Genomic analysis of bacteriophage epsilon 34 of Salmonella enterica serovar Anatum (15+)

Abstract

Background: 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 epsilon 34, 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.

Results: The sequence shows that epsilon 34 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.

Conclusion: 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 epsilon 34 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.

Figure 1

O-antigen modifying operons in Salmonella bacterial and phage genome sequences. A. Examples of GtrABC operons. Operons are shown for phage ε³⁴ and P22, as well as for a possibly intact P22-like prophage, which we call Scho1, in the genome of Salmonella enterica serovar Choleraesuis strain SC-B67 (where the putative gtrC gene is named SC0368); genes are indicated as colored rectangles, and rectangles of the same color are recognizably homologous to one another. The transcription of the P22 operon is indicated by a horizontal arrow. B. A CLUSTAL X2 [80] generated neighbor-joining tree of fifty-one known Salmonella GtrC proteins. Horizontal distances in the tree are proportional to sequence differences, and bootstrap values (out of 1000 trials) are shown above the lines and sequence differences are shown below the lines for the long branches. Each GtrC protein is named on the right of the tree according to its location with its "phage" or "Salmonella serovar" name followed by and underlined space and the numeric portion of its GenBank "locus_tag"; "+ number" in parentheses indicates the number of additional identical sequences currently known in other Salmonella strains. On the far right, colored bars indicate the type of prophage that is associated with the gtrC gene; red, P22-like; yellow, P2-like; green, λ-like; and blue, prophage remnant of uncertain ancestry.

Figure 2

Genomes of the P22-like phages of Salmonella. The genomes of four Salmonella temperate phages (P22, ε³⁴, ST104 and ST64T) and two apparently intact Salmonella prophages (Scho1 and Para1; see text) are shown with the open reading frames indicated as colored rectangles. Similar rectangle colors indicate homology and these homologies are connected by yellow trapezoids between adjacent genomes; different open reading frame colors indicate apparent nonhomologies. The circular genome sequences are arbitrarily opened at the start of the small terminase gene. Above, the constant (among this type of phage) order of gene functions are indicated and think black lines between the genomes denote the apparent boundaries between these regions. Asterisks (*) mark genes where homology breaks clearly occur within genes (see text) and daggers (†) indicate the presences of tRNA genes (which read Asn GTT and Thr TGT codons in Scho1 and Asn GTT in Para1). The site of integration into the host genome is indicated at the attachment site (att) of each genome. Finally, the experimentally determined transcription pattern of phage P22 is indicated above the P22 genome (the gray arrowhead on the rightmost mRNA indicates that this transcript extends across the artificial break in the genome and continues at the other "end").

Figure 3

The "ant morons" of the P22-like phages. The genomic regions between the homologues of P22 genes 16 and 9 are shown for the currently known P22-like phages and prophages (the 16 and 9 homologues are indicated in dark orange and marked with the P22 gene name). Genes with the ant moron (see text) are shown as boxes with one end pointing the direction of transcription, and previously given gene names are indicated on the genes for phages P22 and ε³⁴; gene colors and names in the other phages indicate which P22 and/or ε³⁴ they are similar to. Blue genes are each unique sequence; that is, the different blue genes are unrelated to one another. The gray triangle indicated the presence of a IS3 transposon. Asterisks (*) denote genes that appear to be damaged by truncation or frame breaking mutations relative to the P22 genes. Sources for the sequences of the phages and prophages shown in the figure, that are not given in the text, are E. coli HS (accession No. CP000802) and E. coli B7A (accession No. NZ_AAJT01000004).