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At least 217 records · Page 12Linked to original sources
PubMed2004

Comparative genomics of the KdgR regulon in Erwinia chrysanthemi 3937 and other gamma-proteobacteria.

In the plant-pathogenic enterobacterium Erwinia chrysanthemi, almost all known genes involved in pectin catabolism are controlled by the transcriptional regulator KdgR. In this study, the comparative genomics approach was used to analyse the KdgR regulon in completely sequenced genomes of eight enterobacteria, including Erw. chrysanthemi, and two Vibrio species. Application of a signal recognition procedure complemented by operon structure and protein sequence analysis allowed identification of new candidate genes of the KdgR regulon. Most of these genes were found to be controlled by the cAMP-receptor protein, a global regulator of catabolic genes. At the next step, regulation of these genes in Erw. chrysanthemi was experimentally verified using in vivo transcriptional fusions and an attempt was made to clarify the functional role of the predicted genes in pectin catabolism. Interestingly, it was found that the KdgR protein, previously known as a repressor, positively regulates expression of two new members of the regulon, phosphoenolpyruvate synthase gene ppsA and an adjacent gene, ydiA, of unknown function. Other predicted regulon members, namely chmX, dhfX, gntB, pykF, spiX, sotA, tpfX, yeeO and yjgK, were found to be subject to classical negative regulation by KdgR. Possible roles of newly identified members of the Erw. chrysanthemi KdgR regulon, chmX, dhfX, gntDBMNAC, spiX, tpfX, ydiA, yeeO, ygjV and yjgK, in pectin catabolism are discussed. Finally, complete reconstruction of the KdgR regulons in various gamma-proteobacteria yielded a metabolic map reflecting a globally conserved pathway for the catabolism of pectin and its derivatives with variability in transport and enzymic capabilities among species. In particular, possible non-orthologous substitutes of isomerase KduI and a new oligogalacturonide transporter in the Vibrio species were detected.

Artificial Gene Fusion↗
PubMed2006

Exploitation of a beta-lactamase reporter gene fusion in the carbapenem antibiotic production operon to study adaptive evolution in Erwinia carotovora.

Erwinia carotovora subsp. carotovora strain ATTn10 produces the beta-lactam antibiotic 1-carbapen-2-em-3-carboxylic acid (carbapenem) by expressing the carABCDEFGH operon. Mutants exhibiting increased carbapenem gene transcription were positively selected using an engineered strain with a functional beta-lactamase translational fusion in carH, the last gene of the operon. However, spontaneous ampicillin-resistant mutants were isolated even when transcription of carH : : blaM was blocked by a strongly polar mutation in carE. The mechanism of resistance was shown to be due to cryptic IS10 elements transposing upstream of carH : : blaM, thereby providing new promoters enabling carH : : blaM transcription. Southern blots showed that IS10 was present in multicopy in ATTn10. In addition, a Tn10 genetic remnant was discovered. The results offer insights into the genetic archaeology of strain ATTn10 and highlight the powerful impacts of cryptic IS elements in bacterial adaptive evolution.

Adaptation, Biological↗
PubMed2003

Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila.

Studies in mammals uncovered important signaling roles of nitric oxide (NO), and contributions to innate immunity. Suggestions of conservation led us to explore the involvement of NO in Drosophila innate immunity. Inhibition of nitric oxide synthase (NOS) increased larval sensitivity to gram-negative bacterial infection, and abrogated induction of the antimicrobial peptide Diptericin. NOS was up-regulated after infection. Antimicrobial peptide reporters revealed that NO triggered an immune response in uninfected larvae. NO induction of Diptericin reporters in the fat body required immune deficiency (imd) and domino. These findings show that NOS activity is required for a robust innate immune response to gram-negative bacteria, NOS is induced by infection, and NO is sufficient to trigger response in the absence of infection. We propose that NO mediates an early step of the signal transduction pathway, inducing the innate immune response upon natural infection with gram-negative bacteria.

Amino Acid Sequence↗
PubMed2002

Inhibition of the plastidic ATP/ADP transporter protein primes potato tubers for augmented elicitation of defense responses and enhances their resistance against Erwinia carotovora.

Tubers of transgenic potato (Solanum tuberosum) plants with decreased activity of the plastidic ATP/ADP transporter AATP1 display reduced levels of starch, modified tuber morphology, and altered concentrations of primary metabolites. Here, we demonstrate that the spontaneous production of hydrogen peroxide, the endogenous content of salicylic acid, and the levels of mRNAs of various defense-related genes are similar in tuber discs of wild-type and AATP1(St) antisense plants. However, upon challenging the tissue with fungal elicitors or culture supernatants of the soft rot-causing pathogen Erwinia carotovora subsp. atroseptica, the AATP1(St) antisense tubers exhibit highly potentiated activation of defense responses when compared with wild-type tissue. The augmented defense responses comprise enhanced accumulation of transcripts of five defense-related genes (beta-1,3-GLUCANASE B2 and A1, CHITINASE B3 and A2, and Phe AMMONIA-LYASE) and enhanced elicitation (up to 21-fold) of the early hydrogen peroxide burst. The potentiated activation of cellular defense responses in AATP1(St) antisense tubers is not accompanied by a precedent increase in endogenous salicylic acid levels, but is associated with a strongly enhanced resistance of the tissue to E. carotovora. From these results, we conclude that inhibition of primary metabolic reactions induces a primed state that sensitizes the potato tubers for improved elicitation of various cellular defense responses, which likely contribute to enhanced E. carotovora resistance.

DNA, Antisense↗
PubMed2006

EARLY RESPONSIVE TO DEHYDRATION 15, a negative regulator of abscisic acid responses in Arabidopsis.

EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) is rapidly induced in response to various abiotic and biotic stress stimuli in Arabidopsis (Arabidopsis thaliana). Modulation of ERD15 levels by overexpression or RNAi silencing altered the responsiveness of the transgenic plants to the phytohormone abscisic acid (ABA). Overexpression of ERD15 reduced the ABA sensitivity of Arabidopsis manifested in decreased drought tolerance and in impaired ability of the plants to increase their freezing tolerance in response to this hormone. In contrast, RNAi silencing of ERD15 resulted in plants that were hypersensitive to ABA and showed improved tolerance to both drought and freezing, as well as impaired seed germination in the presence of ABA. The modulation of ERD15 levels not only affected abiotic stress tolerance but also disease resistance: ERD15 overexpression plants showed improved resistance to the bacterial necrotroph Erwinia carotovora subsp. carotovora accompanied with enhanced induction of marker genes for systemic acquired resistance. We propose that ERD15 is a novel mediator of stress-related ABA signaling in Arabidopsis.

Abscisic Acid↗
PubMed2000

Linear beta-1,3 glucans are elicitors of defense responses in tobacco.

Laminarin, a linear beta-1,3 glucan (mean degree of polymerization of 33) was extracted and purified from the brown alga Laminaria digitata. Its elicitor activity on tobacco (Nicotiana tabacum) was compared to that of oligogalacturonides with a mean degree of polymerization of 10. The two oligosaccharides were perceived by suspension-cultured cells as distinct chemical stimuli but triggered a similar and broad spectrum of defense responses. A dose of 200 microg mL(-1) laminarin or oligogalacturonides induced within a few minutes a 1.9-pH-units alkalinization of the extracellular medium and a transient release of H(2)O(2). After a few hours, a strong stimulation of Phe ammonia-lyase, caffeic acid O-methyltransferase, and lipoxygenase activities occurred, as well as accumulation of salicylic acid. Neither of the two oligosaccharides induced tissue damage or cell death nor did they induce accumulation of the typical tobacco phytoalexin capsidiol, in contrast with the effects of the proteinaceous elicitor beta-megaspermin. Structure activity studies with laminarin, laminarin oligomers, high molecular weight beta-1, 3-1,6 glucans from fungal cell walls, and the beta-1,6-1,3 heptaglucan showed that the elicitor effects observed in tobacco with beta-glucans are specific to linear beta-1,3 linkages, with laminaripentaose being the smallest elicitor-active structure. In accordance with its strong stimulating effect on defense responses in tobacco cells, infiltration of 200 microg mL(-1) laminarin in tobacco leaves triggered accumulation within 48 h of the four families of antimicrobial pathogenesis-related proteins investigated. Challenge of the laminarin-infiltrated leaves 5 d after treatment with the soft rot pathogen Erwinia carotovora subsp. carotovora resulted in a strong reduction of the infection when compared with water-treated leaves.

Cells, Cultured↗
PubMed2001

Jasmonate-dependent induction of indole glucosinolates in Arabidopsis by culture filtrates of the nonspecific pathogen Erwinia carotovora.

Elicitors from the plant pathogen Erwinia carotovora trigger coordinate induction of the tryptophan (Trp) biosynthesis pathway and Trp oxidizing genes in Arabidopsis. To elucidate the biological role of such pathogen-induced activation we characterized the production of secondary defense metabolites such as camalexin and indole glucosinolates derived from precursors of this pathway. Elicitor induction was followed by a specific increase in 3-indolylmethylglucosinolate (IGS) content, but only a barely detectable accumulation of the indole-derived phytoalexin camalexin. The response is mediated by jasmonic acid as shown by lack of IGS induction in the jasmonate-insensitive mutant coi1-1. In accordance with this, methyl jasmonate was able to trigger IGS accumulation in Arabidopsis. In contrast, ethylene and salicylic acid seem to play a minor role in the response. They did not trigger alterations in IGS levels, and methyl jasmonate- or elicitor-induced IGS accumulation in NahG and ethylene-insensitive ein2-1 mutant plants was similar as in the wild type. The breakdown products of IGS and other glucosinolates were able to inhibit growth of E. carotovora. The results suggest that IGS is of importance in the defense against bacterial pathogens.

Arabidopsis↗
PubMed2004

Chlorophyllase 1, a damage control enzyme, affects the balance between defense pathways in plants.

Accumulation of reactive oxygen species (ROS) is central to plant response to several pathogens. One of the sources of ROS is the chloroplast because of the photoactive nature of the chlorophylls. Chlorophyllase 1 (encoded by AtCLH1) of Arabidopsis thaliana is quickly induced after tissue damage (e.g., caused by the bacterial necrotroph Erwinia carotovora or the necrotrophic fungus Alternaria brassicicola). RNA interference silencing of AtCLH1 resulted in failure to degrade free chlorophyll after tissue damage and in resistance to E. carotovora. Both inoculation with E. carotovora and exposure to high light caused elevated accumulation of hydrogen peroxide in AtCLH1 silenced plants. This was accompanied by expression of marker genes for systemic acquired resistance and induction of antioxidant defenses. Interestingly, downregulation of AtCLH1 resulted in increased susceptibility to A. brassicicola, resistance to which requires jasmonate signaling. We propose that AtCLH1 is involved in plant damage control and can modulate the balance between different plant defense pathways.

Antioxidants↗
PubMed1991

Differential activation of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge.

Potato genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were expressed in response to pathogen, elicitor, and wounding. HMGR catalyzes the rate-limiting step in isoprenoid biosynthesis leading to accumulation of phytoalexins and steroid glycoalkaloids. Wounding caused increases in HMGR mRNA levels. A rapid and transient peak occurred 30 minutes after wounding, followed by a slower peak at 14 hours; both were correlated with increased enzyme activity. Induction of HMGR mRNA by the soft rot pathogen Erwinia carotovora subsp carotovora or arachidonic acid began 8 hours after challenge and continued through 22 hours. Potato HMGR is encoded by a gene family. An HMGR gene-specific probe was used to demonstrate that one isogene of the HMGR family is pathogen activated and is distinct from isogene(s) that are wound activated. This provides evidence that defense-related increases in HMGR activity are due to mRNA level increases and that HMGR isogenes are activated differentially by wounding or pathogen challenge.

Arachidonic Acids↗
PubMed1995

Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.

Base Sequence↗
PubMed1999

Crystallization and preliminary crystallographic analysis of the endo-polygalacturonase from Erwinia carotovora ssp. carotovora.

Crystals of endo-polygalacturonase from Erwinia carotovora ssp. carotovora have been grown from polyethylene glycol 6000 by the hanging-drop method. Polygalacturonase is important in the virulence of this plant pathogen. The protein crystallizes in space group C2 with unit-cell parameters a = 81.3, b = 53.0, c = 103.1 A, beta = 112.6 degrees and with a single molecule in the asymmetric unit. The crystals diffract to 1.9 A.

Bacillus subtilis↗
PubMed2005

Crystallization and preliminary crystallographic analysis of L-asparaginase from Erwinia carotovora.

Bacterial L-asparaginases have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia for over 30 y. However, their use is limited owing to the glutaminase activity of the administered enzymes, which results in serious side effects. In contrast, L-asparaginase from Erwinia carotovora exhibits low glutaminase activity at physiological concentrations of L-asparagine and L-glutamine in the blood. Recombinant Er. carotovora L-asparaginase was crystallized in the presence of L-glutamate by the hanging-drop vapour-diffusion method using 10 mg ml(-1) purified enzyme, 16-18%(w/v) PEG 3350 and 0.2 M NaF. X-ray diffraction data were collected to 2.6 A at 293 K using an in-house rotating-anode generator. The crystals belong to the monoclinic P2(1) space group, with unit-cell parameters a = 78.0, b = 112.3, c = 78.7 A, beta = 101.9 degrees and a homotetramer in the crystallographic asymmetric unit. A molecular-replacement solution has been found and refinement is currently in progress. The crystal structure may provide leads towards protein-engineering efforts aimed at safer asparaginase administration in leukaemia treatment.

Asparaginase↗
PubMed1993

A note on the primary structure and expression of an Erwinia carotovora polygalacturonase-encoding gene (peh1) in Escherichia coli and Saccharomyces cerevisiae.

A 1209-base pair (bp) DNA fragment containing the endopolygalacturonase-encoding gene (peh1) from Erwinia carotovora subsp. carotovora was amplified by the polymerase chain reaction (PCR) technique and expressed in Escherichia coli. The nucleotide sequence of the PCR product was determined and found to be highly homologous to the primary structures of other polygalacturonase-encoding genes. The peh1 DNA fragment encoding the mature polygalacturonase was inserted between two different yeast expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMS10 and pAMS11. These YIp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae. Transcription initiation signals present in these expression-secretion cassettes were derived from the yeast alcohol dehydrogenase (ADC1P) or mating pheromone alpha-factor (MF alpha 1P) gene promoters. The transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of polygalacturonase was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S). Northern blot analysis revealed the presence of peh1 mRNA in the yeast transformants and a polypectate agarose test was used to monitor polygalacturonase production.

Amino Acid Sequence↗
PubMed1994

Verification of ELISA results by immunomagnetic isolation of antigens from extracts and analysis with SDS-PAGE and western blotting, demonstrated for Erwinia spp. in potatoes.

Isolation of antigens on immunomagnetic beads and subsequent analysis with SDS-PAGE and Western blotting (immunomagnetic isolation-Western blotting (IMI-WB)) was used to verify positive ELISA results for Erwinia chrysanthemi and Erw. carotovora subsp. atroseptica in potato peel extracts. Direct analysis of highly contaminated extracts by Western blotting without previous immuno-isolation resulted in background reactions, whereas immunomagnetic isolation resulted in distinct bands of specific antigens. Target cells as well as antigenic cell products were captured in IMI-WB. Band patterns on IMI-WB of cell-free culture filtrates and cell suspensions were highly similar, but the removal of cells lowered the detection level by 10- to 100-fold. Threshold levels of IMI-WB were generally comparable with those of ELISA. No differences in threshold levels and band patterns were found between a direct format and an indirect format of immuno-isolation. In IMI-WB, blotting patterns differed between Erw. chrysanthemi and Erw. carotovora subsp. atroseptica. The patterns were identical for 15 Erw. chrysanthemi strains, isolated from potato peel extracts in The Netherlands. However, one of 15 strains of Erw, carotovora subsp. atroseptica from potato peel extracts in The Netherlands gave an aberrant pattern. Target bacteria could be easily distinguished from those of cross-reacting strains on the basis of band patterns. Potato peel extracts naturally contaminated with Erw. chrysanthemi gave IMI-WB patterns that were similar to pure cultures of the homologous strains.

Antigens, Bacterial↗
PubMed1996

Immunomagnetic separation of Erwinia carotovora subsp. atroseptica from potato peel extracts to improve detection sensitivity on a crystal violet pectate medium or by PCR.

Immunomagnetic separation (IMS) procedures for the selective separation of Erwinia carotovora subsp. atroseptica from potato peel extract were optimized for the recovery of target and removal of non-target bacteria. A streptomycin-resistant strain of Erw. carotovora subsp. atroseptica was used in combination with a crystal violet pectate (CVP) medium supplemented with 100 micrograms ml-1 of streptomycin to determine the recovery level of the target bacterium. Recovery obtained with a polyclonal antiserum against Erw. carotovora subsp. atroseptica at a concentration of 6 micrograms IgG ml-1 was greater than that obtained with two monoclonal antibodies against lipopolysaccharides of Erw. carotovora subsp. atroseptica at a concentration of 10 micrograms IgG ml-1. A linear relationship was found between particle concentration ranging from 12 to 200 micrograms ml-1 and recovery level. When the Advanced Magnetics (AM) protein A and anti-rabbit IgG particles in the AM separation system and the Dynal anti-rabbit IgG particles in the Dynal separation system were examined, the highest recovery level per microgram of particles (66%) was obtained with the Advanced Magnetics protein A particles, followed by AM anti-rabbit particles (37%). Without IMS, detection of Erw. carotovora subsp. atroseptica in tuber peel extracts on a CVP-medium without streptomycin was impossible when the ratio of Erw. carotovora subsp. carotovora to Erw. carotovora subsp. atroseptica was greater than 100 or when large numbers of other saprophytic bacteria were present, because of overcrowding. IMS, using the AM anti-rabbit IgG particles, ensured that Erw. carotovora subsp. atroseptica could be enumerated in tuber peel extract consistently, to a detection level of 100 cells ml-1. Similarly, the IMS procedure lowered the detection level of Erw. carotovora subsp. atroseptica in a twofold diluted peel extract by PCR to ca 2.0 x 10(3) cells ml-1 or 50 cells per reaction tube. In contrast, positive results in PCR without IMS were obtained only when the peel extract was diluted 100 times and when the concentration of Erw. carotovora subsp. atroseptica was at least 10(5) cell ml-1.

Animals↗
PubMed2004

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil.

AIMS: To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. METHODS AND RESULTS: Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. CONCLUSIONS: The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.

Brazil↗
PubMed2005

Lethal effect of the gliding arc discharges on Erwinia spp.

AIMS: To compare the decontamination performances of glidarc on strains of Erwinia of industrial interest. METHODS AND RESULTS: Cultures of Erwinia carotovora carotovora, Erwinia carotovora atroseptica and Erwinia chrysanthemi taken in stationary phase were exposed to the plasma generated by electric discharges in a gliding arc reactor prototype. The kinetics of destruction of bacteria were followed by direct platting. All bacterial strains presented a three-phase destruction kinetics leading to an apparent sterilization within 10 min. Epifluorescent observations using life/dead probes revealed the absence of viable but not cultivable resistant forms. Measurement of the physical parameters of the medium confirmed that the technique was nonthermal but that reactive species responsible for a decrease of the pH were generated. However, even after neutralization the medium did not allow bacterial growth. CONCLUSIONS: The results demonstrate that glidarc allows a rapid and complete destruction of planctonic strains of Erwinias without formation of resistant forms. SIGNIFICANCE AND IMPACT OF THE STUDY: The reduction rate obtained by this technique shows the great industrial interest of glidarc for decontamination and suggests that it can be used for sterilization of industrial water effluents.

Bacteriological Techniques↗