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Query: UNIPROT:P06889 (Mol)
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The metA gene encoding homoserine acetyltransferase, the first enzyme of the methionine biosynthetic pathway, was isolated from a pMT1-based corynebacterium glutamicum gene library via complementation of an Escherichia coli metA mutant. A DNA-sequence analysis of the cloned DNA is identified an open-reading frame of 1,137 bp which encodes a protein with the molecular weight of 41,380 comprising 379 amino acids. The putative protein product showed good amino acid-sequence homology to its counterpart in other organisms. The internal fragment of the cloned DNA was successfully used to disrupt chromosomal metA, demonstrating the identity of the cloned gene. The C. glutamicum metA mutant lost the ability to grow on glucose minimal medium supplemented with homoserine. However, the mutant could grow on a minimal medium supplemented with cystathionine, demonstrating that C. glutamicum uses the cystathionine route to synthesize methionine. Introduction of a plasmid carrying cloned metA into C. glutamicum resulted in a 10-fold increase in enzyme activities and expression of a protein product of M(r) 41,000, which agrees with the sequence data and is similar in size to those of other homoserine acetyltransferases. Unlike E. coli whose metA product uses succinyl coenzyme A as a substrate, the cloned metA gene produced homoserine acetyltransferase which uses only acetyl coenzyme A as the acyl donor.
Mol Cells 1998 Jun 30
PMID:Isolation and analysis of metA, a methionine biosynthetic gene encoding homoserine acetyltransferase in corynebacterium glutamicum. 966 65

The enterobacterium Erwinia carotovora ssp. carotovora strain 71 (hereafter Ecc71) produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh) and protease (Prt). These enzymes degrade plant cell wall components and are largely responsible for the elicitation of soft-rot diseases in plants and plant products. Ecc71 also produces HarpinEcc, the elicitor of hypersensitive reaction (HR) and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (OHL). OHL controls extracellular enzyme and HarpinEcc production. The levels of these enzymes, as well as the expression of hrpNEcc, the structural gene for HarpinEcc, and ohll, the gene specifying OHL synthesis, are negatively regulated by RsmaA. rsmB, formerly aepH, on the other hand, positively regulates extracellular enzyme production. 6His-RsmA recombinant protein purified from E. coli binds rsmB RNA as indicated by gel mobility shift assays. rsmB comprises 547 bp DNA, which is transcribed from a single start site immediately after a sigma70-like promoter. In Ecc71, two rsmB RNA species are detected: a full-length 479 base rsmB RNA and a 259 base rsmB' RNA. rsmB' DNA hybridizes with the 259 base and the 479 base transcripts. A 3' RNase protection assay revealed that the 259 base and the 479 base RNA species end at the same position immediately after the putative rho-independent terminator. The expression of rsmB-lacZ transcriptional fusions established that the rsmB' RNA is not produced because of the activation of an internal promoter. These data strongly suggest that the 259 base rsmB' RNA is derived by processing of the primary rsmB RNA. In Ecc71, rsmB' expression driven by the lac promoter causes overproduction of Pel, Peh, Cel and Prt, and accumulation of pel-1, peh-1, hrpNEcc and ohll transcripts. By contrast, a plasmid with the rsmB' DNA sequence deleted fails to cause overproduction of the extracellular enzymes in Ecc71. The rsmB' effect also occurs in Escherichia coli as glycogen accumulation is stimulated in the presence of rsmB'. In vivo and in vitro translation as well as mutational analysis of rsmB' have established that rsmB' RNA does not yield a translational product. Therefore, we concluded that the rsmB' RNA itself functions as the regulator. Indeed, the expression rsmB' DNA leads to neutralization of the negative effects of the RNA-binding protein, RsmA, in Ecc71 and Serratia marcescens strain SM274. We propose a model that explains how RsmA and rsmB control the expression of genes for extracellular enzymes.
Mol Microbiol 1998 Jul
PMID:Characterization of a novel RNA regulator of Erwinia carotovora ssp. carotovora that controls production of extracellular enzymes and secondary metabolites. 970 16

The plant pathogen Erwinia chrysanthemi produces three acyl-homoserine lactones (acyl-HSLs). One has been identified as N-(3-oxohexanoyl)-homoserine lactone (OHHL), and the two others were supposed to be N (hexanoyl)-homoserine lactone (HHL) and N-(decanoyl)-homoserine lactone (DHL). The genes for a quorum-sensing signal generator (expI) and a response regulator (expR) were cloned. These genes are convergently transcribed and display high similarity to the expI-expR genes of Erwinia carotovora. ExpI is responsible for both OHHL and HHL production. Inactivation of expl had little effect on pectinase synthesis in E. chrysanthemi, as expression of only two of the pectate lyase genes, pelA and pelB, was decreased. E. chrysanthemi expR mutants still produced acyl-HSL and pectinases. However, gel shift and DNAse I footprinting experiments showed that the purified E. chrysanthemi ExpR protein binds specifically to the promoter regions of the five major pel genes. Addition of OHHL modified the ExpR-DNA bandshift profiles, indicating that ExpR interacts with OHHL and binds to DNA in different ways, depending on the OHHL concentration. Localization of the ExpR binding sites just upstream of promoter regions suggests that ExpR functions as an activator of pel expression in the presence of OHHL. The absence of a phenotype in expR mutants strongly suggests that at least an additional interchangeable ExpR homologue exists in E. chrysanthemi. Finally, transcription of expI::uidA and expR::uidA fusions is dependent on the population density, suggesting the existence of a quorum-sensing hierarchy in E. chrysanthemi. These results suggest that the expI-expR locus is part of a complex autoregulatory system that controls quorum sensing in E. chrysanthemi.
Mol Microbiol 1998 Sep
PMID:Characterization of the Erwinia chrysanthemi expI-expR locus directing the synthesis of two N-acyl-homoserine lactone signal molecules. 978 77

The expI-expR locus drives a quorum-sensing system in the phytopathogenic bacterium, Erwinia chrysanthemi. Purified ExpR, an N-acyl homoserine lactone-responsive regulatory protein, binds to the promoter/operator region of the expI and expR genes. DNase I footprinting experiments showed that ExpR protects the regions between -66 and -40 from the P1 transcription initiation site of expl and between -54 and -18 from the expR transcription initiation site P1. The protected region overlaps the two expR promoters, P1 and P2, suggesting that ExpR exerts a negative control on its own gene expression. This assertion is reinforced by the fact that the addition of OHHL dissociates the ExpR-expR DNA complex. In contrast, the location of the ExpR binding site on the expI gene suggests an activator function, as reported for the pel genes. Moreover, ExpR is able to induce DNA bending. In vivo and in vitro studies revealed that CRP functions as an activator of expR expression, but as a repressor of expI transcription. A second level of control of expR and expI occurs through the PecS repressor, a regulator of pectinase synthesis. PecS represses expI expression, while ExpR activates pecS transcription, suggesting the existence of a mutual control between pecS and the expI-expR system in E. chrysanthemi. Regulation of pectinase synthesis in soft rot Erwinia appears to be a complex network of multiple cross-acting regulatory elements. A model that integrates these regulatory elements is proposed.
Mol Microbiol 1998 Sep
PMID:Integration of the quorum-sensing system in the regulatory networks controlling virulence factor synthesis in Erwinia chrysanthemi. 978 78

Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL). Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.
Mol Plant Microbe Interact 1998 Nov
PMID:Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria. 980 99

A mutant strain (CTNUX23) of Rhizobium etli carrying Tn5 unable to grow with sulfate as the sole sulfur source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a metZ (O-succinylhomoserine sulfhydrylase)-homologous gene. The CTNUX23 mutant strain had a growth dependency for methionine, although cystathionine or homocysteine, but not homoserine or O-succinylhomoserine, allowed growth of the mutant. RNase protection assays showed that the metZ-like gene had a basal level of expression in methionine- or cysteine-grown cells, which was induced when sulfate or thiosulfate was used. The metZ gene was cloned from the parent wild-type strain, CE3, and the resulting plasmid pAR204 relieved, after transformation, the methionine auxotrophy of both strains CTNUX23 of R. etli and PAO503(metZ) of Pseudomonas aeruginosa. Unlike strain CE3 or CTNUX23 (pAR204), strain CTNUX23 showed undetectable levels of O-succinylhomoserine sulfhydrylase activity. Strain CTNUX23 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) or to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris, unless methionine was added to the growth medium. These data and our previous results support the notion that cysteine or glutathione, but not methionine, is supplied by the root cells to bacteria growing inside the plant.
Mol Plant Microbe Interact 1999 Jan
PMID:The Rhizobium etli metZ gene is essential for methionine biosynthesis and nodulation of Phaseolus vulgaris. 988 90

Conjugation of the Agrobacterium Ti plasmid pTiC58 is regulated by a hierarchy involving induction by the opines agrocinopines A and B and a quorum-sensing system. Regulation by the opines is mediated by the repressor AccR, while quorum sensing is effected by the transcriptional activator TraR and its ligand, the acyl-homoserine lactone signal molecule Agrobacterium autoinducer (AAI). These last two elements combine to activate expression of the tra system at high population densities. Sequence analysis indicated that traR is the fourth gene of an operon, which we named arc, that is transcribed divergently from accR. Complementation analysis of mutations in the genes 5' to traR showed that the other members of the arc operon are not required for conjugation. Analysis of lacZ reporter fusions demonstrated that traR expression is regulated directly by AccR. Deletion analysis showed that AccR-regulated expression of traR initiates from a promoter located in the intergenic region between accR and orfA, the first gene of the arc operon. Reverse transcriptase-polymerase chain reaction (RT-PCR) and primer extension analyses indicated that the arc transcript initiates upstream of orfA and proceeds uninterrupted through traR. These results are consistent with a model in which quorum sensing is subordinate to the opine regulon because traR has become associated with an operon controlled by the opine-responsive transcriptional regulator.
Mol Microbiol 1999 Jun
PMID:Hierarchical gene regulatory systems arising from fortuitous gene associations: controlling quorum sensing by the opine regulon in Agrobacterium. 1036 9

Cystathionine gamma-synthase catalyses the committed step of de novo methionine biosynthesis in micro-organisms and plants, making the enzyme an attractive target for the design of new antibiotics and herbicides. The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum has been solved by Patterson search techniques using the structure of Escherichia coli cystathionine gamma-synthase. The model was refined at 2.9 A resolution to a crystallographic R -factor of 20.1 % (Rfree25.0 %). The physiological substrates of the enzyme, L-homoserine phosphate and L-cysteine, were modelled into the unliganded structure. These complexes support the proposed ping-pong mechanism for catalysis and illustrate the dissimilar substrate specificities of bacterial and plant cystathionine gamma-synthases on a molecular level. The main difference arises from the binding modes of the distal substrate groups (O -acetyl/succinyl versusO -phosphate). Central in fixing the distal phosphate of the plant CGS substrate is an exposed lysine residue that is strictly conserved in plant cystathionine gamma-synthases whereas bacterial enzymes carry a glycine residue at this position. General insight regarding the reaction specificity of transsulphuration enzymes is gained by the comparison to cystathionine beta-lyase from E. coli, indicating the mechanistic importance of a second substrate binding site for L-cysteine which leads to different chemical reaction types.
J Mol Biol 1999 Jul 30
PMID:The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum reveals its substrate and reaction specificity. 1043 97

Previous studies have shown that the production of extracellular enzymes (pectate lyase [Pel], polygalacturonase [Peh], cellulase [Cel], and protease [Prt]) and harpin(Ecc) (the elicitor of hypersensitive reaction) in Erwinia carotovora subsp. carotovora is regulated by RsmA, an RNA-binding protein, and rsmB, a regulatory RNA (Rsm stands for regulator of secondary metabolites) (Y. Liu et al., Mol. Microbiol. 29:219-234, 1998). We have cloned and characterized a novel regulatory gene, rsmC, that activates RsmA production and represses extracellular enzyme and harpin(Ecc) production, rsmB transcription, and virulence in E. carotovora subsp. carotovora. In an rsmC knockout mutant of E. carotovora subsp. carotovora Ecc71 carrying the chromosomal copy of the wild-type rsmA(+) allele, the basal levels of Pel, Peh, Cel, Prt, and harpin(Ecc) as well as the amounts of rsmB, pel-1, peh-1, celV, and hrpN(Ecc) transcripts are high, whereas the levels of rsmA transcripts and RsmA protein are low. Furthermore, the expression of an rsmA-lacZ gene fusion is lower in the RsmC(-) mutant than in the RsmC(+) parent. Conversely, the expression of an rsmB-lacZ operon fusion is higher in the RsmC(-) mutant than in the RsmC(+) parent. These observations establish that RsmC negatively regulates rsmB transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC(-) mutant, an RsmA(-) mutant, and an RsmA(-) RsmC(-) double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory effects of RsmC on rsmA and rsmB. Exoprotein production by the RsmC(-) mutant is partially dependent on the quorum sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Southern blot data and analysis of PCR products disclosed the presence of rsmC sequences in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, and E. carotovora subsp. carotovora. These findings collectively support the idea that rsmA and rsmB expression in these plant pathogenic Erwinia species is controlled by RsmC or a functional homolog of RsmC.
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PMID:rsmC of the soft-rotting bacterium Erwinia carotovora subsp. carotovora negatively controls extracellular enzyme and harpin(Ecc) production and virulence by modulating levels of regulatory RNA (rsmB) and RNA-binding protein (RsmA). 1049 17

In cell-free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N-acylhomoserine lactone (AHL) biosensor. Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(DeltaAla-L-Val) and cyclo(L-Pro-L-Tyr) respectively. These compounds were also found in cell-free supernatants from Proteus mirabilis, Citrobacter freundii and Enterobacter agglomerans [cyclo(DeltaAla-L-Val) only]. Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes, we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo(L-Phe-L-Pro). Dose-response curves using a LuxR-based AHL biosensor indicated that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) activate the biosensor in a concentration-dependent manner, albeit at much higher concentrations than the natural activator N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL). Competition studies showed that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) antagonize the 3-oxo-C6-HSL-mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR-binding site. Similarly, DKPs were found to be capable of activating or antagonizing other LuxR-based quorum-sensing systems, such as the N-butanoylhomoserine lactone-dependent swarming motility of Serratia liquefaciens. Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.
Mol Microbiol 1999 Sep
PMID:Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other gram-negative bacteria. 1051 Feb 39


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