transmembrane segments, is a unique type. We note that a substantial majority of the 47 "bacterial" gtrC genes in figure 1B are adjacent to gtrB and gtrA genes and reside in prophages. These include P22-like prophages in the genomes of Salmonella serovars Arizonae, Choleraesuis, Dublin, Hadar, Heidelberg, and Paratyphi A; and; apparently defective phage P2-like prophages in serovars Agona, Kentucky, Newport, Saintpaul, Typhimurium, and Virchow; and, a phage λ-like prophage in serovar Schwarzengrund. The fact that gtrABC operons are associated with three very different temperate phage types (P22-, λ- and P2-like) suggests that these operons are evolutionarily important to the Salmonella temperate phages and the hosts that harbor them. The large majority of these prophage-borne gtrABC operons appear to be intact, but in most cases functionality has not yet been demonstrated experimentally. Nonetheless, it seems clear that Salmonella serotype, like Shigella serotype, is frequently modified by the prophages they carry. Presumably, phage carry these genes because they give their prophage host (and so the resident prophage) a selective advantage.
f. Control of gene expression
Gene 46 encodes a protein that is similar to other lambdoid prophage repressors. Its C-terminal protease domain is about 80% identical to the repressors of phage λ and HK97, for example, but its DNA binding N-terminal portion is only about 50% identical to its closest known relatives in phages L, VT2-Sa and Ni12. Thus, the ε34 repressor almost certainly has the same RecA-mediated inactivation mechanism in its C-terminal domain as phage λ , but likely has a different operator binding specificity; the ε34 Cro (gene 47) protein, which should bind the same operators as the repressor, is ≤ 42% identical to known lambdoid Cro proteins. We have identified putative PL and PR promoters (see the ε34 GenBank annotation), and located just upstream of these promoters is a consensus sequence WTACRAWWTGTAT that may represent the operator sites for ε34.
The λ CII protein activates several promoters required for establishment of lysogeny. The ε34 homologue, gp48, is weakly similar to known CII proteins in its N-terminal portion, but its C-terminal putative RNA polymerase binding domain is highly similar to other CII proteins (e.g., 16 identities in the C-terminal 18 AAs with λ CII). Nonetheless, there are TTGCN6TTGC/T sites in reasonable positions in the predicted repressor establishment (PRE) and gene Q anti-sense (PaQ) promoter sequences that suggest the target sequence for ε34 CII is the same or very similar to that of P22 and λ CII . Interestingly, there are two TTGCN6TTGC sites upstream of gtrA suggesting that CII may also activate transcription of the conversion cassette in ε34.
Our analysis of the wildtype ε34 genes 46 (cI), 48 (cII) and 39 (cIII) and surrounding regions shows four differences from the c99 clear plaque mutant, whose genome was sequenced here. These are single amino acid changes, F46V (A32564G) in gp46, and K14E and N41S (A33236G and A33318G, respectively) in gp48 (in addition, there is a T to C change in position 32736, between genes 46 and 47, which does not appear to alter the rightward early promoter or its overlapping operator). The two changes in gp48 alter positions that should be in the N-terminal DNA-binding domain according to the structure of the parallel region in the phage λ CII protein , so the lysogeny defect(s) in c99 are almost certainly due to the above changes in gene 46 and/or 48. These findings confirm a previous complementation analysis which indicated that the c99 mutation was caused by a defect in two different genes . In addition to the regulatory proteins mentioned above in this section, ε34 encodes unambiguous homologues of the early (λ gpN) and late (λ gpQ) transcriptional antitermination proteins found in other lambdoid phages (above). These are discussed further below.
4. Evolution and diversity of the P22-like phages that infect Salmonella
a. P22-like prophages
Several Salmonella phages and prophages are known to have virion assembly proteins that are highly related to P22 and ε34; phages P22, ε34, ST64T and ST104, and prophages in the fully sequenced genomes of Salmonella enterica serovar Paratyphi A strain ATCC 9150  and serovar Choleraesuis strain SC-B67 . These two prophages extend from genes SPA2385 through SPA2431 in Paratyphi and from SC0324 through SC0370 in Choleraesuis; we call these previously unnamed prophages "Para1" and "Scho1", respectively. Similar prophages are present in Salmonella serovars Arizonae (Accession No. NC_010067), Dublin (No. NZ_ABAP0100000), Hadar (NZ_ABFG01000003), and Heidelberg (NZ_ABEL01000001, NZ_ABEM01000002) that are not analyzed here because their genomes are incomplete (S. Casjens, unpublished observations). Figure 2 shows that these phages have a mosaic genome structure typical of the temperate phages (as well as most other phage types). These two prophages contain apparently intact orthologues of all of the genes that are known to be essential in the very well-studied phage P22. These include all of the genes required for control of phage gene expression, DNA replication, virion assembly and lysis. In theory, point mutations could have inactivated nearly any of these prophage genes, but there are many examples of fully functional genes, even in quite highly degraded prophages that are very likely much older than Scho1 and Para1 .
b. Evolution and diversity of the late operon
The morphogenetic regions of the Salmonella P22-like phages are much more like one another than they are like the parallel regions of P22-like phages that infect E. coli or S. flexneri. For example, the coat proteins (ε34 gp5 and homologues) of the Salmonella-infecting members of the group listed above are all >99% identical proteins except that of Scho1 which is about 75% identical to the others. On the other hand the coat proteins of P22-like phages HK620 and CUS-3 that infect E. coli [51, 52] and Shigella phage Sf6  are much more distantly related, with 14–28% identity to the Salmonella phages. It appears that there has been little if any exchange of genetic material corresponding to the ε34 gene 1–10 region between the Salmonella P22-like phages and the E. coli/Shigella P22-like phages.
The virion assembly genes of the six Salmonella phage genomes are very similar (almost all exhibit >90% amino acid sequence identity), with a few exceptions as follows: (1) Very similar homologues of the phage L Dec protein (described above) are encoded by the rightmost genes of the ε34, ST104, ST64T and Para1 genomes in figure 2, but are not encoded by P22 and Scho1. (2) Unlike the other morphogenetic genes (noted as Terminase, Heads, Tails and Tailspike in figure 2), there are significant differences in the lengths of the homologues of ε34 gene 6. The P22 and Scho1 gene 6 homologues have substantial internal deletions relative to the others, and their amino acid sequences have diverged substantially from the others. The role of this gene is unknown, although in P22 it is not essential in the laboratory . (3) The tail and ejection proteins are more variable than the other virion assembly proteins (figure 2). The tailspike genes show an interesting relationship in which the N-terminal head-binding domains (AAs 1–115) are all quite similar; the ε34 domain is the most divergent with about 75% identity to the others. The remaining C-terminal portions of the tailspikes are present in three forms (ε34, Scho1, and P22/ST104/ST64T/Para1) which are not recognizably similar in sequence; this receptor-recognition domain binds and cleaves the bacterial surface O-antigen polysaccharide [23, 25, 54]. Atomic structures of both domains of the P22 tailspike have been determined, and the C-terminal domain has an unusual β-helix structure [55, 56]. The C-terminal domains of ε34 and Scho1 tailspikes have high β-helix predictions , and the ε34 tailspike is resistant to denaturing agents and proteolysis [[21, 22], R. Villafane, unpublished], suggesting that they may have similar overall structures. (4) Gene 13 of ε34, whose protein product is ejected with the DNA, is very similar to that of E. coli phage CUS-3 (not shown), so gene 13 has clearly enjoyed exchange with phages that currently infect different host species. Thus, even in this small sampling, it is apparent that variation is greater in the genes that are thought to interact with the bacterium during the injection process. Such hyper-variation has been noticed previously in the tail fibers of long-tailed phages [58, 59], but not with the short-tailed ones. Such variations are presumably the result of evolutionary sparring involving the physical interaction between the phage virions and their hosts in the first steps of infection.
Two other parts of the late operon are also of interest, the lysis genes and the "moron" immediately 5' of the tailspike gene. The lysis module of lambdoid phages consists of four genes, a holin, a lysin, and homologues of λ Rz and Rz1 proteins . There are three apparently nonhomologous types of holins and three types of lysins present in the six Salmonella P22-like phages in figure 2, yet each of these "lysis cassettes" functions to lyse Salmonella. The second region, between the tailspike gene (19) and the rest of the late operon, is variable in length, has no function in morphogenesis or lysis, and contains different sets of genes in different phages. By these criteria this region fits the definition of a "moron", a (usually) independently expressed gene or group of genes that appears to be inserted into a phage operon when that operon is compared across various related phages [31, 61]. This region, which we call here the "ant moron" because it often harbors a homologue to the P22 antirepressor gene ant is diagrammed in figure 3. In phage P22 this region has been studied in some depth, and it contains genes for two protein repressors (mnt and arc), an antisense RNA (sar) which control the expression of the antirepressor (ant) gene [62–67] (together these are called the immunity I region ). In addition, this region in P22 includes genes that encode a superinfection exclusion protein (sieA)  and two genes of unknown function (hkcC, 59a). The parallel ε34 ant moron contains an apparently functional immunity I region (genes 16, 17 and 18) and two genes (14 and 15) of unknown function, one of which is a homologue of gene hkcB which lies in a similar position in the E. coli phage HK620 genome . The ST64T, ST104 and Para1 have ant morons that are nearly identical to one another and contain only the mnt repressor gene and a gene of unknown function; the parallel Scho1 region has a different gene of unknown function and what appears to be a degraded (and likely nonfunctional) Arc repressor gene. In order to further understand the variability of the ant moron, we include ten other currently known completely sequenced non-Salmonella P22-like phages and prophages in figure 3. These phages all have morphogenetic genes that are syntenic to P22 (but quite divergent in sequence), and they have ant moron regions that vary from its absence in Sf6 , APSE-1 , APSE-2  and ϕSG1  to the P22 /CUS-3 )/UTI-1 (prophage in E. coli strain UT189 )/APEC-1 (prophage in E. coli strain APEC O1 ) types which each contain six predicted genes.
It is curious that these different morons are present at this location in these phages, but no morons are present in any other locations in their morphogenetic gene clusters. Perhaps this is the only location that can tolerate such an insertion? On the other hand, perhaps successful moron insertion is extremely rare, and since each of the ant morons has some sequence similarity to at least one of the others, it is plausible that a single ancestral moron inserted at this position in the past, and the variation that currently exists is due to more frequent insertions and deletions that modified the original insertion. From the sequence differences, it seems clear that these different morons have non-identical functions at present, and only the roles of the P22 version have been studied. Interestingly, the P22 ant moron is thought to have a (accidental?) role in controlling the level of tailspike protein produced during infection by affecting the frequency which PLate initiated RNA polymerase molecules transcribe through the ant moron into the tailspike gene . The Mnt and Arc repressors may have a role in this process, so the ant morons of Scho1, HK620, Sf6, APSE-1, APSE-2 and ϕSG1 may lack this ability. The fact that the HK620 and Scho1 morons have what appear to be degraded ant genes suggests that they may at one time have had a functional antirepressor gene (and complete immunity I region?) in this location that is no longer needed. The apparent lack of arc genes in the CUS-3 and HS-1 ant type morons, in spite of the presence of putative antirepressor genes, suggests that they have a mechanism for controlling the later that differs from P22. Since all sixteen of the phages shown in figure 3 have syntenic sets of morphogenetic genes, the wide differences observed in the ant morons, ten types in sixteen genomes, indicate that this region has a much higher rate of genetic flux than the rest of the late operon.
c. Evolution and diversity in the early operons
The early left (early antitermination through integration regions in figure 2) and early right (replication through late antitermination regions) operons are highly mosaic, even among the Salmonella P22-like phages that have highly similar virion assembly genes, and there is ample evidence for past genetic exchanges with other members of the larger "lambdoid" phage group. For example, P22, ε34 and Para1 have late antitermination genes that are 99% identical to phage λ gene Q protein, and putative origin recognition DNA replication proteins of ε34 and Scho1 are 96% and 97% identical, respectively, to the ES18 homologue, and the parallel ST104 replication protein is 98% identical to the very different coliphage HK97 homologue (ES18 and HK97 both have long non-contractile tails and head and tail genes that are not recognizably similar to those of the P22-like phages). Clearly some of these gpQ-like and replication proteins have been exchanged independently and quite recently between the Salmonella-infecting P22-like phage and members of the larger lambdoid group, since they have not had time to diverge significantly from their relatives in otherwise very different phages.
d. DNA binding specificity of regulatory proteins
Development of the phage life cycle depends on a number of nucleotide sequence-specific nucleic acid-protein interactions. For example, in the lambdoid phages such specificity is observed in early operon repression, replication origin binding, DNA packaging, integration, and the transcriptional antiterminations that give rise to early and late transcripts . For each of these, different specificities are known in different phages. These biologically important relationships are not indicated by different gene colors in figure 2 because the proteins with different specificities are nonetheless homologous to one another. Overall, the extent of diversity that is required to alter the nucleic acid binding specificity is not known (nor should it be a particular value), and of course laboratory examples are known in which single amino acid changes can alter specificity. Nonetheless, it seems reasonable to estimate that proteins with more than 90% sequence identity might be expected to have the same, or at least similar, target specificity. Table 1 shows the comparison of these six proteins in the P22-like Salmonella phages. Only the packaging specificity appears to be the same in all six phages. The other five proteins show between two and five different predicted specificities (i.e., very different sequence types). For example, each of the six repressors is very different in sequence from the others, except those of ST64T and ST104, which are identical. And the late antitermination proteins (homologues of the phage λ Q protein) of these six phages are likely to have the specificities of phages λ or Sf6, except ST104 which is an apparently "new" type. It is interesting to note that among the six ε34 proteins in Table 1, three are predicted to have specificities that do not to exist in previously analyzed phages. Thus, it seems that current analyses are yet close to having a complete list of the sequence (i.e., apparent specificity) types for these important proteins. This subset of these phages' genes also shows clearly that recent shuffling of these genes has occurred within the Salmonella P22-like phages. For example, Para1 encodes repressor and DNA replication proteins that are 100% and 97% identical to those of ST104 and ST64T, respectively, while both of these proteins are very different from each other in ST104 and ST64T. Similarly, Para1 has a phage λ (99% identity) type late antitermination protein, indicating another difference from phages ST104 and ST64T. We cannot deduce the directionality of these exchange event(s), or whether there were intermediary phages, but clearly these genes have been exchanged among these three phages so recently that one protein has not changed at all and the other only changed 3% since their last common ancestor.
We determined the complete nucleotide sequence of the Salmonella P22-like lambdoid phage ε34, and found that it has novel predicted specificities for host polysaccharide modification enzymes, virion receptor binding, integration, early transcriptional antitermination protein and prophage repressor. We used its sequence to help understand the previously unanalyzed sequences of two Salmonella prophages. These sequences, along with the previously known phage P22, ST64T and ST104 genome sequences, give a much clearer picture of the variation among this rather closely related group of Salmonella phages. In spite of this very close relationship, genome mosaicism is found to be prevalent in the early regions of their genomes.
Purification of phage and isolation of DNA
The original phage strain, ε34, a generous gift from Dr. Andrew Wright (to RV) and Dr. Horst Schmieger (to SC), was subjected to hydroxylamine mutagenesis to produce the clear plaque and highly lytic variant, ε34 c99 [48, 77]. The mutant and wildtype DNAs were prepared and purified as described [21, 78] or by using QIAGEN Lambda DNA Purification Kit (QIAGEN, Valencia CA).
The DNA was sequenced commercially by Fidelity System Inc. (Gaithersburg MD) with the final sequence opened immediately upstream of the small terminase subunit gene to be in conformity with the presentation of other similar phages. The completely annotated sequence for this phage can be obtained from GenBank under Accession No. EU570103. This sequence agrees with previously determined sequences of the ε34 tailspike gene  and scaffolding protein gene (P. Weigele and S. Casjens, unpublished).
Protein-encoding genes were identified using Kodon (Applied Maths, Austin, TX) with the proteins being screened for homologues using BLASTP and PSI-BLAST  against the nonredundant protein database at NCBI. Terminators, promoters and operator sequences were defined on the basis of their position and sequence relatedness to similar sites in Salmonella phage P22. The Betawrap http://groups.csail.mit.edu/cb/betawrap/, Softberry http://www.softberry.com/berry.phtml, tRNAscan-SE http://lowelab.ucsc.edu/tRNAscan-SE/, http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html web sites were used for beta-helix structure, phage promoter, tRNA gene and transmembrane helix identification, respectively.
gene product of gene X
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We thank Andrew Wright and Horst Schmieger for phage strains. SC was supported in part by NIH grant R01AI074825. AK is supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada. RV was supported by funds from the Dean of the Ponce School of Medicine.
RV, AK and SC jointly conceived of the project, EG, MZ and RV carried out preliminary and finalization sequencing work, and AK completed the nucleotide sequence, and RV, SC and AK cooperated to perform the sequence analysis and draft the manuscript. All authors have read and approved the final manuscript.