Vibrio Phage KVP40 Encodes a Functional NAD ⁺ Salvage Pathway

Abstract

The genome of T4-type Vibrio bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, nadV and natV, would suffice for an NAD ϩ salvage pathway. NadV is an apparent nicotinamide phosphoribosyltransferase (NAmPRTase), and NatV is an apparent bifunctional nicotinamide mononucleotide adenylyltransferase (NMNATase) and nicotinamideadenine dinucleotide pyrophosphatase (Nudix hydrolase). Genes encoding the predicted salvage pathway were cloned and expressed in Escherichia coli, the proteins were purified, and their enzymatic properties were examined. KVP40 NadV NAmPRTase is active in vitro, and a clone complements a Salmonella mutant defective in both the bacterial de novo and salvage pathways. Similar to other NAmPRTases, the KVP40 enzyme displayed ATPase activity indicative of energy coupling in the reaction mechanism. The NatV NMNATase activity was measured in a coupled reaction system demonstrating NAD ϩ biosynthesis from nicotinamide, phosphoribosyl pyrophosphate, and ATP. The NatV Nudix hydrolase domain was also shown to be active, with preferred substrates of ADP-ribose, NAD ϩ , and NADH. Expression analysis using reverse transcriptionquantitative PCR (qRT-PCR) and enzyme assays of infected Vibrio parahaemolyticus cells demonstrated nadV and natV transcription during the early and delayed-early periods of infection when other KVP40 genes of nucleotide precursor metabolism are expressed. The distribution and phylogeny of NadV and NatV proteins among several large double-stranded DNA (dsDNA) myophages, and also those from some very large siphophages, suggest broad relevance of pyridine nucleotide scavenging in virus-infected cells. NAD ϩ biosynthesis presents another important metabolic resource control point by large, rapidly replicating dsDNA bacteriophages.IMPORTANCE T4-type bacteriophages enhance DNA precursor synthesis through reductive reactions that use NADH/NADPH as the electron donor and NAD ϩ for ADPribosylation of proteins involved in transcribing and translating the phage genome. We show here that phage KVP40 encodes a functional pyridine nucleotide scavenging pathway that is expressed during the metabolic period of the infection cycle. The pathway is conserved in other large, dsDNA phages in which the two genes, nadV and natV, share an evolutionary history in their respective phage-host group.

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“…The array of genes for a nicotinamide riboside transporter, a nicotinamidenucleotide adenylyltransferase and ADP-ribose pyrophosphatase may be involved in a potential pyridine nucleotide (NAD+) salvage pathway. In vitro tests to support an analogous observation have been recently performed for a Vibrio bacteriophage [52]. A gene producing a nicotinamide mononucleotide transporter, present in all the phages studied here, may be another unit of this pathway.…”

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“…Among these genes are proteins that build the replisome, such as DNA polymerase (PhiOC_p004), DNA polymerase sliding clamp (PhiOC_p010), and its subunits (PhiOC_p008-9), as well as DNA helicase (PhiOC_p001), DNA primase (PhiOC_p384), and single-stranded DNA-binding protein (PhiOC_p084). Moreover, RNase H (PhiOC_p092) and DNA ligase (PhiOC_p091) are involved in the removal of the initiator RNA primers of Okazaki fragments, and the ligation of those primers during lagging-strand DNA synthesis have also been indicated [26,30]. Potentially, vB_OspM_OC's modes of initiating replication are similar to those of the T4 phage and are origin-and recombination-dependent [26].…”

“…This protein may convert nucleoside triphosphate (dNTP) into a nucleotide (dNMP) and diphosphate (PP i ). Moreover, genes encoding two polynucleotide kinases (PhiOC_p107 and PhiOC_p110) and a putative nucleotidyltransferase (PhiOC_p140) were also identified, which together take part in the nucleotide salvage pathway [30,31].…”

“…Moreover, it was shown that at least 169 different bacteriophages encode the NadV-like NAmPRTase. However, only 50 documented phages encode a bifunctional NatV-like enzyme, and all of these also contain a nadV gene in their genomes [30]. Amongst them, 21 phages have genomes with sizes above 200 kb, including the previously mentioned KVP40 phage.…”

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“…In addition to proteins already annotated as structural proteins, several enzymes or proteins with other functions were also detected by LC-MS/MS (Tables S3 and S4). These included DNA polymerase (Stab21 gene g145, Figure 6), glycerophosphoryl diester phosphodiesterase (g193) likely involved in cell wall teichoic acid hydrolysis [45,46], nicotinamide phosphosribosyltransferase (g207) that augments nucleotide synthesis [47], ribonucleotide reductases (g138-g140) that are also involved in dNTP synthesis [48], endo-and exonucleases (g130, g132) playing role in host DNA degradation and provision of nucleoside 5 -monophosphate precursors for synthesis of phage DNA progeny [49]. The sigma factor (g151) will quide the host RNA-polymerase to start transcription from phage promoters.…”