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PubMed1985

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora.

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.

Chromosome Mapping↗

PubMed1986

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica.

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)

Erwinia↗

PubMedDate not supplied

[Expression of pectate lyase genes of Erwinia carotovora subsp. carotovora 17A and Erwinia carotovora subsp. atroseptica 36A in Erwinia carotovor substp. atroseptica 36A cells].

E.atroseptica 36A cells were transformed by the recombinant plasmids p27-1 and pEA364 (derivatives of the vector plasmid pUC19) containing pectate lyase genes of E.carotovora 17A and E.atroseptica 36A, respectively. The synthesis of pectate lyases determined by the cloned genes of bacteria of both subspecies, as well as the synthesis of the native enzymes, were induced by sodium poly pectate. Increase of the dose of pectate lyase genes did not result in alteration of pectate lyase secretion by E.atroseptica 36ApEA364 cells. At the same time, the efficiency of secretion of heterologous pectate lyases by E.atroseptica 36Ap27-1 cells was lower. The synthesis and secretion of the resident isoenzymes are as efficient as those of the parental cells. The results indicate a high specificity of the pectinase secretory system in Erwinia of different species and, moreover, subspecies.

Cloning, Molecular↗

PubMed1998

Characterization of a gene locus from Erwinia amylovora with regulatory functions in exopolysaccharide synthesis of Erwinia spp.

In a genomic library of Erwinia amylovora, a locus has been identified that can suppress an Erwinia stewartii rcsA mutant. In addition, the locus induced a mucoid sticky phenotype of colonies in a wild-type strain of Erwinia stewartii and increased exopolysaccharide synthesis in several species of bacteria belonging to the genus Erwinia. An open reading frame was identified at this locus encoding a 225 amino acid protein that contained a helix-turn-helix motif typical of transcriptional regulators. The corresponding gene was subsequently named rcsV (regulator of capsular synthesis affecting viscosity). A mutant of rcsV in wild-type Erwinia amylovora had no detectable phenotype and produced typical levels of amylovoran under laboratory conditions. The rcsV gene on a high copy number plasmid under the control of its own promoter did not alter amylovoran production, in contrast to in-frame fusions of the structural gene in expression vectors. Since even the lac promoter was inert in the expression of rcsV, a DNA-binding protein could inhibit transcription of the gene in Erwinia amylovora. On the other hand, an Erwinia amylovora rcsA mutant was suppressed by rcsV when its promoter was replaced and the structural gene fused in-frame with lacZ' or malE. Northern blots, with total RNA from Erwinia amylovora, or promoter analysis using the GUS reporter gene did not show expression of rcsV in Erwinia amylovora, although primer extension analysis did. RcsV could be a component involved in the regulation of amylovoran synthesis, and gene expression may require an unknown external signal during the life cycle or pathogenesis of Erwinia amylovora.

Amino Acid Sequence↗

PubMed2002

Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi.

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.

Bacterial Typing Techniques↗

PubMed1999

Reclassification of non-pigmented Erwinia herbicola strains from trees as Erwinia billingiae sp. nov.

Twenty-two Erwinia-like strains, isolated from trees since the late fifties and belonging to a distinct phenotypic group with resemblance to Pantoea agglomerans, were further characterized by conventional biochemical tests, the BIOLOG metabolic fingerprinting system and fatty acid analysis. Their phylogenetic positions were determined by comparing the 16S rRNA gene sequence of a representative strain to available sequences of Erwinia, Pantoea, Pectobacterium and Brenneria species. The strains were shown to belong to the genus Erwinia, with Erwinia rhapontici and Erwinia persicina as the closest phylogenetic relatives. The name Erwinia billingiae sp. nov. is proposed (type strain LMG 2613T) and a description of the species is given.

Bacterial Typing Techniques↗

PubMed1980

Acceptance by Erwinia spp. of R plasmid R68.45 and its ability to mobilize the chromosome of Erwinia chrysanthemi.

R plasmid R68.45 was transferred in broth matings from Escherichia coli to strains of Erwinia amylovora, E. carotovora subsp. atroseptica, E. chrysanthemi, and E. herbicola (Enterobacter agglomerans); the frequency of transfer ranged from 2 x 10(-8) to 5 x 10(-4) per input donor cell depending on the bacterial species. The drug resistance markers tet(+), amp(+), and kan(+) were stable in these Erwinia species. Transconjugants of Erwinia spp., but not of the wild-type parent Erwinia strains, acquired levels of antibiotic resistance (tetracycline, 50 mug/ml; ampicillin, 200 mug/ml; kanamycin 200 mug/ml) similar to those of the donor R68.45-bearing strain of Escherichia coli. Erwinia transconjugants (with one exception of E. carotovora subsp. atroseptica) were donors of the antibiotic resistance markers; the frequency of transfer was consistently higher with an E. coli strain than with Erwinia spp. as recipients, and when matings were done on a solid surface (membranes) rather than in liquid. Transfer of chromosomal markers ade(+), gal(+), gtu(+) (utilization of galacturonate), his(+), leu(+), lys(+), thr(+), and trp(+) occurred in crosses between E. chrysanthemi strains harboring R68.45 and appropriate recipient strains; the frequency of transfer ranged from 9.0 x 10(-8) to 2.0 x 10(-6) depending on the selective marker. Analysis of the coinheritance of unselected markers among various classes of recombinants revealed linkage between thr-leu-lys-ade and between trp and his, thus confirming earlier findings with the Hfr-type donor cells. Since R68.45 mobilized an array of chromosomal markers in the wild-type as well as genetically marked strains of E. chrysanthemi, the system, used in conjunction with the existing Hfr strains, should provide a useful tool to study the genetics of plant pathogenicity of this bacterial species. In contrast to E. chrysanthemi, R68.45 did not mobilize chromosomal markers ilv(+), his(+), rbs(+), ser(+), and thr(+) in E. amylovora EA178.

Ampicillin↗

PubMedDate not supplied

[Expression of pel genes of Erwinia chrysanthemi ENA49 in Erwinia carotovora var. atroseptica 36A cells].

Erwinia atroseptica 36A cells were transformed by the recombinant plasmid pPL5-1 (a derivative of the vector plasmid pUC19) containing pelb and pelc genes which encode pectate lyases of Erwinia chrysanthemi ENA49. Synthesis of pectate lyases PLB and PLC determined by the cloned pel genes is constitutive in Erwinia atroseptica 36ApPL5-1 cells and not inducible by sodium polypectate. The major part of these enzymes was accumulated in the periplasmic fraction of Erwinia atroseptica and cells were unable to efficiently secrete the enzymes into the cultural medium. Synthesis and secretion of the native pectate lyases by Erwinia atroseptica harboring the plasmid were as efficient as by the parental cells. The obtained results suggest the high specificity of pectate lyase secretory systems of kindred Erwinias.

Cloning, Molecular↗

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↗

PubMed1986

Enterobacter asburiae sp. nov., a new species found in clinical specimens, and reassignment of Erwinia dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacter dissolvens comb. nov. and Enterobacter nimipressuralis comb. nov.

Enterobacter asburiae sp. nov. is a new species that was formerly referred to as Enteric Group 17 and that consists of 71 strains, 70 of which were isolated from humans. Enterobacter asburiae sp. nov. strains gave positive reactions in tests for methyl red, citrate utilization (Simmons and Christensen's), urea hydrolysis, L-ornithine decarboxylase, growth in KCN, acid and gas production from D-glucose, and acid production from L-arabinose, cellobiose, glycerol (negative in 1 to 2 days, positive in 3 to 7 days), lactose, D-mannitol, alpha-methyl-D-glucoside, salicin, D-sorbitol, sucrose, trehalose, and D-xylose. They gave negative reactions in the Voges-Proskauer test and in tests for indole, H2S production, phenylalanine, L-lysine decarboxylase, motility, gelatin, utilization of malonate, lipase, DNase, tyrosine clearing, acid production from adonitol, D-arabitol, dulcitol, erythritol, i(myo)-inositol, melibiose, and L-rhamnose. They gave variable reactions in tests for L-arginine dihydrolase (25% positive after 2 days) and acid production from raffinose (69% positive after 2 days). Thirty-four Enterobacter asburiae sp. nov. strains were tested for DNA relatedness by the hydroxyapatite method with 32PO4-labeled DNA from the designated type strain (1497-78, ATCC 35953). The strains were 69 to 100% related in 60 degrees C reactions and 63 to 100% related in 75 degrees C reactions. Divergence within related sequences was 0 to 2.5%. Relatedness of Enterobacter asburiae sp. nov. to 84 strains of members of the Enterobacteriaceae was 5 to 63%, with closest relatedness to strains of Enterobacter cloacae, Erwinia dissolvens, Enterobacter taylorae, Enterobacter agglomerans, Erwinia nimipressuralis, and Enterobacter gergoviae. All strains tested were susceptible to gentamicin and sulfdiazine, and most were susceptible to chloramphenicol, colistin, kanamycin, nalidixic acid, carbenicillin and streptomycin. All strains were resistant to ampicillan, cephalothin, and penicillin, and most were resistant or moderately resistant to tetracycline. Enterobacter asburiae sp. nov strains were isolated from a variety of human sources, most prevalent of which were urine (16 strains), respiratory sources (15 strains), stools (12 strains), wounds (11 strains), and blood (7 strains). The clinical significance of Enterobacter aburiae is not known. As a result of this and previous studies, proposals are made to transfer Erwinia dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacter dissolvens comb. nov. and Enterobacter nimipressuralis comb. nov., respectively.

Adult↗

PubMed1995

Identification of a global repressor gene, rsmA, of Erwinia carotovora subsp. carotovora that controls extracellular enzymes, N-(3-oxohexanoyl)-L-homoserine lactone, and pathogenicity in soft-rotting Erwinia spp.

The production of extracellular enzymes such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt) is activated by the cell density (quorum)-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (HSL); plant signals; and aep genes during postexponential growth of Erwinia carotovora subsp. carotovora 71. Studies with mutants of E. carotovora subsp. carotovora 71 derepressed in exoenzyme production led to the identification of a negative regulator gene, rsmA (rsm, repressor of secondary metabolites). Nucleotide sequencing, transcript assays, and protein analysis established that a 183-bp open reading frame encodes the 6.8-kDa RsmA. rsmA has extensive homology with the csrA gene of Escherichia coli, which specifies a negative regulator of carbon storage. Moreover, the suppression of glycogen synthesis in E. coli by rsmA indicates that the Erwinia gene is functionally similar to csrA. Southern hybridizations revealed the presence of rsmA homologs in soft-rotting and non-soft-rotting Erwinia spp. and in other enterobacteria such as Enterobacter aerogenes, E. coli, Salmonella typhimurium, Shigella flexneri, Serratia marcescens, and Yersinia pseudotuberculosis. rsmA suppresses production of Pel, Peh, Cel, and Prt, plant pathogenicity, and synthesis of HSL in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, and E. chrysanthemi. In the E. carotovora subsp. carotovora 71, rsmA reduces the levels of transcripts of hslI, a luxI homolog required for HSL biosynthesis. This specific effect and the previous finding that HSL is required for extracellular enzyme production and pathogenicity in soft-rotting Erwinia spp. support the hypothesis that rsmA controls these traits by modulating the levels of the cell density (quorum)-sensing signal.

4-Butyrolactone↗

PubMed2003

Instability of short-sequence DNA repeats of pear pathogenic Erwinia strains from Japan and Erwinia amylovora fruit tree and raspberry strains.

An array of short-sequence DNA repeats (SSRs) occurs in the plasmid pEA29 of the fire blight pathogen Erwinia amylovora. A large number of "fruit tree" strains, mainly from Central and Western Europe, were screened for their SSR numbers, and the analyses were extended to five raspberry strains from North America and six pear pathogenic Erwinia strains from Japan. The repeat ATTACAGA present in all E. amylovorastrains was found to be reiterated 3 to 15 times. The Japanese strains contained the major repeat sequence GGATTCTG, which was reiterated 16 to 24 times. ATTACAGG, which resembles the SSR of E. amylovora, was reiterated two or three times. In a novel approach, sequencing gels were used to visualize the rare occurrence of shorter arrays (down to three repeats) in E. amylovoraand the Japanese Erwinia strains. Changes in the repeat numbers in E. amylovora were observed repeatedly when the bacteria had been exposed to stress conditions. The repeat structures of homo- and heteroduplices of PCR-amplified repeats were also analyzed by cleavage of annealed molecules with the single-strand-specific endonuclease from bacteriophage T4. Not only heteroduplexes, but also homoduplexes showed non-matching regions in the SSRs, which could arise from transient formation of loops due to strand slippage during the assays.

Base Sequence↗

PubMed1986

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora.

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).

Asparaginase↗

PubMed1990

Sequence of the peh gene of Erwinia carotovora: homology between Erwinia and plant enzymes.

Polygalacturonase (Peh) and other pectolytic enzymes play a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have sequenced the peh gene of Erwinia carotovora subsp. carotovora, and identified its product as a precursor of molecular weight 42,639, and a mature protein of molecular weight 42,200. A putative KdgR-binding site was identified in the region 5' to the peh gene. The Peh protein showed significant homology with Peh from tomato. In addition, we have found homologies between pectin methylesterase and pectate lyase from Erwinia and their counterparts in tomato. These homologies are described, and their significance discussed.

Amino Acid Sequence↗

PubMed1972

Deoxyribonucleic acid relatedness among species of Erwinia and between Erwinia species and other enterobacteria.

Relatedness in species of Erwinia was assessed by determining the extent of reassociation in heterologous deoxyribonucleic acid preparations. Thermal elution chromatography on hydroxyapatite was used to separate reassociated nucleotide sequences from nonreassociated sequences and to determine the thermal stability of related nucleotide sequences. An apparent 15% core of relatedness is present between fire blight, soft-rot, and "atypical" Erwinia species. All Erwinia species showed low to moderate reaction with representative enteric bacteria.

Biological Evolution↗

PubMed1990

Characterization of transposon insertion out- mutants of Erwinia carotovora subsp. carotovora defective in enzyme export and of a DNA segment that complements out mutations in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi.

Soft-rotting Erwinia spp. export degradative enzymes to the cell exterior (Out+), a process contributing to their ability to macerate plant tissues. Transposon (Tn5, Tn10, Tn10-lacZ) insertion Out- mutants were obtained in Erwinia carotovora subsp. carotovora 71 by using plasmid and bacteriophage lambda delivery systems. In these mutants, pectate lyases, polygalacturonase, and cellulase, which are normally excreted into the growth medium, accumulated in the periplasm. However, localization of the extracellular protease was not affected. The Out- mutants were impaired in their ability to macerate potato tuber tissue. Out+ clones were identified in a cosmid library of E. carotovora subsp. carotovora 71 by their ability to complement mutants. Localization of cyclic phosphodiesterase in the periplasm indicated that the Out+ plasmids did not cause lysis or a nonspecific protein release. The Out+ derivatives of the E. carotovora subsp. carotovora 71 mutants regained the ability to macerate potato tuber tissue. Our data indicate that a cluster of several genes is required for the Out+ phenotype. While one plasmid, pAKC260, restored the Out+ phenotype in each of the 31 mutants of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi, it failed to render Escherichia coli export proficient. Homologs of E. carotovora subsp. carotovora 71 out DNA were detected by Southern hybridizations in subspecies of E. carotovora under high-stringency conditions. In contrast, E. chrysanthemi sequences bearing homology to the E. carotovora subsp. carotovora 71 out DNA were detectable only under low-stringency hybridization. Thus, although the out genes are functional in these two soft-rotting bacterial groups, the genes appear to have diverged.

Biological Transport↗

PubMed1996

Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway.

The type II or Sec-dependent secretion system is used by diverse Gram-negative bacteria for secretion of extracellular proteins. Of the 12-15 proteins involved in secretion, the requirement for many has not been demonstrated and little is known about their functions in the secretion process. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete extra-cellular pectate lyases (Pels) using the type II or Out pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed genes from the other species, suggesting the presence of species-specific recognition factors in the Out systems of the two Erwinia species. We previously reported the isolation of a cosmid clone, pCPP2OO6, from E. chrysanthemi EC16, which enables Escherichia coil to secrete heterologously expressed E. chrysanthemi Pels. Sequencing in a region required for secretion revealed the presence of 12 genes, outC-M and outO. We report here the construction of functionally non-polar mutations in each gene in the outC-M operon and outS and outB using a polA(ts) strain of E. coli to facilitate homologous recombination between out genes carrying deletions and their wild-type copies on pCPP2006. By testing for complementation of each deletion with wild-type out genes from E. chrysanthemi EC16 and E. carotovora SCRI193 we have demonstrated that: (i) each out gene is required for secretion of E. chrysanthemi PelE from E. coli with the exception of outH; (ii) each mutation can be complemented by its homologue from E. carotovora, except for outC and outD; (iii) outC and outD from E. carotovora do not confer secretion of Pel1 on the E. chrysanthemi Out system; and (iv) Pel1 secretion can be conferred on the E. chrysanthemi Out system by the presence of outC-M, S and B from E. carotovora. The data suggest that OutC and OutD are gatekeepers of the Out system involved in recognition of Pels targeted for secretion but that OutC and OutD from E. carotovora cannot be successfully assembled into the E. chrysanthemi Out system.

Bacterial Proteins↗

PubMed1993

A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii.

A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned. Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants. Five complementation groups, essential for amylovoran synthesis and virulence in E. amylovora, were identified and designated ams A-E. The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii. Mucoidy and virulence were restored to E. stewartii mutants in four cps complementation groups by the cloned E. amylovora ams genes. Conversely, the E. stewartii cps gene cluster was able to complement mutations in E. amylovora ams genes. Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different. EPS production and virulence were also restored to E. amylovora amsE and E. stewartii cpsD mutants by clones containing the Rhizobium meliloti exo A gene.

Cloning, Molecular↗