Extracellular and periplasmic isoenzymes of pectate lyase from Erwinia carotovora subspecies carotovora belong to different gene families

Pectate lyase (Pel) plays a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have characterized the four Pel isoenzymes of Erwinia carotovora subspecies carotovora strain SCRI193. In this paper we concentrate on two isoenzymes which have different locations in SCRI193: PLb is periplasmic and PLc is extracellular. Comparison of the gene products and nucleotide sequences of pelB and pelC allowed us to assign them to different gene families. In addition, we have identified a number of conserved amino acid residues that are common to all extracellular Pel isoenzymes.     

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.     

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)     

The study of Erwinia carotovora bacteriocins in the nalidixic acid resistant Erwinia carotovora

A novel approach is proposed for the study of the macromolecular bacteriocins of Erwinia carotovora (MCTVs). The approach lies in that the bacteriocinogeny of pectolytic erwinia is studied using a lawn of a bacterial mutant resistant to nalidixic acid, an inducer of MCTVs. The high efficiency of this approach was demonstrated by studying carotovoricins in 104 different E. carotovora strains, 88% of which bear MCTVs, distinguished by the morphology of zones of induced lysis on a lawn of susceptible cells, the lysis pattern, and some other characteristics. Preliminary studies by this approach showed that there is no correlation between the occurrence of MCTVs in particular E. carotovora strains and the habitat of the host plants from which these strains were isolated. There are grounds to believe that the approach proposed can also be used for investigating bacterial lysogeny.     

Fluoride-induced filaments of Erwinia carotovora

Sodium fluoride induces filamentous growth of Erwinia carotovora when it is grown in liquid media containing aspartic acid only as the sole source of nitrogen. It is proposed that a stable complex of F(-)-Mg2+-enzyme and PO2(4) resulting in Mg2+ deficiency and consequent inability of E. carotovora cells to oxidize aspartic acid normally, is responsible for the formation of filaments in the presence of fluoride ions.     

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.     

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.     

Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora

Soft rot Erwinia species secrete a range of enzymes into the extracellular environment. Therefore, the genetically amenable Erwinia system is a useful model for the study of protein secretion by Gram-negative bacteria. We have used a λ-sensitive derivative of Erwinia carotovora subspecies carotovora (Ecc) and the transposon TnphoA, to isolate a range of extracellular enzyme mutants. The use of TnphoA provides an enrichment for extracellular enzyme mutants over other transposon-based systems. In these mutants, the alkaline phosphatase activity of the hybrid protein Is found in the periplasm, and is under the control of the Ecc promoters. Three TnphoA-induced extracellular enzyme mutants were studied in detail. One proved to be an enzyme structural gene mutant, whilst the other two appeared to be secretory mutants.     

Oligomerization of L-asparaginase from Erwinia carotovora

Bacterial L-asparaginases catalyzing hydrolysis of L-asparagine to L-aspartate and ammonia, are used in medical practice for treatment of acute lymphoblastic leukemia. The long-term therapy with these preparations is accompanied by a number of side effects, which are attributed to glutaminase activity of L-asparaginase. Substrate specificity and activity of L-asparaginases are directly associated with the oligomerization process of this enzyme, which is active only as the tetramer because its active sites are located in the contact areas between monomers. The present work is devoted to homology modeling of spatial structure of L-asparaginase from Erwinia carotovora, the comparative molecular-graphic analysis of subunit interfaces, and the development of a new experimental approach for studies of enzyme oligomerization. L-Asparaginase was immobilized on a surface of CM5 optical chip of biosensor Biacore 3000, which is based on the surface plasmon resonance technology. The dissociation process of enzyme tetrameric complexes up to monomers and subsequent oligomerization process have been registered.     

Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora

Soft rot Erwinia species secrete a range of enzymes into the extracellular environment. Therefore, the genetically amenable Erwinia system is a useful model for the study of protein secretion by Gram-negative bacteria. We have used a lambda-sensitive derivative of Erwinia carotovora subspecies carotovora (Ecc) and the transposon TnphoA, to isolate a range of extracellular enzyme mutants. The use of TnphoA provides an enrichment for extracellular enzyme mutants over other transposon-based systems. In these mutants, the alkaline phosphatase activity of the hybrid protein is found in the periplasm, and is under the control of the Ecc promoters. Three TnphoA-induced extracellular enzyme mutants were studied in detail. One proved to be an enzyme structural gene mutant, whilst the other two appeared to be secretory mutants.     

Response of endophytic bacterial communities in potato plants to infection with Erwinia carotovora subsp. atroseptica

The term endophyte refers to interior colonization of plants by microorganisms that do not have pathogenic effects on their hosts, and various endophytes have been found to play important roles in plant vitality. In this study, cultivation-independent terminal restriction fragment length polymorphism analysis of 16S ribosomal DNA directly amplified from plant tissue DNA was used in combination with molecular characterization of isolates to examine the influence of plant stress, achieved by infection with the blackleg pathogen Erwinia carotovora subsp. atroseptica, on the endophytic population in two different potato varieties. Community analysis clearly demonstrated increased bacterial diversity in infected plants compared to that in control plants. The results also indicated that the pathogen stress had a greater impact on the bacteria population than the plant genotype had. Partial sequencing of the 16S rRNA genes of isolated endophytes revealed a broad phylogenetic spectrum of bacteria, including members of the alpha, beta, and gamma subgroups of the Proteobacteria, high- and low-G+C-content gram-positive organisms, and microbes belonging to the Flexibacter-Cytophaga-Bacteroides group. Screening of the isolates for antagonistic activity against E. carotovora subsp. atroseptica revealed that 38% of the endophytes protected tissue culture plants from blackleg disease.     

Response of Endophytic Bacterial Communities in Potato Plants to Infection with Erwinia carotovora subsp. atroseptica

The term endophyte refers to interior colonization of plants by microorganisms that do not have pathogenic effects on their hosts, and various endophytes have been found to play important roles in plant vitality. In this study, cultivation-independent terminal restriction fragment length polymorphism analysis of 16S ribosomal DNA directly amplified from plant tissue DNA was used in combination with molecular characterization of isolates to examine the influence of plant stress, achieved by infection with the blackleg pathogen Erwinia carotovora subsp. atroseptica, on the endophytic population in two different potato varieties. Community analysis clearly demonstrated increased bacterial diversity in infected plants compared to that in control plants. The results also indicated that the pathogen stress had a greater impact on the bacteria population than the plant genotype had. Partial sequencing of the 16S rRNA genes of isolated endophytes revealed a broad phylogenetic spectrum of bacteria, including members of the α, β, and γ subgroups of the Proteobacteria, high- and low-G+C-content gram-positive organisms, and microbes belonging to the Flexibacter-Cytophaga-Bacteroides group. Screening of the isolates for antagonistic activity against E. carotovora subsp. atroseptica revealed that 38% of the endophytes protected tissue culture plants from blackleg disease.     

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 .     

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.