Effects of a mutation that eliminates UDP glucose-pyrophosphorylase on the pathogenicity of Erwinia carotovora subsp. carotovora.

A nonpathogenic mutant of Erwinia carotovora obtained by Mu d1 mutagenesis was defective in the ability to utilize several carbon sources. The basis of the mutation was analyzed biochemically and shown to be a defect in the ability to form UDP glucose-pyrophosphorylase. The nonpathogenic phenotype of the mutant was caused by its sensitivity to galactose.     

Chromosomal mapping in Erwinia carotovora subsp. carotovora with the IncP plasmid R68::Mu.

Conjugational gene transfer was established in Erwinia carotovora subsp. carotovora SCRI193 by using plasmid R68::Mu c+ to mobilize the chromosome into multiply mutant recipients. It was observed that although the plasmid alone mobilized markers randomly at a frequency of ca. 10(-5) chromosomal recombinants per donor, the presence of a Mu prophage on the chromosome of the donor increased the frequency of mobilization of markers adjacent to the prophage by up to 10-fold. Using this system it was possible to order 17 chromosomal mutations. The behavior of Mu in E. carotovora subsp. carotovora was also studied.     

Nonsense-suppressor mutants of Erwinia carotovora subsp. carotovora

Abstract A promiscuous plasmid (pLM2) carrying amber mutations in two antibiotic-resistance genes was transferred to a derivative of Erwinia carotovora subsp. carotovora strain SCRI193. Following mutagenesis, two putative amber-suppressing mutants of this strain were isolated. The genotype of these mutants was confirmed by use of rep _am plasmid-specific phage. This constitutes the first isolation of amber-suppressing mutants in Erwinia spp.     

Transposon Tn5 mutagenesis in Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica

In matings between Escherichia coli 2492(pJB4JI) and Erwinia carotovora subsp. carotovora Ecc71 and E. carotovora subsp. atroseptica Eca12, Kmr Gms transconjugants were obtained at high frequencies, indicating instability of the Mu-containing plasmid pJB4JI and transposition of Tn5 into the recipient genome. This was verified by Southern blot hybridization with pRZ102 DNA containing Tn5 as the 32P-labeled probe. Examination of Kmr Gms transconjugants of Ecc71 and Eca12 disclosed that a proportion (2 to 3%) were either auxotrophic or defective in catabolism of specific carbohydrates. Spontaneous prototrophic revertants were obtained for all markers with the exception of ilv, tyr, and suc. Genetic and physical data indicate that scattered insertions of Tn5 from pJb4JI into the chromosome of Ecc71 and Eca12 produced a variety of altered phenotypes due mostly to single insertions of Tn5 not accompanied by Mu DNA.     

Defective lysogeny in Erwinia carotovora

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128-192 nm in length and 13-21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55-59, 66-75, and 92-98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid-induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.     

Plasmid ColVBtrp maintenance in Erwinia carotovora.

Plasmid ColVBtrp maintenance in Erwinia carotovora cells was followed by measuring kinetics of elimination of plasmid genetic markers and loss of plasmid deoxyribonucleic acid. An E. carotovora mutant stably carrying plasmid ColVBtrp was isolated. Besides stable plasmid maintenance, the mutant showed altered sensitivity to male-specific phage MS2, sensitivity to drugs, and colony morphology.     

Survival of the phytopathogen Erwinia carotovora subsp. carotovora in sterile and non-sterile soil, sand and their admixture

Survival of phytopathogens in irrigation water and irrigated soil is of major concern to the agricultural community. In the present study, an Erwinia carotovora subsp. carotovora strain was tested for survival capability in three non-sterile and heat-sterilized soil matrices (soil, sand and soil + sand) over 35 d. In all non-sterile soil matrices, Erw. carotovora subsp. carotovora numbers declined below the detection limit over 35 d, the main variance being the decline rate related to nutrients available in the different matrices (soil, soil + sand and sand respectively). In heat-sterilized soil and soil + sand matrices Erw. carotovora subsp. carotovora revealed a regrowth, while in sterile sand matrix its decline was lower over the same time period. In previous published reports, when soil was sterilized by irradiation, such a regrowth was not observed. Application of an initial single load of sodium nitrate solution (70 mg l⁻¹) was found to extend bacteria survival rate in non-sterile and sterile soil columns. In sterile soil columns supplemented with sodium nitrate, Erw. carotovora subsp. carotovora did survive well for up to 60 d, with a major regrowth over the first 12 d and decline up to day 60, reaching initial loading numbers. The information on the potential survival of Erw. carotovora subsp. carotovora in soil for up to 35 d and regrowth in sterile soil should be of concern, especially when irrigation is performed with poor quality water.     

Novel mutants of Erwinia carotovora subsp. carotovora defective in the production of plant cell wall degrading enzymes generated by Mu transpososome-mediated insertion mutagenesis

As in Erwinia carotovora subsp. carotovora the regulation details of the main virulence factors, encoding extracellular enzymes that degrade the plant cell wall, is only rudimentally understood, we performed a genetic screen to identify novel candidate genes involved in the process. Initially, we used Mu transpososome-mediated mutagenesis approach to generate a comprehensive transposon insertion mutant library of ca. 10000 clones and screened the clones for the loss of extracellular enzyme production. Extracellular enzymes production was abolished by mutations in the chromosomal helEcc, trkAEcc yheLEcc, glsEcc, igaAEcc and cysQEcc genes. The findings reported here demonstrate that we have isolated six new representatives that belong to the pool of genes modulating the production of virulence factors in E. carotovora.     

Efficient transformation of Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica.

We used a modified version of the method of Hanahan (D. Hanahan, J. Mol. Biol. 166:557-580, 1983) to transform Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica with the plasmids pBR322, pBR325, and pAT153. The transformation frequency ranged from 1 X 10(2) to 4 X 10(4) colonies per micrograms of plasmid DNA. The nature of these transformants was confirmed by plasmid analysis. ColE1-based plasmids make potentially useful cloning vectors for the study of genes involved in the pathogenesis of this species.     

Studies on Inc-P plasmids in Erwinia carotovora subsp. carotovora

Abstract Inc-P plasmids, RP4, R751, pMO850, and pRK2013 were transferred to Erwinia carotovora . These plasmids were stably maintained in E. carotovora and the transconjugants were efficient donors of respective plasmids to other strains of E. carotovora and Escherichia coli . These plasmids were not able to mobilize chromosomal markers from one strain of E. carotovora to another strain of E. carotovora even in the presence of homologous DNA sequences on the plasmid and the bacterial chromosome. The presence of Inc-P plasmid does not affect the pathogenic phenotype of E. carotovora . A broad host range Inc-P cosmid, pLAFR1, was transferred to E. carotovora with the help of pRK2013, suggesting the potential use of a binary plasmid system for genetic complementation in E. carotovora .     

Structure of Erwinia carotovora temperate bacteriophage 59 and its DNA.

The temperate phage 59 from Erwinia carotovora and its DNA were studied. The phage particles have an icosahedral head and a long noncontractile tail with a base plate. The virus DNA makes up 50% of the total virus and exists as a linear molecule (molecular weight, 2.6 X 10(7)). A model of virus structural organization is presented.     

Temperate bacteriophage ZF40 of Erwinia carotovora: phage particle structure and DNA restriction analysis

Structural organization of the temperate bacteriophage ZF40 of Erwinia carotovora was studied. Phage ZF40 proved to be a typical member of the Myoviridae family (morphotype A1). Phage particles consist of an isometric head 58.3 nm in diameter and a contractile 86.3-nm-long tail with a complex basal plate and short tail fibers (31.5 nm). Phage tail sheath, a truncated cone in shape, is characterized by specific packaging of structural subunits. The ZF40 phage genome is 45.8 kb in size, as determined by restriction analysis, and contains DNA cohesive ends. The ZF40 phage of Erwinia carotovora is assumed to be a new species of bacteriophages specific for enterobacteria.     

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

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

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.     

Study of Erwinia carotovora phage resistance with the use of temperate bacteriophage ZF40

The causes of the unique phage resistance of the pectinolytic phytopathogenic strains of Erwinia carotovora were studied with the use of temperate bacteriophage ZF40. It was shown that, in these bacteria, the bacteriophage-cell interaction can be substantially blocked at the adsorption level. An adequate indicator for studying the temperate bacteriophages of erwinias was developed on the basis of mutants resistant to macromolecular bacteriocins. Various restriction-modification systems, which influence cell resistance to bacteriophages, were revealed for the first time in E. carotovora. The phage resistance was shown to be determined by the wide occurrence of homoimmune temperate viruses in pectinolytic erwinias.     

The ineffectuality of potato protease inhibitor on the extracellular protease from Erwinia carotovora subsp. carotovora

Growth conditions are described for optimum production of extracellular protease in batch cultures of the soft rot pathogen Erwinia carotovora subsp. carotovora . This protease was inhibited by approximately 96% by 1 mmol/1 EDTA and by 55–6% by 10 mmol/l cysteine thereby classifying it as a metalloprotease. It was not inhibited by a chymotrypsin inhibitor extracted from potato tubers. This evidence suggests that the potato chymotrypsin inhibitor is not associated with resistance of potatoes to E. carotovora subsp. carotovora .     

Restriction map of permuted DNA of Erwinia carotovora temperate bacteriophage 59

The cyclic permutation and terminal redundancy were found in the genomes of Erwinia carotovora temperate bacteriophage 59 by electron microscopic studies. The headful mechanism for bacteriophage DNA cleavage and packaging during the phage morphogenesis was confirmed by the restriction analysis technique. Restriction map of the bacteriophage 59 DNA was constructed for restriction endonucleases BamHI, Bg1II, Eco31, Sa1I, SmaI, EcoRI.     

Structure of the O-polysaccharide of Erwinia carotovora ssp. carotovora GSPB 436

The O-polysaccharide of a phytopathogenic bacterium, Erwinia carotovora ssp. carotovora GSPB 436, was studied by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure in text] The O-polysaccharide contains a minor proportion of 4-O-methylrhamnose, which is suggested to terminate the polymer main chain.