Vibrio Phage KVP40 Encodes a Functional NAD+Salvage Pathway.
The genome of T4-typeVibriobacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two,nadVandnatV, would suffice for an NAD+salvage pathway. NadV is an apparent nicotinamide phosphoribosyltransferase (NAmPRTase), and NatV is an apparent bifunctional nicotinamide mononucleotide adenylyltransferase (NMNATase) and nicotinamide-adenine dinucleotide pyrophosphatase (Nudix hydrolase). Genes encoding the predicted salvage pathway were cloned and expressed inEscherichia coli, the proteins were purified, and their enzymatic properties were examined. KVP40 NadV NAmPRTase is activein vitro, and a clone complements aSalmonellamutant defective in both the bacterialde novoand 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+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+, and NADH. Expression analysis using reverse transcription-quantitative PCR (qRT-PCR) and enzyme assays of infectedVibrio parahaemolyticuscells demonstratednadVandnatVtranscription 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+biosynthesis presents another important metabolic resource control point by large, rapidly replicating dsDNA bacteriophages.IMPORTANCET4-type bacteriophages enhance DNA precursor synthesis through reductive reactions that use NADH/NADPH as the electron donor and NAD+for ADP-ribosylation 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,nadVandnatV, share an evolutionary history in their respective phage-host group.
Department of Plant & Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA.
Jae Yun
JY
Lee
Department of Plant & Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA.
Zhiqun
Z
Li
Department of Plant & Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA eric_miller@ncsu.edu.
Eric S
ES
Miller
D017122:Bacteriophage T4 / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D023281:Genomics; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009243:NAD / Q000378:metabolism*; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000235:genetics / Q000378:metabolism; D009711:Nucleotides; D011725:Pyridines / Q000737:chemistry*; D011755:Pyrophosphatases / Q000378:metabolism; D014736:Vibrio parahaemolyticus / Q000821:virology*
D009711:Nucleotides; D011725:Pyridines; D009243:NAD; D009612:Nicotinamide-Nucleotide Adenylyltransferase; D011755:Pyrophosphatases; C024289:NAD pyrophosphatase
NAD; bacteriophages; genomics; metabolism