Application of Amplified Fragment Length Polymorphism Fingerprinting for Taxonomy and Identification of the Soft Rot Bacteria Erwinia carotovora and Erwinia chrysanthemi

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.     

External Loops at the C Terminus of Erwinia chrysanthemi Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.     

Role of Antibiosis in Competition of Erwinia Strains in Potato Infection Courts

Erwinia carotovora subsp. betavasculorum strains produced a bactericidal antibiotic in vitro that inhibited a wide spectrum of gram-negative and gram-positive bacteria. The optimum temperature for production was 24°C, and the addition of glycerol to culture media enhanced antibiotic production. Antibiotic production by these strains in the infection court of potato was the principal determinant enabling it to gain ascendancy over competing antibiotic-sensitive Erwinia carotovora subsp. carotovora strains. There was a complete correlation between antibiotic production by E. carotovora subsp. betavasculorum in vitro and inhibition of competing E. carotovora subsp. carotovora strains in planta. Inhibition of the latter by the former was apparent after 10 h of incubation in potato tuber wounds. Population densities of sensitive E. carotovora subsp. carotovora strains in mixed potato tuber infections with E. carotovora subsp. betavasculorum were approximately 10⁶-fold lower after 48 h of incubation than in corresponding single sensitive strain infections. E. carotovora subsp. carotovora were not inhibited in tuber infections that were incubated anaerobically. This correlated with the absence of antibiotic production during anaerobic incubation in vitro. Antibiotic-resistant strains of E. carotovora subsp. carotovora were not inhibited in planta or in vitro by E. carotovora subsp. betavasculorum. Moreover, isogenic antibiotic-negative (Ant⁻) mutant E. carotovora subsp. betavasculorum strains were not inhibitory to sensitive E. carotovora subsp. carotovora strains in tuber infections.     

Yeastlike fungi from greater wax moth larvae (Galleria mellonella) fed antibiotics

The effect of the antibiotics oxytetracycline, chloramphenicol, and penicillin on the gut flora of Galleria mellonella larvae was not only to suppress Streptococcus faecalis, a typical gut organism of all stages of the moth, but also simultaneously to cause an increase in the number of yeastlike fungi, Candida guilliermondi, Candida krusei, and Geotrichum candidum. Nystatin prevented or minimized yeast multiplication. Most pupae and adults were sterile or contained only S. faecalis, even when prepupae had contained many fungi. A combination of oxytetracycline-nystatin in a total dosage of 1 and 3 × MIC/cm² of honeycomb surface, respectively, reduced both S. faecalis and fungal counts, so that after a 3-day incubation, most of the larvae were sterile or near sterile.     

Bacteriostatic effects of cerium-humic acid complex

The bacteriostatic potency of the cerium-humic acid complex was evaluated by experimental measurement of this complex interaction with E. coli, Bacillus pyocyaneus, Staphylococcus aureus, Leuconostoc and Streptococcus faecalis, and by comparison bacteriostatic effects with the cerium-citrate complex. The experimental results indicated that the cerium-humic acid complex strongly inhibited growth of all five bacterial strains, and its diameter of bacteriostatic circles were more than 30 mm. The minimal bacteria-inhibiting concentration were 1×10⁻³, 2×10⁻³ and 1×10⁻² mol/L for E. coli and Bacillus pyocyaneus, Staphylococcus aureus, and Leuconostoc and Streptococcus faecalis individually, and the measured minimal bactericidal concentrations were 2×10⁻³ and 1×10⁻² mol/L for Bacillus pyocyaneus, E. coli, and Leuconostoc. To kill Staphylococcus aureus and Streptococcus faecalis, the concentration had to be more than 1×10⁻² mol/L. On the contrary, we found that cerium-citrate complex did not inhibit the growth of the above five bacteria, but stimulated bacterial growth. The completely different bacteriostatic results of two cerium complexes may hint that the association and chemical properties of the two complexes were different.     

Biosynthetic features and properties of xylose isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovora subsp. atroseptica

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovora subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria produced the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovora subsp. atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the D-xylose-containing nutrient medium of 0.1–1.5% D-glucose did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovora subsp. atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.     

Genetically Engineered Erwinia carotovora in Aquatic Microcosms: Survival and Effects on Functional Groups of Indigenous Bacteria

The survival of genetically engineered Erwinia carotovora L-864, with a kanamycin resistance gene inserted in its chromosome, was monitored in the water and sediment of aquatic microcosms. The density of genetically engineered and wild-type E. carotovora strains declined at the same rate, falling in 32 days below the level of detection by viable counts. We examined the impact of the addition of genetically engineered and wild-type strains on indigenous bacteria belonging to specific functional groups important in nutrient cycling. For up to 16 days, the densities of total and proteolytic bacteria were significantly higher (P < 0.05) in microcosms inoculated with genetically engineered or wild-type E. carotovora, but by 32 days after inoculation, they had decreased to densities similar to those in control microcosms. Inoculation of genetically engineered or wild-type E. carotovora had no apparent effect on the density of amylolytic and pectolytic bacteria in water and sediment. Genetically engineered and wild-type E. carotovora did not have significantly different effects on the densities of specific functional groups of indigenous bacteria (P > 0.05).     

Gut flora of Galleria mellonella suppressing ingested bacteria.

Streptococcus faecalis, the only bacterium occurring almost invariably at high populations in guts of Galleria mellonella larvae, suppresses bacteria ingested with food by producing bacteriocin, an antibioticlike substance having a narrow range of bactericidal activity, and by releasing a lysozymelike enzyme, especially in the presence of proteolytic enzymes. The insect intestinal fluid apparently increases the activity of S. faecalis lytic enzyme. Unlike other organisms tested, S. faecalis has shown a strong bactericidal action against various species of unrelated bacteria. Microscopical examination of the sensitive organisms used as indicators has revealed changes resembling formation of protoplasts, gradually leading to destruction of bacterial cells. The insect guts could not be infected, even when the larvae had ingested a high dose of Pseudomonas aeruginosa, Proteus mirabilis, or Bacillus thuringiensis. The mechanism by which S. faecalis could suppress the ingested bacteria is suggested.     

Derivation of Mutants of Erwinia carotovora subsp. betavasculorum Deficient in Export of Pectolytic Enzymes with Potential for Biological Control of Potato Soft Rot

Erwinia carotovora subsp. betavasculorum Ecb168 produces an antibiotic(s) that suppresses growth of the related bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. Strain Ecb168 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet. Genes (out) involved in secretion of pectolytic enzymes by Ecb168 were localized to two HindIII fragments (8.5 and 10.5 kb) of Ecb168 genomic DNA by hybridization to the cloned out region of E. carotovora subsp. carotovora and by complementation of Out^- mutants of E. carotovora subsp. carotovora. Out^- mutants of Ecb168, which did not secrete pectate lyase into the culture medium, were obtained when deletions internal to either HindIII fragment were introduced into the genome of Ecb168 through marker exchange mutagenesis. Out^- mutants of Ecb168 were complemented to the Out⁺ phenotype by introduction of the corresponding cloned HindIII fragment. Out^- mutants of Ecb168 were less virulent than the Out⁺ parental strain on potato tubers. Strain Ecb168 and Out^- derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating that the uncharacterized antibiotic(s) responsible for antagonism was exported through an out-independent mechanism. Strain Ecb168 and Out^- derivatives reduced the establishment of large populations of E. carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovora subsp. carotovora.     

Characterization of Monoclonal Antibodies Specific for Erwinia carotovora subsp. atroseptica and Comparison of Serological Methods for Its Sensitive Detection on Potato Tubers

Seven monoclonal antibodies (MAbs) to Erwinia carotovora subsp. atroseptica have been produced. One, called 4G4, reacted with high specificity for serogroup I of E. carotovora subsp. atroseptica, the most common serogroup on potato tubers in different serological assays. Eighty-six strains belonging to different E. carotovora subsp. atroseptica serogroups were assayed. Some strains of serogroup XXII also reacted positively. No cross-reactions were observed against other species of plant pathogenic bacteria or 162 saprophytic bacteria from potato tubers. Only one strain of E. chrysanthemi from potato cross-reacted. A comparison of several serological techniques to detect E. carotovora subsp. atroseptica on potato tubers was performed with MAb 4G4 or polyclonal antibodies. The organism was extracted directly from potato peels of artificially inoculated tubers by soaking or selective enrichment under anaerobiosis in a medium with polypectate. MAb 4G4 was able to detect specifically 240 E. carotovora subsp. atroseptica cells per ml by indirect immunofluorescence and immunofluorescence colony staining and after soaking by ELISA-DAS (double-antibody sandwich enzyme-linked immunosorbent assay) after enrichment. The same amount of cells was detected by using immunolectrotransfer with polyclonal antibodies, and E. carotovora subsp. atroseptica and subsp. carotovora were distinguished by the latter technique. ELISA-DAS using MAb 4G4 with an enrichment step also efficiently detected E. carotovora subsp. atroseptica in naturally infected tubers and plants.     

Association of Bacteria with Marine Invertebrates: Implications for Ballast Water Management

Bacteria associated with plankton are of importance in marine bioinvasions and the implementation of ship’s ballast water treatment technologies. In this study, epibiotic and endobiotic bacteria associated with zooplankton, including barnacle nauplii, veliger larvae, and adults of the copepod Oithona sp., were characterized and quantified. Barnacle nauplius and veliger larva harbored ~4.4 × 10⁵ cells ind^?1 whereas Oithona sp. had 8.8 × 10⁵ cells ind^?1. Computation of bacterial contribution based on biovolume indicated that despite being the smallest zooplankton tested, veliger larvae harbored the highest number of bacteria, while barnacle nauplii, the largest of the zooplankton, tested in terms of volume contributed the least. Pulverization of zooplankton led to an increase in bacterial numbers; for example, Vibrio cholerae, which was initially 3.5 × 10³, increased to 5.4 × 10⁵ CFU g^?1; Escherichia coli increased from 5.0 × 10² to 1.3 × 10⁴ CFU g^?1; and Streptococcus faecalis increased from 2.1 × 10² to 2.5 × 10⁵ CFU g^?1, respectively. Pulverized zooplankton was aged in the dark to assess the contribution of bacteria from decaying debris. Aging of pulverized zooplankton led to emergence of Chromobacterium violaceum, which is an opportunistic pathogen in animals and humans.     

A single gene that promotes interaction of a phytopathogenic bacterium with its insect vector,Drosophila melanogaster

Insects are major vectors of plant and animal disease, and bacterial phytopathogens are often disseminated by flies. We have previously reported that some isolates of the phytopathogenic bacterial species Erwinia carotovora infect Drosophila and activate an immune response. Using a genetic screen, we have now identified two genes that are required by E. carotovora to infect Drosophila. One of these genes has a regulatory role whereas the other, evf, confers an infectious phenotype: its transfer to non-infectious Erwinia strains or to several enterobacteria improves survival in the gut and triggers the immune response. Overexpression of Erwinia virulence factor (evf) allowed bacteria to colonize the apical side of the gut epithelium and in some cases to spread to the body cavity. Our results demonstrate a specific interaction between plant pathogens and flies that promote their dissemination.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Two Genomic Regions Involved in Catechol Siderophore Production by Erwinia carotovora

Two regions involved in catechol biosynthesis (cbs) of Erwinia carotovora W3C105 were cloned by functional complementation of Escherichia coli mutants that were deficient in the biosynthesis of the catechol siderophore enterobactin (ent). A 4.3-kb region of genomic DNA of E. carotovora complemented the entB402 mutation of E. coli. A second genomic region of 12.8 kb complemented entD, entC147, entE405, and entA403 mutations of E. coli. Although functions encoded by catechol biosynthesis genes (cbsA, cbsB, cbsC, cbsD, and cbsE) of E. carotovora were interchangeable with those encoded by corresponding enterobactin biosynthesis genes (entA, entB, entC, entD, and entE), only cbsE hybridized to its functional counterpart (entE) in E. coli. The cbsEA region of E. carotovora W3C105 hybridized to genomic DNA of 21 diverse strains of E. carotovora but did not hybridize to that of a chrysobactin-producing strain of Erwinia chrysanthemi. Strains of E. carotovora fell into nine groups on the basis of sizes of restriction fragments that hybridized to the cbsEA region, indicating that catechol biosynthesis genes were highly polymorphic among strains of E. carotovora.     

Effects of fluorescent pseudomonads on the potato blackleg syndrome

In four field trials in 1983 and 1984 potato tubers were inoculated by dipping them at planting in a suspension of Erwinia carotovora subsp. atroseptica and then treated with a powder formulation of two strains of fluorescent pseudomonads (B 10 and I 13) isolated from potatoes. lnoculation with E. carotovora increased blackleg and reduced emergence, plant growth, tuber size and weight compared with uninoculated controls. These effects were partially reversed by treatment of tubers with fluorescent pseudomonads which also reduced contamination by E. carotovora and the soft-rot potential of progeny tubers. In some trials a mixture of both pseudomonad isolates delayed the breakdown of the mother tuber although individual treatments did not.     

Streptococcus faecalis,Bacillus alvei, and sacbrood virus in European foulbrood of the honey bee

Contrary to reports from elsewhere, Streptococcus faecalis or Bacillus alvei did not cause European foulbrood in bee larvae also inoculated with sacbrood virus. The larvae died of sacbrood, by which time S. faecalis had mostly disappeared, although B. alvei multiplied saprophytically, as in European foulbrood, in some of the remains. Larvae that died of sacbrood already contained much sacbrood virus before they were sealed in their cells, when they appeared unaffected by the virus, but when they are most likely to die of European foulbrood, which is caused by Streptococcus pluton, often accompanied by secondary invaders, such as S. faecalis. Therefore, larvae killed by European foulbrood can be expected sometimes to contain much sacbrood virus, particularly as this virus is common.     

Saprozoic Nematodes as Carriers and Disseminators of Plant Pathogenic Bacteria

The plant pathogenic bacteria Agrobacterium tumefaciens (Smith and Townsend) Conn. (strain 5-14 Deep), Erwinia amylovora (Burill) Winslow et al., E. carotovora (Jones) Holland and Pseudornonas phaseolicola (Burk.) Dows. (ICPB-PM3) and the red-pigmented non-pathogen Serratia marcescens Bizio were hosts for the saprozoic nematode Pristionchus Iheritieri (Maupas, 1919) Paramonov. Viable bacteria survived passage through the nematode and produced typical colonies on nutrient agar plates. Female nematodes ingested more bacterial cells and retained them longer than did males. It was hypothesized saprozoic nematodes may disseminate pathogenic bacteria to new infection courts.     

Rapid Identification and Differentiation of the Soft Rot Erwinias by 16S-23S Intergenic Transcribed Spacer-PCR and Restriction Fragment Length Polymorphism Analyses

Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the α-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten “atypical” E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two “atypical” E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica.     

Lysozymelike lytic enzyme of Streptococcus faecalis and its role in the larval development of wax moth, Galleria mellonella

The predominant type of bacteria present in the gut of larval Galleria mellonella were streptococci group D identified as Streptococcus faecalis which showed bacteriolytic activity. Young larvae usually contained mixed populations with a marked dominance of fecal streptococci while normally developed mature larvae most frequently contained large uniform populations of S. faecalis. Pupal stages were found to contain the highest percentage of individuals with pure cultures of fecal streptococci.The author suggests a hypothesis that, owing to its bacteriolytic properties, S. faecalis can be considered as a component of the natural, nonspecific defense mechanism of G. mellonella against bacterial infections. The lytic enzyme released in the exponential growth phase of S. faecalis participates in the selection process stabilizing the microbial flora of wax moth larvae; it limits the population of other forms of bacteria. Larval resistance to bacterial infections to a large extent depends on the magnitude of the populations and thus on S. faecalis muramidase concentration. Bacterial lysozyme inhibited the growth of the ingested organisms and in consequence it prevented the proliferation of undesired bacteria in the digestive tract of Galleria larvae.The lytic enzyme proved to be identical with autolysin, a β-N-acetylmuramide glycanhydrolase (EC 3.2.1.17) which has been isolated from trypsin-speeded wall autolysates of S. faecalis by Shockman and Cheney (1969).     

The araC gene of Citrobacter freundii

The araC gene of Citrobacter freundii was cloned into plasmid pBR322 and expressed in Escherichia coli and Salmonella typhimurium. The nucleotide sequence and the predicted translational product were determined and compared to those of E. coli, S. typhimurium and Erwinia carotovora. The predicted translational product is 281 amino acids (aa) long, identical in size to that of S. typhimurium, and is 11 and 29 aa shorter than that of E. coli and E. carotovora, respectively. The nucleotide sequence of the araC gene of C. freundii is 83% homologous to the araC genes of both E. coli and S. typhimurium, but only 60% homologous to that of E. carotovora with respect to the regions they share. The predicted amino acid sequence is highly conserved and shows 96% and 94% homology to S. typhimurium and E. coli, respectively. E. carotovora shows only a 58% aa homology. The activator and autoregulatory activities of each plasmid encoded AraC protein in a S. typhimurium araC::lacZ protein fusion strain were examined.