Arabidopsis thaliana AtNUDT7 Nudix pyrophosphatase hydrolyzes NADH and ADP-ribose in vitro and is an important factor in the cellular response to diverse biotic and abiotic stresses. Several studies have shown that loss-of-function Atnudt7 mutant plants display many profound phenotypes. However the molecular mechanism of AtNUDT7 function remains elusive. To gain a better understanding of this hydrolase cellular role, proteins interacting with AtNUDT7 were identified. Using AtNUDT7 as a bait in an in vitro binding assay of proteins derived from cultured Arabidopsis cell extracts we identified the regulatory protein RACK1A as an AtNUDT7-interactor. RACK1A-AtNUDT7 interaction was confirmed in a yeast two-hybrid assay and in a pull-down assay and in Bimolecular Fluorescence Complementation (BiFC) analysis of the proteins transiently expressed in Arabidopsis protoplasts. However, no influence of RACK1A on AtNUDT7 hydrolase catalytic activity was observed. In vitro interaction between RACK1A and the AGG1 and AGG2 gamma subunits of the signal transducing heterotrimeric G protein was also detected and confirmed in BiFC assays. Moreover, association between AtNUDT7 and both AGG1 and AGG2 subunits was observed in Arabidopsis protoplasts, although binding of these proteins could not be detected in vitro. Based on the observed interactions we conclude that the AtNUDT7 Nudix hydrolase forms complexes in vitro and in vivo with regulatory proteins involved in signal transduction. Moreover, we provide the initial evidence that both signal transducing gamma subunits bind the regulatory RACK1A protein.

The PA0336 protein from Pseudomonas aeruginosa belongs to the family of widely distributed Nudix pyrophosphohydrolases, which catalyze the hydrolysis of pyrophosphate bonds in a variety of nucleoside diphosphate derivatives. The amino acid sequence of the PA0336 protein is highly similar to that of the RppH Nudix RNA pyrophosphohydrolase from Escherichia coli, which removes pyrophosphate from 5'-end of triphosphorylated RNA transcripts. Trans-complementation experiments showed that the P. aeruginosa enzyme can functionally substitute for RppH in E. coli cells indicating that, similar to RppH, the Pseudomonas hydrolase mediates RNA turnover in vivo. In order to elucidate the biological significance of the PA0336 protein in Pseudomonas cells, a PA0336 mutant strain was constructed. The mutated strain considerably increased level of the virulence factor pyocyanin compared to wild type, suggesting that PA0336 could be involved in downregulation of P. aeruginosa pathogenicity. This phenotype was reversed by complementation with the wild type but not catalytically inactive PA0336, indicating that the catalytic activity was indispensable for its biological function. Pathogenesis tests in Caenorhabditis elegans showed that the PA0336 mutant of P. aeruginosa was significantly more virulent than the parental strain, confirming further that the P. aeruginosa RNA pyrophosphohydrolase PA0336 modulates bacterial pathogenesis by down-regulating production of virulence-associated factors. To study the role of PA0336 further, transcriptomes of the PA0336 mutant and the wild-type strain were compared using RNA sequencing. The level of 537 transcripts coding for proteins involved in a variety of cellular processes such as replication, transcription, translation, central metabolism and pathogenesis, was affected by the lack of PA0336. These results indicate that the PA0336 RNA pyrophosphohydrolase functions as a global regulator that influences many of transcripts including those involved in P. aeruginosa virulence.

Nudix proteins catalyze the hydrolysis of pyrophosphate bonds in a variety of substrates and are ubiquitous in all domains of life. The genome of an important opportunistic human pathogen, Pseudomonas aeruginosa , encodes multiple Nudix proteins. To determine the role of nine Nudix hydrolases of the P. aeruginosa PAO1161 strain in its fitness, virulence or antibiotic resistance mutants devoid of individual enzymes were constructed and analyzed for growth rate, motility, biofilm formation, pyocyanin production, and susceptibility to oxidative stress and different antibiotics. The potential effect on bacterial virulence was studied using the C aenorhabditis elegans – P. aeruginosa infection model. Of the nine mutants tested, five had an altered phenotype in comparison with the wild‐type strain. The ΔPA3470, ΔPA3754, and ΔPA4400 mutants showed increased pyocyanin production, were more resistant to the β‐lactam antibiotic piperacillin, and were more sensitive to killing by H 2 O 2 . In addition, ΔPA4400 and ΔPA5176 had impaired swarming motility and were less virulent for C. elegans . The ΔPA4841 had an increased sensitivity to oxidative stress. These changes were reversed by providing the respective nudix gene in trans indicating that the observed phenotype alterations were indeed due to the lack of the particular Nudix protein.

SummaryNudix pyrophosphatases, ubiquitous in all organisms, have not been well studied. Recent implications that some of them may be involved in response to stress and in pathogenesis indicate that they play important biological functions. We have investigated NudC Nudix proteins from the plant pathogen Pseudomonas syringae pv. tomato str. DC3000 and from the human pathogen Pseudomonas aeruginosa PAO1161. We found that these homologous enzymes are homodimeric and in vitro preferentially hydrolyse NADH. The P. syringae mutant strain deficient in NudC accumulated NADH and displayed significant defects in growth, motility and biofilm formation. The wild type copy of the nudC gene with its cognate promoter delivered in trans into the nudC mutant restored its fitness. However, introduction of the P. syringae nudC gene under the control of the strong tacp promoter into either P. syringae or P. aeruginosa cells had a toxic effect on both strains. Opposite to P. syringae NudC, the P. aeruginosa NudC deficiency as well as its overproduction had no visible impact on cells. Moreover, P. aeruginosa NudC does not compensate the lack of its counterpart in the P. syringae mutant. These results indicate that NudC from P. syringae, but not from P. aeruginosa is vital for bacteria.