Complete genome sequence of the broad-host-range vibriophage KVP40: comparative genomics of a T4-related bacteriophage

Abstract

The complete genome sequence of the T4-like, broad-host-range vibriophage KVP40 has been determined. The genome sequence is 244,835 bp, with an overall G+C content of 42.6%. It encodes 386 putative protein-encoding open reading frames (CDSs), 30 tRNAs, 33 T4-like late promoters, and 57 potential rho-independent terminators. Overall, 92.1% of the KVP40 genome is coding, with an average CDS size of 587 bp. While 65% of the CDSs were unique to KVP40 and had no known function, the genome sequence and organization show specific regions of extensive conservation with phage T4. At least 99 KVP40 CDSs have homologs in the T4 genome (Blast alignments of 45 to 68% amino acid similarity). The shared CDSs represent 36% of all T4 CDSs but only 26% of those from KVP40. There is extensive representation of the DNA replication, recombination, and repair enzymes as well as the viral capsid and tail structural genes. KVP40 lacks several T4 enzymes involved in host DNA degradation, appears not to synthesize the modified cytosine (hydroxymethyl glucose) present in T-even phages, and lacks group I introns. KVP40 likely utilizes the T4-type sigma-55 late transcription apparatus, but features of early- or middle-mode transcription were not identified. There are 26 CDSs that have no viral homolog, and many did not necessarily originate from Vibrio spp., suggesting an even broader host range for KVP40. From these latter CDSs, an NAD salvage pathway was inferred that appears to be unique among bacteriophages. Features of the KVP40 genome that distinguish it from T4 are presented, as well as those, such as the replication and virion gene clusters, that are substantially conserved.

FIG. 1.

KVP40 CDS map. CDSs are labeled with the name of the closest homolog, and thus T4 nomenclature is primarily indicated (bold italics); nonitalics are primarily bacterial orthologs. Numbering below the CDS arrow is that used in the text, tables, and GenBank. Colors correspond to protein functional categories, as follows: orange, DNA metabolism; yellow, DNA replication; red, transcription; brown, translation; light blue, tail and tail fibers; dark blue, head; green, host interactions; turquoise, NAD salvage; pink, bacterial-type genes; gray, hypothetical genes in T4; hatched, conserved hypothetical; white, unique hypothetical. Stem-loops are predicted transcription terminators (Table 5) or other RNA structures; the clover leafs mark the positions of tRNAs, and the circle-squiggle symbols denote predicted transmembrane domains.

FIG. 2.

KVP40 tRNA gene cluster. tRNAs were identified with tRNAscan-SE (36). ps, possible pseudo-tRNA. The codon recognized is shown below each tRNA. The tRNA cluster extends from nucleotides 173138 to 181214, all encoded on the negative strand.

FIG. 3.

Probable KVP40 nucleotide metabolism pathway. KVP40 lacks genes for several enzymes for host DNA breakdown and synthesis of modified cytosine (glucosylated hydroxymethyl [Hm]-deoxycytosine), which are marked by X. The anaerobic ribonucleotide reductase (Nrd) likely acts on ribonucleoside triphosphates (rNTP), like the T4 enzyme. KVP40 encodes a host-like dUTPase (CDS021) rather than the bifunctional T4-like dCTPase/dUTPase. Other abbreviations: Cd, dCMP deaminase; Endo, endonuclease; dNMP, deoxynucleoside monophosphate; dNDP, deoxynucleoside diphosphate; Td, thymidylate synthase; Tk, thymidine kinase. All other nomenclature is as for T4. CDS assignments are listed in Table 3.

FIG. 4.

Inferred pyridine nucleotide salvage cycle encoded by KVP40. The components of the NAD salvage pathway (solid lines) are most related to enzymes of bacteria, none of which have been previously identified in phages. PnuC (CDS215) and NadR (CDS211) are shown as a complex for active transport of nicotinamide mononucleotide (NMN) across the bacterial membrane (hatched bar). Nicotinamide (NAm) is assimilated, but an active transport system has not been described. NadV (CDS264) is a nicotinamide phosphoribosyltransferase (NAmPRT) resembling the enzyme from Haemophilus ducreyi that catalyzes the formation of nicotinamide mononucleotide from nicotinamide. NadR is bifunctional, having an apparent nicotinamide mononucleotide adenylyltransferase domain similar to that of bacterial enzymes. The two-domain Nudix hydrolase (here designated NatV; CDS162) resembles one found in the marine cyanobacterial species Synechocystis, which also has a nicotinamide mononucleotide adenylyltransferase domain. Hydrolysis of NADH (dashed lines) can occur by the NatV Nudix hydrolase and by a KVP40 enzyme (CDS043) that is a Sir2/CobB-like enzyme prevalent in eukaryotes and bacteria (also not previously seen in a phage). See references , , , , and for details on the pathway and related enzymes.