Erwinia Strains Research Articles

Charakterisierung von Erwinia-stämmen insbesondere der Herbicola-Gruppe durch Chinone der Atmungskette und Enzyme des Fumarat-Stoffwechsels

Summary Phenotypical characterization of 50 strains of the genus Erwinia including the species Enterobacter agglomerans on the basis of 80 conventional characters does not enable an unequivocal differentiation of the various Erwinia strains. With respect to respiratory quinones and fumarate metabolism, which are considered to be criteria of great taxonomic significance, the Erwinia/Enterobacter species could be devided into four groups. The members of groups 1 and 2, containing a soluble NADH-fumarate reductase or no and only ubiquinone do not represent the “true” genus Erwinia . These groups are different from the family Enterobacteraceae and may characterize a new taxon. The homogeneity of group 1 was confirmed by DNA-DNA hybridization of some strains with the type strain of Enterobacter agglomerans (NCTC 9381). The members of groups 3 and 4, which contain both ubiquinone and menaquinone and a membrane-bound NADH-fumarate reductase resp. NAD-fumarate oxidase, resemble the members of the family Enterobacteraceae including the genus Pectobacterium with respect to these criteria.

The spread ofErwinia carotovora var.atroseptica (blackleg) and var.carotovora (tuber soft rot) from degenerating seed to progeny tubers in soil

In 1977, spread of serologically identifiable strains ofErwinia carotovora var.atroseptica and var.carotovora were traced from inoculated ‘seed’ through soil to progeny tubers of cv. Pentland Crown by placing inoculated tubers against the seed tubers of field plants at four different dates from late June to September. Progeny tubers were dug and soil samples taken 2, 4 and 8 weeks after placement and at a common final harvest, and tubers induced to rot and the causal organism identified by immunodiffusion serology. Early placements coincided with the onset of predominantly dry weather which lasted until early August and although bacteria of both varieties were detected in soil none were found in induced progeny tuber rots. Much of August was wet and resulted in successful spread of both pathogens to progeny tubers. Most of September was dry and spread to progeny from the last placement date was only detected in early October following late September rain.

Taxonomy and Emended Description of Strains of Erwinia Isolated from Musa paradisiaca Linnaeus

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Acceptance by Erwinia spp. of R plasmid R68.45 and its ability to mobilize the chromosome of Erwinia chrysanthemi.

R plasmid R68.45 was transferred in broth matings from Escherichia coli to strains of Erwinia amylovora, E. carotovora subsp. atroseptica, E. chrysanthemi, and E. herbicola (Enterobacter agglomerans); the frequency of transfer ranged from 2 x 10(-8) to 5 x 10(-4) per input donor cell depending on the bacterial species. The drug resistance markers tet(+), amp(+), and kan(+) were stable in these Erwinia species. Transconjugants of Erwinia spp., but not of the wild-type parent Erwinia strains, acquired levels of antibiotic resistance (tetracycline, 50 mug/ml; ampicillin, 200 mug/ml; kanamycin 200 mug/ml) similar to those of the donor R68.45-bearing strain of Escherichia coli. Erwinia transconjugants (with one exception of E. carotovora subsp. atroseptica) were donors of the antibiotic resistance markers; the frequency of transfer was consistently higher with an E. coli strain than with Erwinia spp. as recipients, and when matings were done on a solid surface (membranes) rather than in liquid. Transfer of chromosomal markers ade(+), gal(+), gtu(+) (utilization of galacturonate), his(+), leu(+), lys(+), thr(+), and trp(+) occurred in crosses between E. chrysanthemi strains harboring R68.45 and appropriate recipient strains; the frequency of transfer ranged from 9.0 x 10(-8) to 2.0 x 10(-6) depending on the selective marker. Analysis of the coinheritance of unselected markers among various classes of recombinants revealed linkage between thr-leu-lys-ade and between trp and his, thus confirming earlier findings with the Hfr-type donor cells. Since R68.45 mobilized an array of chromosomal markers in the wild-type as well as genetically marked strains of E. chrysanthemi, the system, used in conjunction with the existing Hfr strains, should provide a useful tool to study the genetics of plant pathogenicity of this bacterial species. In contrast to E. chrysanthemi, R68.45 did not mobilize chromosomal markers ilv(+), his(+), rbs(+), ser(+), and thr(+) in E. amylovora EA178.

Taxonomy and Emended Description of Strains of Erwinia Isolated from Musa paradisiaca Linnaeus

The phytopathogenic bacterium which causes soft rot of the pseudostem of Musa paradisiaca Linnaeus has been designated previously as Erwinia carotovora subsp. paradisiaca, E. paradisiaca, E. carotovora subsp. chrysanthemi, and E. chrysanthemi. Thirty strains were examined and compared with strains of closely related Erwinia species and their subspecies. The strains from M. paradisiaca were found to be phenotypically similar to E. chrysanthemi strains on the basis of the following characteristics: pectate degradation; production of phosphatase, indole, acetoin, and acid from ethanol; growth at 36 to 37°C; susceptibility to erythromycin; gas from glucose; utilization of malonate; no growth in 5% NaCl; and no production of acid from α-methyl-d-glucoside, trehalose, maltose, lactose, or palatinose. They could be distinguished from 322 strains of E. chrysanthemi isolated from 22 other plant hosts by the following phenotypic properties: production of acid from d-arabinose and raffinose; utilization of sodium tartrate; no production of lecithinase or of acid from inulin, mannitol, or sorbitol; and inability to liquefy gelatin.

Characterization of intracellular polygalacturonic acidtrans-eliminase fromKlebsiella oxytoca, Yersinia enterocolitica, andErwinia chrysanthemi

Intracellular polygalacturonic acidtrans-eliminase (PATE) was purified and characterized fromKlebsiella oxytoca andYersinia enterocolitica, enterobacteria unable to macerate plant tissue. The well-studied PATE from a strain ofErwinia chrysanthemi, a phytopathogen able to macerate plant tissue and cause soft-rot disease, was included for comparison. PATE from all strains displayed endo-splitting activity with pH optima between pH 8.5 and 9.0E. chrysanthemi had three isozymes (pls at pH 9.4, 9.0, and 7.8),K. oxytoca had two isozymes (pIs at pH 5.9 and 5.3), andY. enterocolitica had one isozyme (pI at pH 5.8). Molecular weights for theKlebsiella andYersinia PATEs were 71,000 and 55,000, respectively, compared with 33,000 for theErwinia PATE. Unlike theErwinia enzyme, theKlebsiella andYersinia PATEs did not require divalent cations for activity and could not macerate plant tissue without addition of pectinmethylesterase. The polygalacturonic acid-degrading enzymes found inK. oxytoca andY. enterocolitica appear to represent a separate type of PATE enzyme. It is unlikely that these organisms are phytopathogens; however, their ability to degrade polygalacturonic acid is probably advantageous to their survival in environments containing decomposing plant residues.

A method of testing the effect of antibacterial compounds on bacterial soft rot of potatoes, and results for preparations of dichlorophen and sodium hypochlorite

A product containing dichlorophen as the main active ingredient was bacteriostatic against, strains ofErwinia carotovora and pectolytic clostridia in culture at concentrations equivalent to 25 mg/litre and 30 mg/litre respectively of dichlorophen. An aqueous solution of the product containing 200 mg/litre dichlorophen was rapidly bactericidal toE. carotovora var.atroseptica but a solution containing 2.3 g/litre a.i. failed to kill spores of a pectolytic clostridium. Immersion of tubers in a solution containing 200 mg/litre dichlorophen gave a concentration of about 53 mg/kg in the peel. Formulations applied as dips or sprays at concentrations of up to 2.3 g/litre dichlorophen failed to reduce the incidence of soft rot in tubers stored under <1% O2 in N2 at 100% relative humidity and 20 C for up to 11 days. BothE. carotovora and pectolytic clostridia were isolated from rots occurring after treatment of tubers with 2 g litre dichlorophen. Solutions of sodium hypochlorite (0.6–2 g litre free chlorine, pH 9.4–9.7) reduced the incidence of soft rot in unwounded tubers stored in the above conditions.

Synthesis and excretion of polygalacturonic acid trans-eliminase in Erwinia, Yersinia, and Klebsiella species

Various enterobacteria exhibit several patterns of synthesis and excretion of polygalacturonic acid trans-eliminase (PATE), one of the enzymes involved in degradation of pectic substances. In a strain of Erwinia chrysanthemi, PATE is an extracellular enzyme and is almost totally excreted. In strains of Yersinia enterocolitica and Yersinia pseudotuberculosis, PATE is a periplasmic and cytoplasmic enzyme. In a strain of Erwinia carotovora, PATE activity is extracellular, periplasmic, and cytoplasmic. In an "oxytocum" strain of Klebsiella pneumoniae, PATE is entirely cytoplasmic; none is found in the periplasm and almost none in culture supernatants. Cells (but not supernatants, because PATE is not excreted) of K. pneumoniae show much higher levels of PATE activity when grown on polygalacturonate than when grown on other carbon sources. A clinical strain of Y. enterocolitica synthesizes high levels of PATE activity (almost all cell-bound) when grown in the presence of gluconate, glycerol, or polygalacturonate, and considerably less activity when grown on glucose. A clinical strain of Y. pseudotuberculosis synthesizes barely detectable quantities of cell-bound PATE regardless of the carbon source. The phytopathogenic Erwinia strains synthesize and excrete high levels of PATE activity when grown on polygalacturonate, galacturonate, gluconate, or glycerol. Growth on glucose represses PATE activity in the Erwinia strains; the extent of repression differs between the two strains. The different patterns of synthesis and excretion are discussed in the light of the possible catabolic and (or) cytolytic (plant tissue-macerating) functions and the related ecological significance of PATE in these enterobacteria.

Bacterial Foot Rot of Rice Caused by a Strain ofErwinia chrysanthemi

Bacterial Foot Rot of Rice Caused by a Strain of<i>Erwinia chrysanthemi</i>

Continuous-flow enrichment of a strain ofErwinia carotovora having specificity for highly methylated pectin

A pectinolytic bacterium was isolated from a mixed microbial population by means of a chemostat enrichment procedure. The bacterium, which was identified asErwinia carotovora, grew only on highly methylated pectin and produced a pectin lysase which released unsaturated monomer and dimer from 71% esterified citrus pectin. The pectin lyase was inducible only by pectins having a high methyl content and in pectin-limited chemostats its synthesis passed through a maximum at a dilution rate close to 0.04h-1.

Donor strains of the soft-rot bacterium Erwinia chrysanthemi and conjugational transfer of the pectolytic capacity.

Donor strains of Erwinia chrysanthemi ICPB EC16, a member of the soft-rot (pectolytic) section of the enterobacterial genus Erwinia, were obtained by chromosomal integration of an F'lac(+) plasmid originating from Escherichia coli. These stable donor strains, selected from an unstable F'lac(+) heterogenote by repeated platings of single Lac(+) colonies on lactose minimal agar, do not segregate (as does the parent F'lac(+) heterogenote) into Lac(-) or F(-) clones, in either the presence or absence of acridine orange. One representative donor strain (from the 12 that have been selected) has been examined in more detail; it can transfer ade(+), gal(+), gtu(+) (utilization of galacturonate), his(+), lac(+), leu(+), lys(+), mcu(+) (multiple carbohydrate utilization), pat(+) (production of polygalacturonic acid trans-eliminase), thr(+), and trp(+) in a polarized manner to appropriate recipient strains of E. chrysanthemi; the frequencies of ade(+), leu(+), and thr(+) transfer were higher than those of the other markers tested to date. This donor strain transfers lac(+) genes during a 6-h mating on membranes; most of the Lac(+) recombinants are donors of chromosomal markers. The kinetics of entry as well as the frequencies of transfer of chromosomal markers indicate that thr(+) and leu(+) enter the recipient as proximal markers and that lac(+) enters as a distal marker. Analysis of the recombinants demonstrates close linkage between thr and leu, ade and thr, his and pat, and his and trp loci. The results suggest that the integration of F'lac(+) into the chromosome of E. chrysanthemi has occurred at a region adjacent to the leu-thr loci, and that the chromosome is transferred in the following sequence: origin----leu--thr--ade--lys--mcu--pat--his--trp--gal--gtu--lac--F. Plant-tissue maceration occurs in Pat(+) recombinants and not in Pat(-) recombinants, even though both form another pectolytic enzyme, hydrolytic polygalacturonase. This genetic evidence supports the idea that the E. chrysanthemi polygalacturonic acid trans-eliminase plays an essential role in bringing about plant-tissue maceration.

The spread ofErwinia carotovora var.atroseptica (blackleg) from degenerating seed to progeny tubers in soil

In 1976, spread of a serologically identifiable strain ofErwinia carotovora var.atroseptica was traced from inoculated ‘seed’ to progeny tubers of cv. Pentland Crown by placing inoculated tubers against seed tubers of plants in the field at four different dates from June to September. Progeny tuber samples were dug 2, 4 and 8 weeks after placement, induced to rot, and the bacterium responsible identified by immunodiffusion tests. Spread was not detected until 7 September following the onset of rain and cooler temperatures. Bacteria placed in the field in June were detected in November having survived the hot dry summer in the remains of inoculated tubers.

Genetic transfer of Pseudomonas aeruginosa R factors to plant pathogenic Erwinia species.

The R factors RP1, R68 and R91 were freely transmissible to and from Pseudomonas aeruginosa, Salmonella typhimurium, and various plant pathogenic Erwinia spp. The antibiotic resistance spectrum of R+ Erwinia recipients was similar to those of other bacteria harboring these R factors, but maximum resistance levels differed with each recipient. The sponstaneous elimination of these factors from the Erwinia strains and the ability to transfer multiple antibiotic resistance suggest that these exist as plasmids in these hosts. Several, but not all, RP1-carrying Erwinia strains were sensitive to the RP1 specific phage PRR1. The R factor R18-1 was also transferred from P. aeruginosa to Erwinia spp. R18-1 was unstable in all Erwinia strains. Stable strains were isolated in which R18-1 could not be eliminated by sodium dodecyl sulfate and could not be transferred to other strains.

Intrageneric clustering and divergence of Erwinia strains from plants and man in the light of deoxyribonucleic acid segmental homology.

The relationships among Erwinia species (originating from plants and man) and other enterobacteria were examined in terms of DNA–DNA segmental homology. All of the organisms studied show a "core homology" amounting to about 10–15%, thus supporting the inclusion of the erwinias in the taxonomic vicinity of the other enterobacteria, and also revealing that the present genus Erwinia is hardly separable from the other enterobacterial genera solely on the extent of DNA–DNA segmental homology. Genetic clusters based on DNA–DNA segmental homology are found within all of Dye's "natural" (phenotypic) groups of the genus Erwinia. However, these genetic clusters are not coextensive with any entire natural group, and designation of any of them as genera seems implausible at present. Except for a few cases of obvious synonymy, the several Erwinia nomenspecies generally showed significant nomen-species-specific segments of DNA, but a taxonomic revision at the species level based only on DNA–DNA segmental homology would seem premature. The Erwinia strains isolated from human clinical sources are essentially indistinguishable on the basis of DNA–DNA segmental homology from the epiphytic and related Erwinia strains derived from plants.

Estimating the density of individual bacterial populations introduced into natural ecosytems.

The abundance of individual strains of Xanthomonas, Erwinia, and Rhizobium added to soil was determined by a method that allows for the enumeration of surviving cells of the introduced population in the presence of many other soil microorganisms. The method should be useful for measuring numbers of other bacteria in heterogeneous communities.

Transmission of lac by the sex factor E in Erwinia strains from human clinical sources.

Lactose-utilizing (Lac(+)) strains of Erwinia spp. from human clinical material transfer lac by conjugation to plant strains of Erwinia herbicola and Erwinia amylovora, to other Erwinia strains from human clinical sources, and also to Escherichia coli, Paracolobactrum arizonae, Salmonella typhimurium, and Shigella dysenteriae. The frequency of this transfer varies with the donor and recipient strains employed. The lac genes appear stable in these exconjugants, and they are not cured by acridine orange. The Lac(+) exconjugants transfer lac to an Escherichia coli F(-) Lac(-) strain; the frequency of this transfer is high with E. herbicola and S. typhimurium exconjugants and relatively low with other exconjugants. The most studied Erwinia donor strain from human clinical material (EH133) and its Lac(+) exconjugants are insensitive to the F-specific phage, M13. P1-mediated transduction of lac, by using a Lac(+) exconjugant of E. coli as the donor and an E. coli F(-) Lac(-) strain as the recipient, revealed that all 50 Lac(+) transduced clones tested also inherited donor ability, suggesting a close linkage between the Erwinia sex factor (designated as E) and the lac genes. The E. coli culture harboring E-lac (E and the lac genes linked to it) does not restrict phages T1, T7, and lambdavir. E-lac is compatible with F'his, R100 drd-56 (F-like), and R64 drd-11 (I-like); cells harboring F'his or one of the R factors do not show super-infection immunity to the incoming E-lac, and E-lac plus one of the other plasmids can coexist stably in the same cell. The fertility of cells harboring F'his or R100 drd-56-as determined by the frequency of conjugal transfer of his or of the resistance determinant (Tet(r) in case of R100 drd-56) and also by sensitivity to F-specific phage (M13)-is not altered by the presence of E-lac, and this suggests that the sex factor E might belong to the fi(-) class.

Genetic transfer of episomic elements among Erwinia species and other enterobacteria: F'Lac+.

The episomic element F'lac(+) was transferred, probably by conjugation, from Escherichia coli to Lac(-) strains of Erwinia herbicola, Erwinia amylovora, and Erwinia chrysanthemi (but not to several other Erwinia spp. In preliminary trials). The lac genes in the exconjugants of the Erwinia spp. showed varying degrees of stability depending on the strain (stable in E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but markedly unstable in E. chrysanthemi strain EC16). The lac genes and the sex factor (F) were eliminated from the exconjugants by treatment with acridine orange, thus suggesting that both lac and F are not integrated in the Erwinia exconjugants. All of the tested Lac(+) exconjugants of E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but not of E. chrysanthemi strain EC 16, were sensitive to the F-specific phage M13. The heterogenotes (which harbored F'lac(+)) of E. herbicola strains Y46 and Y74, E. amylovora strain EA178, and E. chrysanthemi strain EC16 were able to transfer lac genes by conjugation to strains of E. herbicola, E. amylovora, E. chrysanthemi, Escherichia coli, and Shigella dysenteriae. The frequency of such transfer from Lac(+) exconjugants of Erwinia spp. was comparable to that achieved by using E. coli F'lac(+) as donors, thus indicating the stability, expression, and restriction-and-modification properties of the sex factor (F) in Erwinia spp.

Factors governing pectate lyase synthesis in soft rot and non-soft rot bacteria

Regulation of pectate lyase synthesis was examined in Erwinia and Pseudomonas species. Soft rot bacteria produced 100- to 1000-fold more enzyme per cell under optimal conditions than did other bacterial pathogens or saprophytes. Among pseudomonads, only Pseudomonas marginalis and a few other fluorescent strains isolated from plant tissue synthesized pectate lyase. Constitutive synthesis was common in this group. In contrast, lyase synthesis in most Erwinia species examined was inducible. Polypectate and pectin were effective carbon sources for lyase induction in some Erwinia strains while other strains showed much higher inducible synthesis on pectin than on polypectate. Vegetable extracts had profound effects on lyase synthesis. Potato extract, which was previously reported as exerting a synergistic effect on induction of pectate lyase in Erwinia carotovora ATCC 8061 grown on pectin, repressed lyase synthesis in other Erwinia strains. At high concentrations, extracts of carrot, lettuce and onion severely repressed constitutive and inducible synthesis. At low concentrations, lettuce and onion extracts stimulated constitutive lyase synthesis in Pseudomonas marginalis. The wide variety of regulatory controls governing lyase synthesis and the repressive as well as synergistic effects of vegetable extracts on synthesis suggest that the etiology of bacterial soft rot diseases can be influenced by such regulatory mechanisms.

Erwinia infection from environmental sources

To the Editor.— The recent findings of intravenous fluid contamination with Erwinia organisms has focused attention on these bacteria. At Yale-New Haven Hospital, Erwinia strains were isolated from blood cultures in 1966, 1969, and 1970. The three patients, two males and one female, were 17 (patients 1 and 2) and 28 (patient 3) years old, and had been admitted for reasons other than septicemia, as follows: transverse arrest (patient 1), diabetic ketoacidosis (patient 2), and injuries from a motorcycle accident (patient 3). Within 12 hours after admission, the first two patients experienced chills, tachycardia, and temperatures which rose rapidly to 102 to 103 F. The third patient had two temperature spikes to 105 F on two successive days. All had been given fluids intravenously (without catheter) prior to the temperature spikes, and all were treated promptly with antibiotics, as follows: patient 1 with streptomycin and penicillin, patient 2 with cephalothin,

Cephapirin: In Vitro Antibacterial Spectrum

Cephapirin, a new semisynthetic cephalosporin derivative, was found to have an antibacterial spectrum similar to that of cephalothin. Staphylococcus aureus was inhibited by cephapirin concentrations of 0.09 to 12.5 mug/ml. S. epidermidis, S. viridans, S. pyogenes, and Diplococcus pneumonia isolates were inhibited by less than 1 mug/ml. The Enterococcus required a concentration of 25 mug of antibiotic per ml for inhibition. Approximately 65% of Escherichia coli, and all Klebsiella, indole-negative Proteus, and Salmonella strains tested were inhibited by the drug. Serratia, Pseudomonas, indole-positive Proteus, and Erwinia strains were highly resistant. Inoculum size was not an important factor in determining the level of sensitivity of S. aureus to cephapirin. The antibiotic does not appear to be significantly bound to serum protein. In vitro development of resistance to the drug was demonstrated with two isolates of S. aureus.