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PubMed2003

Comparative study of regulatory mechanisms for pectinase production by Erwinia carotovora subsp. carotovora and Erwinia chrysanthemi.

The production of pectinase, the major virulence determinant of soft-rot Erwinia species, is controlled by many regulatory factors. We focused on the major regulatory proteins, KdgR, CRP, Pir, and PecS, characterized mainly in E. chrysanthemi, and tested for their presence and function in the control of pectate lyase (Pel) and polygalacturonase (Peh) production in E. carotovora subsp. carotovora. Homologues of kdgR and crp but not of pir and pecS were detected by Southern blot analyses in E. carotovora subsp. carotovora. In fact, KdgR and CRP homologues of E. carotovora subsp. carotovora had high amino acid identities to those of E. chrysanthemi, including a complete match of the hypothetical helix-turn-helix DNA-binding motif. However, in Western blot analyses using anti-Pir (E. chrysanthemi) antibodies, a cross-reacting protein was present in both Erwinia species, although Pel production in E. carotovora subsp. carotovora was not further stimulated by adding plant extract into the medium containing PGA (polygalacturonic acid) in which hyperinduction by Pir has been reported in E. chrysanthemi EC16. When plasmids that contained each of these regulatory genes from E. chrysanthemi were introduced into E. carotovora subsp. carotovora, Pel production was controlled as predicted from their roles in E. chrysanthemi, except for PecS. PecS exerted a positive control in E. carotovora subsp. carotovora, in contrast to a negative control in E. chrysanthemi. DNA-binding assays demonstrated that KdgR, CRP, Pir, and PecS of E. chrysanthemi and KdgR and CRP homologues of E. carotovora subsp. carotovora could bind to the promoter regions of pel-1, pel-3, and peh of E. carotovora subsp. carotovora. Taken together, KdgR and CRP homologues of E. carotovora subsp. carotovora may regulate Pel and Peh production as in E. chrysanthemi. However, the presence of Pir and PecS homologues in E. carotovora subsp. carotovora was not identified in this study, though these proteins of E. chrysanthemi were functional on the promoter regions of the pectinase genes of E. carotovora subsp. carotovora.

Amino Acid Sequence↗
PubMed2003

Peh production, flagellum synthesis, and virulence reduced in Erwinia carotovora subsp. carotovora by mutation in a homologue of cytR.

Erwinia carotovora subsp. carotovora is a causal agent of soft-rot diseases in a wide variety of plants. Here, we have isolated a new regulatory factor involved in the virulence of E. carotovora subsp. carotovora by in vivo insertional mutagenesis using a transposon Tn5. The gene was homologous to cytR encoding a transcriptional repressor of nucleoside uptake and catabolism genes in Escherichia coli, Salmonella typhimurium, and Vibrio cholerae. Phenotypic characterization of a nonpolar deletion mutant of the cytR homologue (delta cytR) revealed that the delta cytR mutant produced a reduced level of polygalacturonase (Peh) and lost its motility compared to that in the parental strain. With electron microscopy, the delta cytR mutant was shown to be aflagellate. Furthermore, the expression of fliA and fliC (encoding sigma28 and flagellin, respectively) was also reduced in delta cytR mutant. The virulence of delta cytR mutant was reduced in Chinese cabbage and potato compared to that of the parental strain. These results suggest that the CytR homologue of E. carotovora subsp. carotovora positively controls Peh production and flagellum synthesis and plays an important role in its pathogenicity.

Amino Acid Sequence↗
PubMed2004

Identification and characterization of Nip, necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora is a gram-negative bacterium that causes soft rot disease of many cultivated crops. When a collection of E. carotovora subsp. carotovora isolates was analyzed on a Southern blot using the harpin-encoding gene hrpN as probe, several harpinless isolates were found. Regulation of virulence determinants in one of these, strain SCC3193, has been characterized extensively. It is fully virulent on potato and in Arabidopsis thaliana. An RpoS (SigmaS) mutant of SCC3193, producing elevated levels of secreted proteins, was found to cause lesions resembling the hypersensitive response when infiltrated into tobacco leaf tissue. This phenotype was evident only when bacterial cells had been cultivated on solid minimal medium at low pH and temperature. The protein causing'the cell death was purified and sequenced, and the corresponding gene was cloned. The deduced sequence of the necrosis-inducing protein (Nip) showed homology to necrosis- and ethylene-inducing elicitors of fungi and oomycetes. A mutant strain of E. carotovora subsp. carotovora lacking the nip gene showed reduced virulence in potato tuber assay but was unaffected in virulence in potato stem or on other tested host plants.

Amino Acid Sequence↗
PubMed2004

Potato plants genetically modified to produce N-acylhomoserine lactones increase susceptibility to soft rot erwiniae.

Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing [QS]). In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid. We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant. In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E. carotovora. Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.

Carboxylic Ester Hydrolases↗
PubMed2004

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins.

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.

Bacterial Outer Membrane Proteins↗
PubMed1997

Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnlA: a role for RdgB.

The activation of pectin lyase (Pnl) production in Erwinia carotovora subsp. carotovora strain 71 occurs upon DNA damage via a unique regulatory circuit involving recA, rdgA and rdgB. In a similar Pnl-inducible system reconstituted in Escherichia coli, the rdgB product was found to activate the expression of pnlA, the structural gene for pectin lyase. The kinetic data presented here also show that transcription of pnlA followed that of rdgB in Er. carotovora subsp. carotovora, indicating a temporal order of gene expression. By deletion analysis we have localized the promoter/regulatory region within a 66 bp DNA segment upstream of the pnlA transcriptional start site. This region contains the -10 consensus sequence but not the sequences corresponding to the E. coli -35 region. For DNA-binding studies, rdgB was overexpressed in E. coli and a 14 kDa polypeptide was identified as the gene product. RdgB from crude extracts or a purified preparation caused an identical gel mobility shift of a 164 bp DNA segment containing the pnlA promoter/regulatory region. Utilizing DNase I protection assay the RdgB-binding site was localized between nucleotides -29 and -56, i.e. overlapping the position of the putative -35 box. The findings reported here, taken along with our previous observation that the rdgE product is required for pnlA expression, establishes that rdgB encodes a transcriptional factor which specifically interacts with the pnlA promoter/regulatory region.

Bacterial Proteins↗
PubMed1997

The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF.

The out gene cluster of Erwinia carotovora subsp. carotovora (Ecc) encodes the proteins of the type II or general secretory pathway (GSP) apparatus which is required for secretion of pectinase and cellulase. In this study, fusions between Ecc out genes and the topology probe blaM were constructed. The ability of Out protein domains to export BlaM across the cytoplasmic membrane in both Escherichia coli and the cognate host was utilized to confirm the computer-predicted cytoplasmic membrane topology of OutC and OutF, When outC was fused to blaM, the resulting phenotype suggested that the majority of OutC is targeted to the periplasm, typical of a type II bitopic conformation in the cytoplasmic membrane. In contrast, for the outF gene product, three transmembrane regions were identified which connect a large N-terminal cytoplasmic domain, a smaller periplasmic domain, and a large cytoplasmic loop. Fusions between blaM and outD and outE were used to further substantiate the locations of these gene products in the outer membrane and the cytoplasm respectively. The data derived suggest that a number of the Out apparatus components possess domains in the cytoplasm and/or the periplasm with potential for protein-protein interactions which facilitate the secretion of periplasmic enzyme intermediates across the outer membrane to the external milieu.

Bacterial Outer Membrane Proteins↗
PubMed1998

Cryptic carbapenem antibiotic production genes are widespread in Erwinia carotovora: facile trans activation by the carR transcriptional regulator.

Few strains of Erwinia carotovora subsp. carotovora (Ecc) make carbapenem antibiotics. Strain GS101 makes the basic carbapenem molecule, 1-carbapen-2-em-3-carboxylic acid (Car). The production of this antibiotic has been shown to be cell density dependent, requiring the accumulation of the small diffusible molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) in the growth medium. When the concentration of this inducer rises above a threshold level, OHHL is proposed to interact with the transcriptional activator of the carbapenem cluster (CarR) and induce carbapenem biosynthesis. The introduction of the GS101 carR gene into an Ecc strain (SCRI 193) which is naturally carbapenem-negative resulted in the production of Car. This suggested that strain SCRI 193 contained functional cryptic carbapenem biosynthetic genes, but lacked a functional carR homologue. The distribution of trans-activatable antibiotic genes was assayed in Erwinia strains from a culture collection and was found to be common in a large proportion of Ecc strains. Significantly, amongst the Ecc strains identified, a larger proportion contained trans-activatable cryptic genes than produced antibiotics constitutively. Southern hybridization of the chromosomal DNA of cryptic Ecc strains confirmed the presence of both the car biosynthetic cluster and the regulatory genes. Identification of homologues of the transcriptional activator carR suggests that the cause of the silencing of the carbapenem biosynthetic cluster in these strains is not the deletion of carR. In an attempt to identify the cause of the silencing in the Ecc strain SCRI 193 the carR homologue from this strain was cloned and sequenced. The SCRI 193 CarR homologue was 94% identical to the GS101 CarR and contained 14 amino acid substitutions. Both homologues could be expressed from their native promoters and ribosome-binding sites using an in vitro prokaryotic transcription and translation assay, and when the SCRI 193 carR homologue was cloned in multicopy plasmids and reintroduced into SCRI 193, antibiotic production was observed. This suggested that the mutation causing the silencing of the biosynthetic cluster in SCRI 193 was leaky and the cryptic Car phenotype could be suppressed by multiple copies of the apparently mutant transcriptional activator.

Base Sequence↗
PubMed1999

Role of RpoS in virulence and stress tolerance of the plant pathogen Erwinia carotovora subsp. carotovora.

The plant-pathogenic bacterium Erwinia carotovora subsp. carotovora causes plant disease mainly through a number of extracellular plant-cell-wall-degrading enzymes. In this study, the ability of an rpoS mutant of the Er. carotovora subsp. carotovora strain SCC3193 to infect plants and withstand environmental stress was characterized. This mutant was found to be sensitive to osmotic and oxidative stresses in vitro and to be deficient in glycogen accumulation. The production of extracellular enzymes in vitro was similar in the mutant and in the wild-type strains. However, the rpoS mutant caused more severe symptoms than the wild-type strain on tobacco plants and also produced more extracellular enzymes in planta, but did not grow to higher cell density in planta compared to the wild-type strain. When tested on plants with reduced catalase activities, which show higher levels of reactive oxygen species, the rpoS mutant was found to cause lower symptom levels and to have impaired growth. In addition, the mutant was unable to compete with the wild-type strain in planta and in vitro. These results suggest that a functional rpoS gene is needed mainly for survival in a competitive environment and during stress conditions, and not for effective infection of plants.

Bacterial Proteins↗
PubMed2000

cDNA-AFLP analysis of differential gene expression in the prokaryotic plant pathogen Erwinia carotovora.

For studies of differential gene expression in prokaryotes, methods for synthesizing representative cDNA populations are required. Here, a technique is described for the synthesis of cDNA from the potato pathogens Erwinia carotovora subsp. atroseptica (Eca) and Erwinia carotovora subsp. carotovora (Ecc) using a combination of short oligonucleotide (11-mer) primers that were known to anneal to conserved sequences in the 3' regions of enterobacterial genes. Specific PCR amplifications with primers designed to anneal to 14 known genes from either Eca or Ecc revealed the presence of the corresponding transcripts in cDNA, suggesting that the cDNA represented a broad genomic coverage. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used to identify differentially expressed genes in Eca, including one that shows significant similarity, at the protein level, to an avirulence gene from Xanthomonas campestris pv. raphani. Northern analysis was used to confirm that differentially amplified cDNA fragments were derived from differentially expressed genes. This is the first report of the use of cDNA-AFLP to study differential gene expression in prokaryotes.

Blotting, Northern↗
PubMed2002

Regulation of the expression of prtW::gusA fusions in Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora, a Gram-negative phytopathogenic bacterium, secretes an extracellular metalloprotease, PrtW. Previous results demonstrated that protease activity is necessary for the normal progression of disease symptoms caused by this bacterium. The present study revealed that the prtW gene constitutes an independent transcriptional unit. It is demonstrated that introduction of the prtW(+) plasmid in trans into the prtW(-) mutant restores the protease activity in this strain. Gene fusions to the gusA (beta-glucuronidase) reporter were employed to analyse the transcription of prtW. The transcription of prtW is dependent on many environmental signals. When the bacteria were grown in the presence of potato extract, the expression of the protease gene was markedly higher at the beginning of the exponential phase of growth than that observed when cells were grown in the presence of polygalacturonate (PGA). Analysis of the promoter revealed that an essential regulatory region resided between 371 and 245 bp 5' of the translational start site. As this sequence showed no homology to the KdgR box it may be involved in the binding of an unknown negative regulator protein in E. carotovora subsp. carotovora. The differential responses of prtW expression to potato extract and to PGA appeared to be dependent on the KdgR repressor and the response regulator ExpA. According to the results presented here, it is conceivable that the multiple regulatory network allows flexibility in the expression of the prtW gene during different stages of infection.

Base Sequence↗
PubMed2002

Sample sequencing of a selected region of the genome of Erwinia carotovora subsp. atroseptica reveals candidate phytopathogenicity genes and allows comparison with Escherichia coli.

Genome sequencing is making a profound impact on microbiology. Currently, however, only one plant pathogen genome sequence is publicly available and no genome-sequencing project has been initiated for any species of the genus Erwinia, which includes several important plant pathogens. This paper describes a targeted sample sequencing approach to study the genome of Erwinia carotovora subsp. atroseptica (Eca), a major soft-rot pathogen of potato. A large insert DNA (approx. 115 kb) library of Eca was constructed using a bacterial artificial chromosome (BAC) vector. Hybridization and end-sequence data revealed two overlapping BAC clones that span an entire hrp gene cluster. Random subcloning and one-fold sequence coverage (>200 kb) across these BACs identified 25 (89%) of 28 hrp genes predicted from the orthologous hrp cluster of Erwinia amylovora. Regions flanking the hrp cluster contained orthologues of known or putative pathogenicity operons from other Erwinia species, including dspEF (E. amylovora), hecAB and pecSM (E. chrysanthemi), sequences similar to genes from the plant pathogen Xylella fastidiosa, including haemagglutinin-like genes, and sequences similar to genes involved in rhizobacterium-plant interactions. Approximately 10% of the sequences showed strongest nucleotide similarities to genes in the closely related model bacterium and animal pathogen Escherichia coli. However, the positions of some of these genes were different in the two genomes. Approximately 30% of sequences showed no significant similarity to any database entries. A physical map was made across the genomic region spanning the hrp cluster by hybridization to the BAC library and to digested BAC clones, and by PCR between sequence contigs. A multiple genome coverage BAC library and one-fold sample sequencing are an effective combination for extracting useful information from important regions of the Eca genome, providing a wealth of candidate novel pathogenicity genes for functional analyses. Other genomic regions could be similarly targeted.

Chromosomes, Artificial, Bacterial↗
PubMed1995

Carbapenem antibiotic production in Erwinia carotovora is regulated by CarR, a homologue of the LuxR transcriptional activator.

Strain GS101 of Erwinia carotovora makes the carbapenem antibiotic, 1-carbapen-2-em-3-carboxylic acid. Mutants defective in antibiotic production can be assigned to two groups, group 1 and group 2. Group 2 mutants are defective in the carl gene encoding a protein responsible for synthesis of the Lux autoinducer N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), which is required to induce carbapenem synthesis in strain GS101. In this paper we describe the molecular genetic analysis of the group 1 mutants which we presumed were defective in the carbapenem biosynthesis (car) genes. We isolated a cosmid (cWU142) that complemented the group 1 mutants of strain GS101. A small (1.03 kb) subclone of cWU142 complemented most of the group 1 mutants, and the sequence revealed that the relevant gene (carR) encodes a homologue of the Vibrio fischeri LuxR protein. A disproportionately high frequency of carR mutants arose in strain GS101 and this was due to carR acting as a 'hot spot' target for secondary transposition of a Tn5 element in this strain. The CarR protein joins a rapidly growing list of homologues, found in taxonomically unrelated bacteria, which act as positive transcriptional activators of genes encoding diverse metabolic functions, including bioluminescence, exoenzyme virulence factor synthesis, cell division, plasmid conjugation, rhizosphere-specific gene induction, surfactant synthesis and antibiotic production. Most of these LuxR-type regulators have been shown to depend, for their function, on N-acyl homoserine lactones, which act as chemical signals enabling co-ordination of gene expression with cell density.

Amino Acid Sequence↗
PubMed1996

Global regulation in Erwinia species by Erwinia carotovora rsmA, a homologue of Escherichia coli csrA: repression of secondary metabolites, pathogenicity and hypersensitive reaction.

Our previous studies revealed that rsmA of Erwinia carotovora subsp. carotovora strain 71 suppressed the synthesis of the cell density (quorum) sensing signal N-(3-oxohexanoyl)-L-homoserine lactone, the production of extracellular enzymes and tissue macerating ability in soft-rotting Erwinia species and that homologues of this negative regulator gene were present in other Erwinia species. Northern blot data presented here demonstrate that rsmA and rsmA-like genes are also expressed in soft-rotting and non-soft-rotting Erwinia spp. such as E. amylovora, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. chrysanthemi, E. herbicola and E. stewartii. A low-copy plasmid carrying rsmA of E. carotovora subsp. carotovora strain 71 caused suppression of antibiotic production in E. carotovora subsp. betavasculorum, flagellum formation in E. carotovora subsp. carotovora, carotenoid production in E. herbicola and E. stewartii, and indigoidine production in E. chrysanthemi. In E. amylovora, rsmA of E. carotovora subsp. carotovora suppressed the elicitation of the hypersensitive reaction in tobacco leaves and the production of disease symptoms in apple shoots, in addition to repressing motility and extracellular polysaccharide production. We conclude that rsmA homologues function as global regulators of secondary metabolic pathways as well as factors controlling host interaction of Erwinia species.

Anti-Bacterial Agents↗
PubMed1999

Erwinia carotovora has two KdgR-like proteins belonging to the IciR family of transcriptional regulators: identification and characterization of the RexZ activator and the KdgR repressor of pathogenesis.

A novel Erwinia carotovora subsp. carotovora mutant designated RexZ, (regulator of exoenzymes) showed reduced production of the degradative exoenzymes. The rexZ gene product shows similarity of the KdgR regulatory protein from Erwinia chrysanthemi, described as the major repressor of the pectin catabolism pathway genes in the latter species. In vitro DNA-protein interaction experiments demonstrated that the synthesis of the RexZ protein is controlled by the cAMP-CRP (cAMP-receptor protein) complex. Western blot analysis also revealed the presence of a second KdgR homologue (distinct from RexZ) which, like RexZ, was present in all species of the genus Erwinia tested. The corresponding KdgR proteins from both E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica share a high level of sequence identity with the KdgR homologues from E. chrysanthemi and Escherichia coli. Although the E. carotovora subsp. carotovora rexZ regulatory region displayed specific interactions with both the purified E. chrysanthemi KdgR repressor and the partially purified E. carotovora subsp. carotovora KdgR, in vivo quantification revealed that the cellular level of RexZ protein was unaffected by the presence of pectic compounds. This study shows that the complex regulatory network governing virulence in the erwinias involves two totally distinct, but highly conserved, members of the IcIR class of DNA binding proteins: RexZ and KdgR.

Amino Acid Sequence↗
PubMed1999

Erwinia carotovora DsbA mutants: evidence for a periplasmic-stress signal transduction system affecting transcription of genes encoding secreted proteins.

The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approximately two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene expression in diverse ways.

Cellulase↗
PubMed1999

Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity.

Using mini-Tn5CmR::gusA, a transposon that allows transcriptional fusions to a promoterless beta-glucuronidase gene, a mutant of Erwinia carotovora subsp. carotovora SCC3193 deficient in extracellular protease production and soft-rot pathogenicity in plants was isolated. The mutant, designated SCC6004, produced normal levels of pectate lyase, polygalacturonase and cellulase. The region of the transposon insertion was partially sequenced to permit the design of specific oligonucleotide primers to amplify a 2.7 kb Clal fragment from E. carotovora subsp. carotovora SCC3193. The DNA sequence of the cloned fragment contained two complete and one partial ORFs. One of the complete ORFs (ORF1) was designated prtW and encodes a secreted protease. The deduced amino acid sequence of PrtW showed a high overall identify of 60-66% to the previously described Erwinia chrysanthemi proteases, but no homology to other proteases isolated from different E. carotovora strains. Downstream from ORF1, a further complete ORF (ORF2) and a partial ORF (ORF3) were found, with deduced peptide sequences that have significant similarity to the Inh and PrtD proteins, respectively, from E. chrysanthemi, which are involved in protease secretion. Gene fusion to the gusA reporter was employed to charaterize the regulation of prtW. The prtW gene was found to be strongly induced in the presence of plant extracts. The mutant exhibited reduced virulence, suggesting that PrtW enhances the ability of strain SCC3193 to macerate plant tissue.

Artificial Gene Fusion↗
PubMed2003

AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria.

Quorum sensing is a signalling mechanism that controls diverse biological functions, including virulence, via N-acylhomoserine lactone (AHL) signal molecules in Gram-negative bacteria. With the aim of isolating strains or enzymes capable of blocking quorum sensing by inactivating AHL, bacteria were screened for AHL degradation by their ability to utilize N-3-oxohexanoyl-L-homoserine lactone (OHHL) as the sole carbon source. Among four isolates, strain IBN110, identified as Arthrobacter sp., was found to grow rapidly on OHHL, and to degrade various AHLs with different lengths and acyl side-chain substitutions. Co-culture of Arthrobacter sp. IBN110 and the plant pathogen Erwinia carotovora significantly reduced both the AHL amount and pectate lyase activity in co-culture medium, suggesting the possibility of applying Arthrobacter sp. IBN110 in the control of AHL-producing pathogenic bacteria. The ahlD gene from Arthrobacter sp. IBN110 encoding the enzyme catalysing AHL degradation was cloned, and found to encode a protein of 273 amino acids. A mass spectrometry analysis showed that AhlD probably hydrolyses the lactone ring of N-3-hexanoyl-L-homoserine lactone, indicating that AhlD is an N-acylhomoserine lactonase (AHLase). A comparison of AhlD with other known AHL-degrading enzymes, Bacillus sp. 240B1 AiiA, a Bacillus thuringiensis subsp. kyushuensis AiiA homologue and Agrobacterium tumefaciens AttM, revealed 25, 26 and 21 % overall identities, respectively, in the deduced amino acid sequences. Although these identities were relatively low, the HXDH approximately H approximately D motif was conserved in all the AHLases, suggesting that this motif is essential for AHLase activity. From a genome database search based on the conserved motif, putative AhlD-like lactonase genes were found in several other bacteria, and AHL-degrading activities were observed in Klebsiella pneumoniae and Bacillus stearothermophilus. Furthermore, it was verified that ahlK, an ahlD homologue, encodes an AHL-degrading enzyme in K. pneumoniae. Accordingly, the current results suggest the possibility that AhlD-like AHLases could exist in many other micro-organisms.

Agrobacterium tumefaciens↗