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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+ 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 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+ 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 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+ 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+ 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, nadV and natV, share an evolutionary history in their respective phage-host group.

SUBMITTER: Lee JY

PROVIDER: S-EPMC5388814 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Vibrio Phage KVP40 Encodes a Functional NAD+ Salvage Pathway.

Lee Jae Yun JY Li Zhiqun Z Miller Eric S ES

Journal of bacteriology 20170411 9

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+ 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 predicte ...[more]

PMID: 28167526

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Project description:Plant pathogenic fungi are a major threat to food security and impose a severe economic burden, thus there is a continuous need to develop new strategies to manage them. NAD+ is a co-factor in numerous enzymatic activities and determines the metabolic fate of the cell. Therefore, maintenance of NAD+ concentration is important for cellular viability. Consequently, the NAD+ biosynthetic pathway and redox homeostasis was suggested as a target for antifungal development. We aimed to study how Fusarium oxysporum senses and responds to nicotinaldehyde (NA), an inhibitor of Pnc1, a key enzyme in the salvage pathway of NAD+ biosynthesis. We were able to show that NA was inhibitory in high concentrations to several fungal plant pathogens, with much milder effects on tomato growth. Under low nutrient conditions NA reduced the total amounts of NAD+ in the fungal cell, a trend that was also observed in rich media, although without statistical significance. In low and high nutrient availability NA dramatically reduced the NAD+/NADH ratio. After exposure to NA, NADH levels were increased and NAD+ levels and the biomass were greatly reduced. Cells responded to NA by up-regulation of oxidoreductases, with hardly any up-regulation of the classic response to oxidative stress. Direct measurement of oxidative stress response showed that unlike formaldehyde and hydrogen peroxide, NA caused reductive rather than oxidative stress. Surprisingly, alcohol dehydrogenases were significantly up-regulated more than any other dehydrogenases, including aldehyde dehydrogenases. We propose that conidia of F. oxysporum efficiently detoxified the aldehyde group of NA by reducing NAD+ to NADH; the high concentrations of the latter provoked the expression of alcohol dehydrogenases that in yeast can act to reduce NADH and increase NAD+ amounts, respectively. Overall, the results suggest that targeting NAD+ biosynthesis pathway and redox homeostasis can be a potential approach to manage fungal plant pathogens. Many of the natural antifungal compounds produced by bio-control agents or even the natural biome are aldehydes, and thus the results presented here predict the possible response of Fusarium to wide sources of toxicity in the environment.

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Vibrio Phage KVP40 Encodes a Functional NAD+ Salvage Pathway.

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Vibrio Phage KVP40 Encodes a Functional NAD<sup>+</sup> Salvage Pathway.

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