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PubMed1993

The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa.

Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively. E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups. Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N-3-(oxohexanoyl)-L-homoserine, lactone (HSL), and were avirulent. Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta. Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant. Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups. Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P. aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production. HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants. This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes.

4-Butyrolactone↗
PubMed1993

Some implications of structural collapse during freeze-drying using Erwinia caratovora L-asparaginase as a model.

When the enzyme Erwinia caratovora L-asparaginase was freeze-dried in mixtures of lactose and sodium chloride, biological activity and protein structure were preserved during drying. However, by altering the ratios of the excipients in the formulation it was possible to obtain products which were pharmaceutically acceptable or unacceptable as assessed by the criteria of dried cake appearance, moisture content or ease of reconstitution.

Asparaginase↗
PubMed2005

Application of partial least squares discriminant analysis to two-dimensional difference gel studies in expression proteomics.

Two-dimensional difference gel electrophoresis (DIGE) is a tool for measuring changes in protein expression between samples involving pre-electrophoretic labeling ith cyanine dyes. In multi-gel experiments, univariate statistical tests have been used to identify differential expression between sample types by looking for significant changes in spot volume. Multivariate statistical tests, which look for correlated changes between sample types, provide an alternate approach for identifying spots with differential expression. Partial least squares-discriminant analysis (PLS-DA), a multivariate statistical approach, was combined with an iterative threshold process to identify which protein spots had the greatest contribution to the model, and compared to univariate test for three datasets. This included one dataset where no biological difference was expected. The novel multivariate approach, detailed here, represents a method to complement the univariate approach in identification of differentially expressed protein spots. This new approach has the advantages of reduced risk of false-positives and the identification of spots that are significantly altered in terms of correlated expression rather than absolute expression values.

Analysis of Variance↗
PubMed1997

Characterization of Erwinia carotovora subsp. carotovora LY34 endo-1,4-beta-glucanase genes and rapid identification of their gene products.

Genomic DNA of the phytopathogenic Erwinia carotovora subsp. carotovora LY34 was partially digested with Sau3AI, ligated into the BamHI site of pBlue-script II SK+, and introduced into E. coli. Two clones that were able to hydrolyse carboxymethylcellulose were selected. 1.5 kb and 1.2 kb fragments containing the celA and celB genes, respectively, were subcloned and sequenced. The celA and celB genes had open reading frames of 1,161 bp and 792 bp encoding 487 and 264 amino acid residues with calculated molecular weights of 42,003 Da and 29,890 Da, respectively. Each, CelA and CelB, carried a typical prokaryotic signal peptide of 32 and 36 amino acid residues, respectively. The apparent molecular masses of the proteins when expressed in E. coli cells were approximately 39 kDa (CelA) and 26 kDa (CelB) as assessed by CMC-SDS-PAGE. Activity staining of CMCase in an SDS-PAGE gel containing 0.1% CMC revealed that the cloned endoglucanase isozymes comigrated with the corresponding ones present in Erwinia carotovora subsp. carotovora LY34.

Amino Acid Sequence↗
PubMed2001

The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi.

The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete multiple exoproteins by a type II pathway, the Out system. Secretion in Erwinia is species-specific: exoproteins of one species cannot be secreted by the other. We analysed the role of two components of the Out system, the bitopic inner membrane protein OutC and the secretin OutD, in the specific recognition of secreted proteins. We demonstrated that the PDZ domain of OutC determines its secretion specificity towards certain exoproteins. The secretin is the major determinant of specificity of the Out system: OutD of E. carotovora changes the secretion specificity of E. chrysanthemi and enables it to secrete heterologous exoproteins. Construction of chimeric OutD showed that the N-terminal region is the specificity domain of the secretin. Thus, both the PDZ domain of OutC and the N-terminal region of OutD are required for specific recognition of secreted proteins. Systematic analysis of the secretion of several exoproteins demonstrated that different exoproteins secreted by the Out machinery have different requirement for their presumed targeting signals on OutC and OutD. This strongly indicates that diverse exoproteins possess a variable number of targeting signals which are recognised by different regions of OutC and OutD.

Amino Acid Motifs↗
PubMed1999

Saccharomyces cerevisiae cells harboring the gene encoding sarcotoxin IA secrete a peptide that is toxic to plant pathogenic bacteria.

Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly, Sarcophaga peregrina. Similar to other bactericidal small proteins produced by insects, sarcotoxin IA is released into the hemolymph of larvae and nymphs upon mechanical injury or bacterial infection. The gene (sarco) that encodes this toxin was introduced into Saccharomyces cerevisiae yeast cells and was expressed under a constitutive yeast promoter. The transformed yeast cells were grown in a liquid medium, and a peptide with a similar molecular size to that of the mature sarcotoxin IA was detected in the medium by Western blot analysis. The secreted sarcotoxin-like peptide (SLP) had a potent cytotoxic effect against several bacteria, including plant pathogenic bacteria, similar to the toxic effects of the authentic sarcotoxin IA. Erwinia carotovora was more susceptible to the toxic medium than Pseudomonas solanacearum and Pseudomonas syringae pv. lachrymans. Thus, yeast may be used in the production of such proteins for employment against various bacterial pathogens.

Animals↗
PubMed1992

Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production.

In vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75-2, 75-5 and 75-6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 aa PehA protein, respectively, fused to the mature part of the periplasmic beta-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75-5 and 75-6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp- mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR-), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR- mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR- as well as PehA- mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.

Cloning, Molecular↗
PubMed1992

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 bp in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 bp in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84--Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.

Amino Acid Sequence↗
PubMed1993

Molecular analysis of the major cellulase (CelV) of Erwinia carotovora: evidence for an evolutionary "mix-and-match" of enzyme domains.

The structural gene for the major cellulase of Erwinia carotovora subspecies carotovora (Ecc) was isolated and expressed in Escherichia coli. Sequencing of the gene (celV) revealed a typical signal sequence and two functional domains in the enzyme; a catalytic domain linked by a short proline/threonine-rich linker to a cellulose-binding domain (CBD). The deduced amino acid sequence of the catalytic domain showed homology with cellulases of Family A, including enzymes from Bacillus spp. and Erwinia chrysanthemi CelZ, whereas the CBD showed homology with cellulases from several diverse families, supporting a "mix-and-match" hypothesis for evolution of this domain. Analysis of the substrate specificity of CelV showed it to be an endoglucanase with some exoglucanase activity. The pH optimum is about 7.0 and the temperature optimum about 42 degrees C. CelV is secreted by Ecc and by the taxonomically related Erwinia carotovora subspecies atroseptica (Eca) but not by E. coli. Overproduction of the enzyme from multicopy plasmids in Ecc appears to overload the secretory mechanism.

Amino Acid Sequence↗
PubMed1995

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity.

The celV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 bp and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of CelV1 was highly similar to that of CelV of another E. c. subsp. carotovora strain SCR1193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. CelV1 mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.

Amino Acid Sequence↗
PubMed1994

Acute parotitis during induction therapy including L-asparaginase in acute lymphoblastic leukemia.

In a patient affected by acute lymphoblastic leukemia (ALL) and subjected to therapy with Erwinia L-asparaginase, acute parotitis was observed. Microbiological studies excluded any infectious etiology. Regression of parotitis was spontaneous. This complication has not been previously reported and could be due to the same mechanism of pancreatic injury. The occurrence of acute parotitis needs to be promptly recognized in order to avoid the continuation of L-asparaginase.

Acute Disease↗
PubMed2004

Transfer of tuber soft rot and early blight resistances from Solanum brevidens into cultivated potato.

Tuber soft rot and early blight are serious potato diseases. Development of potato varieties resistant to these diseases has been hindered by the scarcity of resistant germplasm. A diploid wild species, Solanum brevidens, shows significant resistance to both diseases. Numerous potato breeding lines have been developed from a potato- S. brevidens somatic hybrid, A206. A BC(3) clone, C75-5+297, derived from this somatic hybrid as well as its BC(1) and BC(2) parental lines showed resistance to both tuber soft rot and early blight. Clone C75-5+297 has consistently out-yielded common varieties under disease stress. Using both molecular and cytogenetic approaches we demonstrated that a single copy of chromosome 8 from S. brevidens replaced a potato chromosome 8 in C75-5+297. Thus, C75-5+297 represents a potato- S. brevidens chromosome substitution line. Our results suggest that the presence of a single chromosome from S. brevidens may significantly impact the resistance to multiple potato diseases. The high yield potential of C75-5+297 makes it an excellent parent for developing potato varieties with resistances to both tuber soft rot and early blight.

Agriculture↗
PubMed2007

Fusion of the genes for AHL-lactonase and S-layer protein in Bacillus thuringiensis increases its ability to inhibit soft rot caused by Erwinia carotovora.

Two genes, ctc and ctc2, responsible for surface layer (S-layer) protein synthesis in Bacillus thuringiensis CTC, were mutated and resulted in B. thuringiensis Tr5. To synthesize and express the N-acyl-homoserine lactonase (AHL-lactonase) in the extracellular space of B. thuringiensis, the aiiA ( 4Q7 ) gene (an AHL-lactonase gene from B. thuringiensis 4Q7), which confers the ability to inhibit plant soft rot disease in B. thuringiensis 4Q7, was fused with the upstream sequence of the ctc gene, which in turn is essential for S-layer protein secretion and anchoring on the cell surface. The resulting fusion gene, slh-aiiA, was expressed in B. thuringiensis Tr5 to avoid competition for the extracellular space with the native S-layer protein. Our results indicate that B. thuringiensis Tr5 containing the fusion gene slh-aiiA displayed high extracellular AHL-degrading activity. When compared with wild-type B. thuringiensis strains, the ability of the constructed strain to inhibit soft rot disease caused by Erwinia carotovora SCG1 was markedly increased. These findings provide evidence for a significant advance in our ability to inhibit soft rot disease caused by E. carotovora.

Antibiosis↗
PubMed2006

Production of soft rot resistant calla lily by expressing a ferredoxin-like protein gene (pflp) in transgenic plants.

An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of calla lily (Zantedeschia elliottiana (W. Wats.) Engl. cultivar 'Florex Gold') is described. Shoot basal discs were co-cultivated with A. tumefaciens C58C1 carrying a plasmid containing neomycin phosphotransferase (nptII) and plant ferredoxin-like protein (pflp) genes. After Agrobacterium co-cultivation, the shoot basal discs were exposed to 100 mg l(-1) kanamycin for selection. Twenty-eight out of 260 discs (10.8%) were found to have survived and produced shoot clusters. Twenty-six of these were confirmed to contain the pflp transgene by PCR, ending up in 10% transformation efficiency. The disease resistance investigation revealed that 18 transgenic plants exhibited resistance to soft rot disease caused by Erwinia carotovora subsp. carotovora. The presence of pflp gene was demonstrated by PCR, and its accumulation and activity was confirmed by Western blot and disease resistance assay. This was the first report to show the successful transformation and resistance to a bacterial pathogen in Zantedeschia. The protocol is useful for the quality improvement of calla lily through genetic transformation.

Agrobacterium tumefaciens↗
PubMed2001

Altered lignin structure and resistance to pathogens in spi 2-expressing tobacco plants.

The physiological role of the Norway spruce [ Picea abies (L.) Karst.] spi 2 gene, encoding a defense-related cationic peroxidase was examined in transgenic tobacco (Nicotiana tabacum L.). Expression of spi 2, under control of the 35S promoter, in tobacco plants resulted in higher total peroxidase activities. The phenotype of the spi 2-transformed lines was normal. The spi 2-transformed lines displayed lignin levels similar to levels in the control line, but with some alteration in lignin histochemistry and structure. These changes were associated with reduced flexibility of the tobacco stems. The defense against pathogenic microorganisms was altered in the transgenic tobacco plants compared with control plants. High peroxidase activities increased the susceptibility to the pathogenic oomycete Phytophthora parasitica var. nicotianae, but increased the ability of the tobacco plants to suppress growth of the pathogenic bacterium Erwinia carotovora.

DNA, Complementary↗
PubMed2002

Natural variability in the Arabidopsis response to infection with Erwinia carotovora subsp. carotovora.

The natural variation in the response of Arabidopsis thaliana (L.) Heynh. to Erwinia carotovora subsp. carotovora has been studied in seven ecotypes and two mutants. The susceptibility of all the plant types was investigated by (i) macroscopic symptoms, (ii) fluorescence microscopy using green fluorescent protein (GFP) and (iii) bacterial growth in planta. Although all the plants were susceptible to the bacterium, there was no correlation in the degree of infection as ascertained by the three methods. The induction, upon infection, of several genes known to be involved in defense was analyzed by RNA blot hybridization. The patterns of expression of these genes differed according to the genotype. These results suggest that both salicylic and jasmonic acid play a role in the response of Arabidopsis to this bacterium.

Arabidopsis↗
PubMed2003

Sweet pepper ferredoxin-like protein ( pflp) gene as a novel selection marker for orchid transformation.

A novel method for selection of transgenic plants utilizing the sweet pepper ( Capsicum annuum L.) ferredoxin-like protein ( pflp) gene as selection marker and Erwinia carotovora as the selection agent has been developed. An expression vector containing a pflp cDNA driven by a cauliflower mosaic virus 35S promoter was successfully transformed into protocorm-like bodies of Oncidium orchid by Agrobacterium tumefaciens and particle bombardment, respectively. Erwinia carotovora was used as a selection agent to screen transformants, thereby obtaining transgenic plants without the use of an antibiotic selection agent. A total of 32 independent transgenic orchid lines were obtained, out of which 9 transgenic lines (beta-glucuronidase positive) were randomly selected and confirmed by Southern and northern blot analyses. The transgenic orchid plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest the novel use of the pflp gene as a resistance selection marker in plant genetic engineering strategies. In the future, the use of the pflp gene as a selection marker may facilitate the use of smaller gene constructs due to removal of bulky antibiotic selection and reporter genes. These constructs can then be used to incorporate additional genes of choice.

Agrobacterium tumefaciens↗