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PubMed2004

Fast screening method for detection of acyl-HSL-degrading soil isolates.

A reliable method was developed for screening of bacteria isolates capable of degrading acyl-HSLs, the signal molecules in quorum-sensing-mediated processes of many Proteobacteria. The microtiter assay was based on the use of a GFP-marked Escherichia coli strain, which fluoresces upon the presence of acyl-HSLs. Measurement of GFP fluorescence with a Molecular Imager FX scanner (fluorometer) detected isolates capable of degrading acyl-HSLs. The potential of this method was demonstrated by isolation of different bacteria from a potato rhizosphere able to inactivate synthetic and natural acyl HSLs produced by Pectobacterium carotovorum subsp. carotovorum (Pcc) (Erwinia carotovora subsp. carotovora (Ecc)).

4-Butyrolactone↗
PubMed2004

Bacterial ghost technology for pesticide delivery.

Bacterial ghosts are nondenaturated empty cell envelopes of Gram-negative bacteria produced by E-mediated lysis. Such envelopes from the plant-adhering bacterium Pectobacterium cypripedii were tested for their ability to adhere to plant material and to be used as carriers for pesticide delivery. We show, using fluorescence-labeled P. cypripedii ghosts, that depending on the target plants 55 or 10% (rice or soya, respectively) of the applied bacterial ghosts was retained on the leaves after heavy simulated rain (84 mm). Furthermore, the bacterial ghosts could be loaded with the lipophilic triazole fungicide tebuconazole. In subsequent plant experiments in the glass house, the efficacy of the loaded bacterial ghost for resistance to rainfall and the protective and curative effects against the pathogens Erysiphe graminis, Leptosphaeria nodorum, and Pyrenophora teres on barley and wheat and against Sphaerotheca fuliginea on cucumber were tested. The bacterial ghosts were compared primarily with a commercial tebuconazole formulation, a wettable powder, as it has similar physical characteristics. The comparison revealed similar effects and showed consistently higher or comparable efficacy against the pathogens. The standard operational comparison with the most protective, cereal specific emulsion of oil in water displayed that the bacterial ghosts had equal to or lower efficacy than the emulsion. This study confirmed the potential of bacterial ghost platform technology as a new alternative carrier system for pesticides.

Bacteria↗
PubMed2004

Isolation and characterisation of a class of carbohydrate oxidases from higher plants, with a role in active defence.

In a search for novel plant-derived antimicrobial proteins, we screened extracts from salicylic acid (SA)-treated lettuce and sunflower leaves. These extracts displayed very potent antimicrobial activity against a set of phytopathogens. Characterisation of these extracts revealed that in both extracts, proteins of approximately 60 kDa were responsible for the antimicrobial activity. Further characterisation of these proteins and cloning of the respective cDNAs revealed close homology to a range of (plant) oxidases. Dissection of the enzymatic activity of both proteins revealed them to be carbohydrate oxidases (Helianthus annuus carbohydrate oxidase (Ha-CHOX) and Lactuca sativa carbohydrate oxidase (Ls-CHOX)) with broad substrate specificity and with hydrogen peroxide (H(2)O(2)) as one of the reaction products. The sunflower transcript, in addition to being SA inducible, was also inducible by fungal pathogens but not by ethylene and jasmonate. To determine whether Ha-CHOX plays a role in pathogen defence, it was transformed into tobacco and the effect of resistance to Pectobacterium carotovorum ssp. carotovorum was examined. Transgenic plants overexpressing Ha-CHOX displayed enhanced resistance to infection by this pathogen, and the resistance level was proportional to enzyme expression.

Amino Acid Sequence↗
PubMed2004

Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweet potato and its expression in response to stress.

A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1, was isolated from cell cultures of sweet potato ( Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated. swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32. The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform. RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweet potato. The level of swAPX1 RNA progressively increased during cell growth in suspension cultures. In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis. swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 microM), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 microM) or exposure to high temperature (37 degrees C). In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen ( Pectobacterium chrysanthemi). These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses.

Abscisic Acid↗
PubMed1977

Bacterial stalk rot of Maize, its symptoms and host-range.

Stalk rot of maize, caused by Erwinia carotovora f. sp. zeae Sabet (re-designated as Pectobacterium chrysanthemi pathovar. zeae by KELMAN 1974) showed first premature withering and drying up of the uppermost leaves which was soon followed by the lower leaves. The rot either extended from the base upwards (basal rot) or from the top downwards (top rot). In the case of basal rot, the leaves become yellow and the infected tissue becomes brown, soft, and water soaked. Internally, the stalk turns into a soft mass of disintegrated tissue. At this stage the plants usually topple over. A foul odour, accompanied with the presence of dipterous larvae on and in decaying tissues, are the characteristic symptoms of this disease. With the advance of the disease the stalk finally dries up into a conglomeration of dry and shredded or disjointed fibrous tissue. The top rot begins with wilting and drying up ot the tips of middle leaves of the whorl. A decay that continues rapidly spreads downwards throughout the stalk and the affected plants soon droop. The host range studies were suggestive of the fact that the maize pathogen, apart from causing the disease on maize, could produce soft rot in potato, carrot, onion, sugarbeet, sweet potato, papaya, cabbage, and many other plants and could also infect Sorghum vulgare, Pennisetum typhoidium, tobacco, and tomato.

Ecology↗
PubMed1998

Phylogenetic position of phytopathogens within the Enterobacteriaceae.

The almost complete 16S rDNA sequences of twenty nine plant-associated strains, representing species of the genera Erwinia, Pantoea and Enterobacter were determined and compared with those of other members of the Enterobacteriaceae. The species of the genus Erwinia may be divided into three phylogenetic groups. Cluster I represents the true erwinias and comprises E. amylovora, E. mallotivora, E. persicinus, E. psidii, E. rhapontici and E. tracheiphila. We propose to unite the species of cluster II, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. carotovora subsp. odorifera, E. carotovora subsp. wasabiae, E. cacticida, E. chrysanthemi and E. cypripedii in the genus Pectobacterium respectively as P. carotovorum subsp. atrosepticum comb. nov., P. carotovorum subsp. betavasculorum comb. nov., P. carotovorum subsp. carotovorum comb. nov., P. carotovorum subsp. odoriferum comb. nov., P. carotovorum subsp. wasabiae comb. nov., P. cacticidum comb. nov., P. chrysanthemi and P. cypripedii. The species E. alni, E. nigrifluens, E. paradisiaca, E. quercina, E. rubrifaciens and E. salicis, comprising cluster III, are being classified into a new genus Brenneria gen. nov. respectively as B. alni comb. nov., B. nigrifluens comb. nov., B. paradisiaca comb. nov., B. quercina comb. nov., B. rubrifaciens comb. nov. and B. salicis comb. nov. The species of the genus Pantoea, included in this study, form a monophyletic unit (cluster IV), closely related with Erwinia, whereas the three phytopathogenic species of the genus Enterobacter are scattered among the genera Citrobacter and Klebsiella.

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↗
PubMed1999

The phylogenetic position of Serratia, Buttiauxella and some other genera of the family Enterobacteriaceae.

The phylogenetic relationships of the type strains of 38 species from 15 genera of the family Enterobacteriaceae were investigated by comparative 16S rDNA analysis. Several sequences of strains from the genera Citrobacter, Erwinia, Pantoea, Proteus, Rahnella and Serratia, analysed in this study, have been analysed previously. However, as the sequences of this study differ slightly from the published ones, they were included in the analysis. Of the 23 enterobacterial genera included in an overview dendrogram of relatedness, members of the genera Xenorhabdus, Photorhabdus, Proteus and Plesiomonas were used as a root. The other genera formed two groups which could be separated, although not exclusively, by signature nucleotides at positions 590-649 and 600-638. Group A contains species of Brenneria, Buttiauxella, Citrobacter, Escherichia, Erwinia, Klebsiella, Pantoea, Pectobacterium and Salmonella. All seven type strains of Buttiauxella share 16S rDNA similarities greater than 99%. Group B embraces two phylogenetically separate Serratia clusters, a lineage containing Yersinia species, Rahnella aquatica, Ewingella americana, and also the highly related pair Hafnia alvei and Obesumbacterium proteus.

DNA, Ribosomal↗
PubMed2001

Samsonia erythrinae gen. nov., sp. nov., isolated from bark necrotic lesions of Erythrina sp., and discrimination of plant-pathogenic Enterobacteriaceae by phenotypic features.

Bacterial strains isolated from diseased erythrina (Erythrina sp.) trees in Martinique (French West Indies) were studied using phenotypic tests, 16S rDNA sequence analysis and DNA-DNA hybridization. Numerical analysis of phenotypic characteristics showed that these strains formed an homogeneous phenon among plant-pathogenic Enterobacteriaceae, and gave useful and updated information for the identification of these bacteria. Results of DNA-DNA hybridization indicated that strains from erythrina belonged to a discrete genomospecies (89-100% hybridization) and had low levels of DNA relatedness (2-33% hybridization) with reference strains of phytopathogenic Erwinia, Brenneria, Pectobacterium, Pantoea and Enterobacter species. 16S rDNA sequence analysis using three different methods revealed that the position of strain CFBP 5236T isolated from erythrina was variable in the different trees, so that strains from erythrina could not be assigned to any recognized genus. It is proposed that these strains are included in a new genus, Samsonia. The name Samsonia erythrinae is proposed for the new species. The G+C content of the DNA of the type strain, CFBP 5236T (= ICMP 13937T), is 57.0 mol%.

Base Composition↗
PubMed2003

Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plant-pathogenic bacteria.

Bacteria degrading the quorum-sensing (QS) signal molecule N-hexanoylhomoserine lactone were isolated from a tobacco rhizosphere. Twenty-five isolates degrading this homoserine lactone fell into six groups according to their genomic REP-PCR and rrs PCR-RFLP profiles. Representative strains from each group were identified as members of the genera Pseudomonas, Comamonas, Variovorax and Rhodococcus: all these isolates degraded N-acylhomoserine lactones other than the hexanoic acid derivative, albeit with different specificity and kinetics. One of these isolates, Rhodococcus erythropolis strain W2, was used to quench QS-regulated functions of other microbes. In vitro, W2 strongly interfered with violacein production by Chromobacterium violaceum, and transfer of pathogenicity in Agrobacterium tumefaciens. In planta, R. erythropolis W2 markedly reduced the pathogenicity of Pectobacterium carotovorum subsp. carotovorum in potato tubers. These series of results reveal the diversity of the QS-interfering bacteria in the rhizosphere and demonstrate the validity of targeting QS signal molecules to control pathogens with natural bacterial isolates.

4-Butyrolactone↗
PubMed1998

PCR-Based detection of the causal agent of watermark disease in willows (Salix spp.)

The watermark disease, caused by Brenneria salicis (formerly Erwinia salicis), is of significant concern wherever tree-forming willows are grown or occur naturally. The movement of infected, asymptomatic cuttings is a major cause of pathogen dispersal. A reliable and sensitive diagnostic procedure is necessary for the safe movement of willow planting material. We derived primers from the nucleotide sequence of the 16S rRNA gene of B. salicis for the development of a PCR to detect this pathogen. One set of primers, Es1a-Es4b, directed the amplification of a 553-bp fragment from B. salicis genomic DNA as well as B. salicis cells. PCR products were not observed when genomic DNA was tested for 27 strains of other, related plant-associated bacteria. Genomic fingerprinting by amplification fragment length polymorphism of B. salicis strains, originating from four different countries, and related Brenneria, Pectobacterium, and Erwinia strains revealed a very high similarity among the B. salicis genomes, indicating that the spread of the pathogen is mainly due to the transportation of infected cuttings. The PCR had to be preceded by a DNA extraction in order to detect the pathogen in the vascular fluid of willows. The minimum number of cells that could be detected from vascular fluid was 20 CFU/ml. The PCR assays proved to be very sensitive and reliable in detecting B. salicis in willow plant material.

Journal Article↗
PubMed2004

The global regulator genes from biocontrol strain Serratia plymuthica IC1270: cloning, sequencing, and functional studies.

The biocontrol activity of various fluorescent pseudomonads towards plant-pathogenic fungi is dependent upon the GacA/GacS-type two-component system of global regulators and the RpoS transcription sigma factor. In particular, these components are required for the production of antifungal antibiotics and exoenzymes. To investigate the effects of these global regulators on the expression of biocontrol factors by plant-associated bacteria other than Pseudomonas spp., gacA/gacS and rpoS homologues were cloned from biocontrol strain IC1270 of Serratia plymuthica, which produces a set of antifungal compounds, including chitinolytic enzymes and the antibiotic pyrrolnitrin. The nucleotide and deduced protein sequence alignments of the cloned gacA/gacS-like genes-tentatively designated grrA (global response regulation activator) and grrS (global response regulation sensor) and of the cloned rpoS gene revealed 64 to 93% identity with matching genes and proteins of the enteric bacteria Escherichia coli, Pectobacterium carotovora subsp. carotovora, and Serratia marcescens. grrA, grrS, and rpoS gene replacement mutants of strain IC1270 were deficient in the production of pyrrolnitrin, an exoprotease, and N-acylhomoserine lactone quorum-sensing signal molecules. However, neither mutant appeared to differ from the parental strain in the production of siderophores, and only grrA and grrS mutants were deficient in the production of a 58-kDa endochitinase, representing the involvement of other sigma factors in the regulation of strain IC1270's chitinolytic activity. Compared to the parental strain, the grrA, grrS, and rpoS mutants were markedly less capable of suppressing Rhizoctonia solani and Pythium aphanidermatum under greenhouse conditions, indicating the dependence of strain IC1270's biocontrol property on the GrrA/GrrS and RpoS global regulators.

Antifungal Agents↗
PubMed1982

[Antibiotic sensitivity of opportunistic bacteria isolated from patients].

Sensitivity of 147 Enterobacteria strains isolated from feces of various patients was determined with the method of serial dilutions on solid nutrient media. 8 antibiotics were tested. By genera (species) the microorganisms were arranged in the following order: E. coli (65 strains), Citrobacter (33 strains), E. cloacae (15 strains), Serratia liquefaciens (9 strains), Hafnia (6 strains), Klebsiella (4 strains), Pectobacterium (3 strains), non-identified organisms (13 strains). The majority of the strains were resistant to levomycetin (chloramphenicol), morphocycline, tetracycline and tetraolean and at the same time sensitive to streptomycin, neomycin, monomycin and kanamycin. 18 combinations of resistance were found. Repeated examinations of the specimens from the same patient revealed changes in the species composition of the cultures and subsequently in the antibiotic sensitivity spectrum.

Anti-Bacterial Agents↗
PubMed1992

Allergic reactions to Erwinia asparaginase in children with acute lymphoblastic leukemia who had previous allergic reactions to Escherichia coli asparaginase.

BACKGROUND: Escherichia coli asparaginase is an active antileukemia agent in the treatment of childhood acute lymphoblastic leukemia. Allergic reactions occurred in 31 of 125 patients (24.8%) treated with weekly high-dose (25,000 IU/m2) intramuscular E. coli asparaginase and necessitated discontinuation of the drug. METHODS: The authors evaluated the toxic effects of Erwinia asparaginase in the 31 children who had allergic reactions to the E. coli preparation. RESULTS: Subsequent allergic reactions to Erwinia asparaginase occurred in 7 of the 31 children (22.6%). In contrast to previous reports with intravenous administration, most allergic reactions to both asparaginase preparations were characterized by mild urticaria that responded to use of diphenhydramine; none of the reactions was life-threatening. CONCLUSIONS: In summary, the authors found Erwinia asparaginase to be an acceptable substitute for E. coli asparaginase for most children who had allergic reactions. Through the use of both E. coli and Erwinia asparaginase, 94% of children could receive their intended asparaginase.

Adolescent↗
PubMed1993

A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.

Virulence of the plant pathogen Erwinia carotovora subsp. carotovora is dependent on the production and secretion of a complex arsenal of plant cell wall-degrading enzymes. Production of these exoenzymes is controlled by a global regulatory mechanism. A virulent mutants in one of the regulatory loci, expI, show a pleiotropic defect in the growth phase-dependent transcriptional activation of exoenzyme gene expression. The expI gene encodes a 26 kDa polypeptide that is structurally and functionally related to the luxI gene product of Vibrio fischeri. Functional similarity of expI and luxI has been demonstrated by reciprocal genetic complementation experiments. LuxI controls bioluminescence in V.fischeri in a growth phase-dependent manner by directing the synthesis of the diffusible autoinducer, N-(3-oxohexanoyl) homoserine lactone. E.c. subsp. carotovora expI+ strains or Escherichia coli harboring the cloned expI gene excrete a small diffusible signal molecule that complements the expI mutation of Erwinia as well as a luxI mutation of V.fischeri. This extracellular complementation can also be achieved by E.coli harboring the luxI gene from V.fischeri or by adding the synthetic V.fischeri autoinducer. Both the production of the plant tissue-macerating exoenzymes and the ability of the bacteria to propagate in planta are restored in expI mutants by autoinducer addition. These data suggest that the same signal molecule is employed in control of such diverse processes as virulence in a plant pathogen and bioluminescence in a marine bacterium, and may represent a general mechanism by which bacteria modulate gene expression in response to changing environmental conditions.

Amino Acid Sequence↗
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↗
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↗