Dataset Information

Identification of a bifunctional UDP-4-keto-pentose/UDP-xylose synthase in the plant pathogenic bacterium Ralstonia solanacearum strain GMI1000, a distinct member of the 4,6-dehydratase and decarboxylase family.

ABSTRACT: The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-beta-l-threo-pentopyranosyl-4''-ulose in the presence of NAD(+). The second activity converts UDP-beta-l-threo-pentopyranosyl-4''-ulose and NADH to UDP-xylose and NAD(+), albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.

SUBMITTER: Gu X

PROVIDER: S-EPMC2838324 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Publications

Identification of a bifunctional UDP-4-keto-pentose/UDP-xylose synthase in the plant pathogenic bacterium Ralstonia solanacearum strain GMI1000, a distinct member of the 4,6-dehydratase and decarboxylase family.

Gu Xiaogang X Glushka John J Yin Yanbin Y Xu Ying Y Denny Timothy T Smith James J Jiang Yingnan Y Bar-Peled Maor M

The Journal of biological chemistry 20100129 12

The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-beta-l-threo-pentopyranosyl-4''-ulose in the presence of NAD(+). The second activity converts UDP-beta-l-threo-pentopyranosyl-4''-ulose and NADH t ...[more]

PMID: 20118241

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Project description:The invasion and colonization of host plants by the destructive pathogen Ralstonia solanacearum rely on its cell motility, which is controlled by multiple factors. Here, we report that the LysR-type transcriptional regulator CrgA (RS_RS16695) represses cell motility in R. solanacearum GMI1000. CrgA possesses common features of a LysR-type transcriptional regulator and contains an N-terminal helix-turn-helix motif as well as a C-terminal LysR substrate-binding domain. Deletion of crgA results in an enhanced swim ring and increased transcription of flhDC In addition, the ΔcrgA mutant possesses more polar flagella than wild-type GMI1000 and exhibits higher expression of the flagellin gene fliC Despite these alterations, the ΔcrgA mutant did not have a detectable growth defect in culture. Yeast one-hybrid and electrophoretic mobility shift assays revealed that CrgA interacts directly with the flhDC promoter. Expressing the β-glucuronidase (GUS) reporter under the control of the crgA promoter showed that crgA transcription is dependent on cell density. Soil-soaking inoculation with the crgA mutant caused wilt symptoms on tomato (Solanum lycopersicum L. cv. Hong yangli) plants earlier than inoculation with the wild-type GMI1000 but resulted in lower disease severity. We conclude that the R. solanacearum regulator CrgA represses flhDC expression and consequently affects the expression of fliC to modulate cell motility, thereby conditioning disease development in host plants.IMPORTANCERalstonia solanacearum is a widely distributed soilborne plant pathogen that causes bacterial wilt disease on diverse plant species. Motility is a critical virulence attribute of R. solanacearum because it allows this pathogen to efficiently invade and colonize host plants. In R. solanacearum, motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum motility is more complex than previously thought and enhances our understanding of flagellum regulation in R. solanacearum.

Project description:BackgroundThe infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules. AHL-acylase is a quorum quenching enzyme and degrades AHLs by removing the fatty acid side chain from the homoserine lactone ring of AHLs. This blocks AHL accumulation and pathogenic phenotypes in quorum sensing bacteria.ResultsAn aac gene of undemonstrated function from Ralstonia solanacearum GMI1000 was cloned, expressed in Escherichia coli; it inactivated four AHLs that were tested. The sequence of the 795 amino acid polypeptide was considerably similar to the AHL-acylase from Ralstonia sp. XJ12B with 83% identity match and shared 39% identity with an aculeacin A acylase precursor from the gram-positive actinomycete Actinoplanes utahensis. Aculeacin A is a neutral lipopeptide antibiotic and an antifungal drug. An electrospray ionisation mass spectrometry (ESI-MS) analysis verified that Aac hydrolysed the amide bond of AHL, releasing homoserine lactone and the corresponding fatty acids. However, ESI-MS analysis demonstrated that the Aac could not catalyze the hydrolysis of the palmitoyl moiety of the aculeacin A. Moreover, the results of MIC test of aculeacin A suggest that Aac could not deacylate aculeacin A. The specificity of Aac for AHLs showed a greater preference for long acyl chains than for short acyl chains. Heterologous expression of the aac gene in Chromobacterium violaceum CV026 effectively inhibited violacein and chitinase activity, both of which were regulated by the quorum-sensing mechanism. These results indicated that Aac could control AHL-dependent pathogenicity.ConclusionThis is the first study to find an AHL-acylase in a phytopathogen. Our data provide direct evidence that the functioning of the aac gene (NP520668) of R. solanacearum GMI1000 is via AHL-acylase and not via aculeacin A acylase. Since Aac is a therapeutic potential quorum-quenching agent, its further biotechnological applications in agriculture, clinical and bio-industrial fields should be evaluated in the near future.

Dataset Information

Identification of a bifunctional UDP-4-keto-pentose/UDP-xylose synthase in the plant pathogenic bacterium Ralstonia solanacearum strain GMI1000, a distinct member of the 4,6-dehydratase and decarboxylase family.

Publications

Identification of a bifunctional UDP-4-keto-pentose/UDP-xylose synthase in the plant pathogenic bacterium Ralstonia solanacearum strain GMI1000, a distinct member of the 4,6-dehydratase and decarboxylase family.

Similar Datasets

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