pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora.

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

Utilization of plasmid pULB113 (RP4::mini-Mu) to construct a linkage map of Erwinia carotovora subsp. chrysanthemi.

We report experimental evidence that pULB113, an RP4::mini-Mu plasmid, mediates chromosome transfer in a strain of Erwinia carotovora subsp. chrysanthemi which does not accept the F episome. This allowed us to construct a genetic map of that strain by measuring the frequencies of cotransfer of different markers (thy, leu, pro, [his, trp], thyA, rpsL, ile).     

Isolation and characterization of Hfr strains of Erwinia amylovora

Hfr strains (Hfr 159 and its derivatives, Hfr 160 and Hfr 161) were constructed from Erwinia amylovora ICPB EA178 by introducing an Escherichia coli F'his+ plasmid and then selecting for integration of F'his+ after treatment with acridine orange. The Hfr strains were relatively stable upon repeated transfers on nonselective media. Interrupted mating experiments and analyses of inheritance of unselected markers showed that his+ is transferred by Hfr 159 as the proximal marker at a relatively high frequency (about 5 x 10(-4) recombinants per input donor cell), followed by ilv+, orn+, arg+, pro+, rbs+, met+, trp+, leu+, ser+, and thr+ (not necessarily in that precise order). The donor strains, previously constructed in E. amylovora by integration of F'lac+ from E. coli transfer cys+ as the proximal marker followed by ser+. Further analysis of one of those earlier donor strains, Hfr99, showed that ser+ is followed by arg+, orn+, met+, pro+, leu+, ilv+, rbs+, his+, trp+, and thr+ (not necessarily in that precise order). Thus, the Hfr strains constructed by integration of F'his+ are different, in terms of origin and direction of transfer, from those derived from integration of F'lac+. The applicability of these Hfr strains to mapping the genes on the E. amylovora chromosome is indicated.     

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.     

Characteristics of transduction in Erwinia by phage 59

A temperate bacteriophage 59 from polylysogenic strain Erwinia carotovora 268 transduces the following genetic markers: arg+, ilv+, leu+, met+, thr+, thy+, trp+, ura+. The transduction frequencies varied from 1 x 10(-8)- to 1 x 10(-6) and dependent on the multiplicity of infection, UV-irradiation of transducing bacteriophage, the nature of phage lysates. The characteristics of single transductants have been studied.Analysis of the obtained results suggests bacteriophage 59 to perform the generalized transduction.     

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.     

Utilization of a thermosensitive episome bearing transposon TN10 to isolate Hfr donor strains of Erwinia carotovora subsp. chrysanthemi

A thermosensitive episome bearing the transposon Tn10, F(Ts)::Tn10 Lac+, has been successfully transferred from Escherichia coli to several wild strains of the enterobacteria Erwinia carotovora subsp. chrysanthemi, which are pathogenic on Saintpaulia ionantha. In one of these strains, all of the characters controlled by this episome (Lac+, Tetr, Tra+) were expressed, and its replication was stopped at 40 degrees C and above. At 30 degrees C, the episome was easily transferred between strains derived from E. carotovora subsp. chrysanthemi 3937j and to E coli. Hfr donor strains were obtained from a F' strain of 3937j by selecting clones which grew at 40 degrees C on plates containing tetracycline. One of these strains, Hfrq, was examined in more detail: the characters Lac+ and Tetr were stabilized and did not segregate higher than its parental F' strain. The mating was most efficient at 37 degrees C on a membrane. Hfrq transferred its chromosome to recipient strains at high frequency and in a polarized fashion, as evidenced by the gradient of transfer frequencies, the kinetics of marker entry (in interrupted mating experiments), and the analysis of linkage between different markers. The chromosome of Hfrq was most probably transferred in the following sequence: origin...met...xyl...arg...ile...leu...thr...cys...pan...ura...gal...trp...his. ..pur... Moreover, this genetic transfer system proved to be efficient in strain construction.     

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.     

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)     

Translocation of transposons Tn10 and Tn5 in the Yersinia pestis chromosome

The possibility of translocation of the transposons Tn5 and Tn10 into the genome of Yersinia pestis, with the subsequent mutagenic effect was demonstrated. We revealed transposon harbouring clones at frequency 10(-4) to 10(-2). Derivatives of P1cml clr100ts phage served as vectors. Insertion of Tn10 transposon induced mutations in ilv, ser, arg, pur, pro, leu, nic, tyr, gua genes. The number of the insertion sites on the chromosome obtained for Tn5 was the same, these being arg, ade, pyr, leu, gua, trp, his, pan, ilv. The majority of auxotrophs did not revert. Occasionally, revertants were observed at frequencies 10(-8) to 10(-6). Unlike Escherichia coli, reversion was not accompanied by the loss of transposons. The rearrangements induced by transposons, presumably, near the insertion site, as well as duplications of transposons followed by incorporation of copies into novel sites, led to the appearance of additional defective genes, which made it possible to select various types of polyauxotrophs. Based on reiteration of coinciding double and triple mutant markers, we proposed a linkage group of genes within a segment of Y. pestis chromosome: lys ... tyr - ser - arg - ilv - leu - gua - ade(pur) - pro ... his ... pyr ... trp. The reasons for peculiarities of the behaviour of transposons in Y. pestis bacteria are discussed.     

Presence and population dynamics of Erwinia carotovora in irrigation water in south central Colorado

The presence of Erwinia carotovora in surface and underground (well) water was studied using filter concentration and anaerobic enrichment techniques. The organism was found in water samples collected at sites in mountainous (over 80 km from potato-producing regions), transitional (upland) and arable regions every month in 1982 and 1983. Filter concentration and anaerobic enrichment of 3-10 1 of water yielded E. carotovora from 82.8% of the water samples collected from streams, canals and lakes. The organism was detected by direct enrichment of 50 ml water samples in 56.3% of surface water samples collected. Erwinia carotovora subsp. carotovora was the predominant subspecies isolated. Of 1029 strains, 999 (97.1%) were identified as E. carotovora subsp. carotovora and 30 (2.9%) as E. carotovora subsp. atroseptica. Erwinia carotovora subsp. atroseptica was found primarily in water samples collected in arable regions during spring months. Erwinia chrysanthemi was never isolated. Quantitative bacteriological methods were used in 1982 and 1983 to monitor populations of E. carotovora in two streams in south central Colorado. These ranged from undetectable levels to 8.5 cfu/ml of water in Rio Grande River and Saguache Creek. Maximum populations were usually reached by August or September in both streams in both years. Erwinia carotovora was isolated from well water samples collected in the San Luis Valley, but only 15.6 and 15.4% of the samples yielded the organism during 1982 and 1983, respectively. Erwinia carotovora subsp. atroseptica was found only once, and E. carotovora subsp. carotovora was the predominant subspecies detected. Filter concentration of 3.4-10.0 1 of water plus anaerobic enrichment of the samples was usually necessary to detect E. carotovora in well water.     

Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp. atroseptica: identification of kduD gene

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-mini-Tn5xylE transposon into the chromosome of phytopathogenic bacteria Erwinia carotovora subsp. atroseptica. The inability of mutant cells to utilize these substrates was caused by a failure to accomplish the catabolism of unsaturated digalacturonic acid (UDA). Study of enzymatic activities has established that mutant bacteria lost the ability to produce 2,5-diketo-3-deoxygluconate dehydrogenase, an enzyme of intracellular UDA utilization. Molecular cloning of the mutant gene was conducted, and its nucleotide sequence was determined. It was shown that the nucleotide sequence of this gene had an 82% homology with the sequence of Erwinia chrysanthemi EC3937 kduD gene encoding 2,5-diketo-3-deoxygluconate dehydrogenase. The intergene kdul-kduD region in bacteria Erwinia carotovora subsp. atroseptica is shorter in length by 98 nucleotides than the corresponding region of Erwinia chrysanthemi and does not contain promoter sequences. The kduD gene was located at 126.8 min of the Erwinia carotovora subsp. atroseptica genetic map.     

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 alpha-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.     

Involvement of N-acylhomoserine lactones throughout plant infection by Erwinia carotovora subsp. atroseptica (Pectobacterium atrosepticum)

Erwinia carotovora subsp. atroseptica is responsible for potato blackleg disease in the field and tuber soft rot during crop storage. The process leading to the disease occurs in two phases: a primary invasion step followed by a maceration step. Bacteria-to-bacteria communication is associated with a quorum-sensing (QS) process based on the production of N-acylhomoserine lactones (HSL). The role of HSL throughout plant infection was analyzed. To this purpose, HSL produced by a specific E. carotovora subsp. atroseptica wild-type strain, which was particularly virulent on potato, were identified. A derivative of this strain that expressed an HSL lactonase gene and produced low amounts of HSL was generated. The comparison of these strains allowed the evaluation of the role of HSL and QS in disease establishment and development. Bacterial growth and motility; activity of proteins secreted by type I, II, and III systems; and hypersensitive and maceration reactions were evaluated. Results indicated that HSL production and QS regulate only those traits involved in the second stage of the host plant infection (i.e., tissue maceration) and hypersensitive response in nonhost tobacco plants. Therefore, the use of QS quenching strategies for biological control in E. carotovora subsp. atroseptica cannot prevent initial infection and multiplication of this pathogen.     

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.     

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.     

Haemagglutinins and fimbriae of soft rot Erwinias.

Strains of phytopathogenic soft rot Erwinia spp. were examined for haemagglutinin (HA) production. Mannose-sensitive HA was found only in five of 15 strains of E. carotovora subsp. carotovora. Mannose-resistant HA (MRHA) was found in 12 of 15 strains of E.c. carotovora, ten of 13 strains of E.c. subsp. atroseptica and the single strain of E.c. subsp. betavasculorum, as well as all seven strains of E. chrysanthemi. MRHA, detectable only in a microtitre tray HA assay was of either broad- or narrow-spectrum activity when examined against blood of seven different animal species and could be inhibited by the beta-galactoside asialofetuin. Fimbriae of ca 10 nm diameter were found on MRHA(+) bacteria E.c. carotovora and E.c. atroseptica.     

Presence of Erwinia carotovora in surface water in North America

Erwinia carotovora was frequently isolated from samples of surface water collected from 66 rivers, springs, creeks, streams, lakes, reservoirs and ponds in 16 states in the US but was not found in the single fresh water sample collected in Canada. The organism was also isolated from water collected from the Pacific Ocean and the Gulf of Mexico. In Colorado and Wyoming, E. carotovora was isolated from water samples nearly every month of the year when monthly samples were collected from several streams. Erwinia carotovora subsp. carotovora represented 98–8% of the strains recovered from the water samples; E. carotovora subsp. atroseptica made up the remainder of the strains; E. chrysanthemi was not found.