Searching for Biological Function of the Mysterious PA2504 Protein from Pseudomonas aeruginosa.

For nearly half of the proteome of an important pathogen, Pseudomonas aeruginosa, the function has not yet been recognised. Here, we characterise one such mysterious protein PA2504, originally isolated by us as a sole partner of the RppH RNA hydrolase involved in transcription regulation of multiple genes. This study aims at elucidating details of PA2504 function and discussing its implications for bacterial biology. We show that PA2504 forms homodimers and is evenly distributed in the cytoplasm of bacterial cells. Molecular modelling identified the presence of a Tudor-like domain in PA2504. Transcriptomic analysis of a ΔPA2504 mutant showed that 42 transcripts, mainly coding for proteins involved in sulphur metabolism, were affected by the lack of PA2504. In vivo crosslinking of cellular proteins in the exponential and stationary phase of growth revealed several polypeptides that bound to PA2504 exclusively in the stationary phase. Mass spectrometry analysis identified them as the 30S ribosomal protein S4, the translation elongation factor TufA, and the global response regulator GacA. These results indicate that PA2504 may function as a tether for several important cellular factors.

Nudix proteins affecting microbial pathogenesis.

Nudix proteins catalyse hydrolysis of pyrophosphate bonds in a variety of substrates and are ubiquitous in all domains of life. Their widespread presence and broad substrate specificity suggest that they have important cellular functions. In this review, we summarize the state of knowledge on microbial Nudix proteins involved in pathogenesis.

Individual Nudix hydrolases affect diverse features of Pseudomonas aeruginosa.

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 Caenorhabditis 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 ß-lactam antibiotic piperacillin, and were more sensitive to killing by H2 O2 . 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.

Nudix-type RNA pyrophosphohydrolase provides homeostasis of virulence factor pyocyanin and functions as a global regulator in Pseudomonas aeruginosa.

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.

NudC Nudix hydrolase from Pseudomonas syringae, but not its counterpart from Pseudomonas aeruginosa, is a novel regulator of intracellular redox balance required for growth, motility and biofilm formation.

Nudix 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.

Arabidopsis thaliana Nudix hydrolase AtNUDT7 forms complexes with the regulatory RACK1A protein and Ggamma subunits of the signal transducing heterotrimeric G protein.

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.

Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quaternary structure formation and activity.

Arabidopsis thaliana AtNUDT7, a homodimeric Nudix hydrolase active on ADP-ribose and NADH, exerts negative control on the major signaling complex involved in plant defense activation and programmed cell death. The structural and functional consequences of altering several amino-acid residues of the AtNUDT7 protein have been examined by site-directed mutagenesis, far-UV circular dichroism (CD), attenuated total reflection-Fourier transform infrared (ATR-FTIR) and photon correlation (PCS) spectroscopy, biochemical analysis and protein-protein interaction studies. Alanine substitutions of F73 and V168 disallowed dimer formation. Both the F73A- and V168A-mutated proteins displayed no observable enzymatic activity. Alanine substitution of the V69 residue did not significantly alter the enzyme activity and had no influence on dimer arrangement. The non-conserved V26 residue, used as a negative control, did not contribute to the enzyme quaternary structure or activity. Detailed biophysical characterization of the wild-type and mutant proteins indicates that the mutations do not considerably alter the secondary structure of the enzyme but they affect dimer assembly. In addition, mutating residues V69, F73 and V168 disrupted the binding of AtNUDT7 to the regulatory 14.3.3 protein. These are the first studies of the structure-function relationship of AtNUDT7, a Nudix hydrolase of important regulatory function.

The plant Nudix hydrolase family.

Nudix hydrolases are a family of proteins defined by a conserved amino-acid sequence GX(5)-EX(7)REUXEEXGU, where U is a hydrophobic residue. These enzymes are widely distributed among all classes of organisms and catalyze, with varying degrees of substrate specificity, the hydrolysis of a variety of nucleoside diphosphate derivatives: nucleoside di- and triphosphates and their oxidized forms, dinucleoside polyphosphates, nucleotide sugars, NADH, coenzyme A and the mRNA cap. Nudix proteins are postulated to control the cellular concentration of these compounds. The genome of the model plant Arabidopsis thaliana contains 29 genes coding for putative Nudix hydrolases. Recently, several Arabidopsis Nudix genes have been cloned and their products characterized. This review summarizes current knowledge on these plant enzymes and discusses their possible cellular functions.

Cloning and characterization of AtNUDT13, a novel mitochondrial Arabidopsis thaliana Nudix hydrolase specific for long-chain diadenosine polyphosphates.

A cDNA corresponding to the At3g26690 gene, which encodes a Nudix protein (AtNUDT13) with predicted mitochondrial localization, was isolated from an Arabidopsis thaliana library. The 202 amino acid AtNUDT13 polypeptide was overexpressed in Escherichia coli and purified to homogeneity. The preferred substrate for this hydrolase was diadenosine hexaphosphate (Ap(6)A), with K(m) and k(cat)/K(m) values of 0.61 mm and 16.0 x 10(3) m(-)1.s(-1), respectively. Optimal activity was at alkaline pH (8.5) with Mg(2+) (5 mm) as the cofactor. MS analysis revealed that the products of diadenosine hexaphosphate hydrolysis were ADP and adenosine tetraphosphate. Diadenosine pentaphosphate and adenosine tetraphosphate were additional substrates, but diadenosine tetraphosphate and diadenosine triphosphate, adenosine nucleotides, diphosphoinositol polyphosphate and phosphoribosyl pyrophosphate were not hydrolyzed. Chemical crosslinking and size exclusion chromatography demonstrated that the protein exists as a monomer in solution. Subcellular localization studies indicated that the AtNUDT13 protein is targeted to the mitochondria. This is the first description of a plant pyrophosphatase catalyzing the hydrolysis of long-chain diadenosine polyphosphates: molecules with multiple biological activities.

Cloning and characterization of an Arabidopsis thaliana Nudix hydrolase homologous to the mammalian GFG protein.

In a search for a plant antimutator MutT protein, an Arabidopsis thaliana Nudix hydrolase with homology to the mammalian GFG protein was expressed as a hexahistidine fusion polypeptide in Escherichia coli and purified to homogeneity. Unlike the GFG protein, the A. thaliana homolog could not complement the mutT mutation in a MutT-deficient E. coli strain nor was it able to hydrolyze 8-oxo-dGTP, the main substrate of the MutT protein. Instead the recombinant protein hydrolyzed a variety of nucleoside diphosphate derivatives showing a preference for ADP-ribose, with Km and k(cat) values of 1.2 mM and 2.7 s(-1) respectively. The products of ADP-ribose hydrolysis were AMP and ribose-5-phosphate. The optimal activity was at alkaline pH (8.5) with Mg2+ (5 mM) ions as the cofactor. The protein exists as a dimmer in solution.

Cloning and characterization of the first member of the Nudix family from Arabidopsis thaliana.

The sequence motif commonly called a Nudix box, represented by (GX(5)EX(7)REVXEEXGU) is the marker of a widely distributed family of enzymes that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we describe the cloning and characterization of an Arabidopsis thaliana cDNA encoding a Nudix hydrolase that degrades NADH. The deduced amino acid sequence of AtNUDT1 contains 147 amino acids. The recombinant AtNUDT1 was expressed in Escherichia coli and purified. In the presence of Mn(2+) and the optimal pH of 7. 0, the recombinant AtNUDT1 catalyzed the hydrolysis of NADH with a K(m) value of 0. 36 mm. A V(max) of 12. 7 units mg (-1) for NADH was determined. The recombinant AtNUDT1 migrated as a dimer on a gel filtration column. Biochemical analysis of recombinant AtNUDT1 indicated that the first characterized member of the Nudix family from A. thaliana is a NADH pyrophosphatase.