Genome analysis of phage JS98 defines a fourth major subgroup of T4-like phages in Escherichia coli

Abstract

Numerous T4-like Escherichia coli phages were isolated from human stool and environmental wastewater samples in Bangladesh and Switzerland. The sequences of the major head gene (g23) revealed that these coliphages could be placed into four subgroups, represented by the phages T4, RB69, RB49, and JS98. Thus, JS98 defines a new major subgroup of E. coli T4-like phages. We conducted an analysis of the 169-kb JS98 genome sequence. Overall, 198 of the 266 JS98 open reading frames (ORFs) shared amino acid sequence identity with the reference T4 phage, 41 shared identity with other T4-like phages, and 27 ORFs lacked any database matches. Genes on the plus strand encoded virion proteins, which showed moderate to high sequence identity with T4 proteins. The right genome half of JS98 showed a higher degree of sequence conservation with T4 and RB69, even for the nonstructural genes, than did the left genome half, containing exclusively nonstructural genes. Most of the JS98-specific genes were found in the left genome half. Two came as a hypervariability cluster, but most represented isolated genes, suggesting that they were acquired separately in multiple acquisition events. No evidence for DNA exchange between JS98 phage and the E. coli host genome or coliphages other than T4 was observed. No undesired genes which could compromise its medical use were detected in the JS98 genome sequence.

FIG. 1.

g23 tree analysis. Major head gene g23 tree analysis was performed with the sequenced PCR products from our field isolates of T4-like phages. The tree is based on nucleotide sequence alignments (corresponding to codons 346 to 1118 in T4). Origins (S, sewage water; and F, fecal content), places (B, Bangladesh; and S, Switzerland), and years of isolation are indicated for the phages from our survey. The numbers at the nodes give the bootstrap probabilities, and the scale above gives percentages of base pair sequence identity. The tree was rooted with the Vibrio T4 phage KVP40 genome sequence. Their codes are indicated at the level of the twigs of the tree.

FIG. 2.

Alignment of genome maps of phages JS98, T4, and RB69. The ORFs are indicated by arrows as follows: white arrows indicate ORFs which share amino acid sequence identity over >70% of the sequence length, and gray arrows indicate ORFs that are specific to the indicated phage genome in this three-phage comparison. ORFs sharing amino acid sequence identity are linked by color shading; the color scale at the bottom of the figure indicates the percentage of amino acid sequence identity between the compared predicted proteins. The JS98 map is shown at the top and bottom to allow comparisons with both T4 and RB69.

FIG. 3.

Alignment and comparison of a 10-kb region showing a high degree of variability between the phages T4 (top), JS98 (middle), and RB69 (bottom). Genes are colored according to their T4 functional assignments (25). Gray indicates genes unique to JS98. Gene 126 is shown hatched, as it shows 40.5% homology to ORF 063 of phage 44RR. The T4 ORFs are annotated with their conventional gene names, JS98 ORFs are numbered or named after the corresponding T4 homologues, and RB69 genes are quoted with the annotations given to them in the GenBank entry (accession number NC_004928). Amino acid sequence identities between genes were determined using STRETCHER and are indicated by connections of red to yellow shading, according to the color key provided at the bottom right. The black ovals indicate homology between T4 and RB69. The top line provides a base pair scale and the positions of the first and last depicted JS98 genes.

FIG. 4.

Annotated genome map of bacteriophage JS98. Following convention, the map starts at the top left with the rIIA gene and ends at the bottom left with the rIIB gene. The genome was divided into 15-kb segments that are to be read from left to right and from top to bottom. The individual ORFs are depicted as arrows, with the orientation of the arrows indicating whether the genes are carried on the Watson or Crick strand. The color of the arrow identifies the functional category into which the homologous T4 gene was classified (25). The color code for gene function and the COG letter code are provided in the bottom center frame of the figure. The 266 predicted JS98 ORFs plus three tRNA genes are annotated above the corresponding arrows with the names of the homologous T4 genes (letter code or horizontal number code). If the JS98 ORF lacks a T4 gene complement, we attributed a number to the gene, starting from rIIA. To distinguish these JS98 ORFs from T4 genes which also carry numbers as gene identifiers (horizontal numbers), we annotated the JS98 genes lacking a T4 homologue with vertical numbers. The first such JS98 ORF without a T4 complement is found in the second line, following the T4 homologue uvsX, and is annotated as JS98 ORF 38. Three tRNA genes are indicated with dark olive arrows, located between JS98 ORFs 136 and 137. Below the arrows are boxes whose colors indicate how many phages from the Tulane database contain a protein which shares protein sequence identity with the JS98 gene. The key to the color code for the prevalence of the JS98 genes is provided in the bottom right frame.