Taxonomy and pathogenicity of Erwinia cacticida sp. nov.

A total of 108 pectolytic, soft-rotting Erwinia strains were collected from 11 types of cacti growing in Arizona, Texas, northern Mexico, and Australia between 1958 and 1989. Four strains were collected from soils beneath or close to naturally rotting saguaro cacti. Collectively, these strains caused soft rots of saguaro, organ pipe, and senita cacti, Opuntia (cactus) fruits and pads, tomato fruits, and potato slices, but only occasionally caused soft rots of slices of carrot roots. A numerical cluster analysis showed that 98 of the 112 strains formed a uniform group (cluster 1A) that was distinguished from other pectolytic erwinias by an API 20E code of 1205131, by negative reactions in API 50CHE tests for L-arabinose, myo-inositol, D-cellobiose, melibiose, and D-raffinose, and, in supplemental tests, by positive reactions for malonate and growth at 43 degrees C. The average levels of DNA relatedness of 22 cluster 1A strains to the proposed type strain (strain 1-12) as determined by the hydroxyapatite method were 88% in 60 degrees C reactions (with 1% divergence within related sequences) and 87% in 75 degrees C reactions. The levels of relatedness to the type strains of other Erwinia spp. were less than or equal to 38% in 75 degrees C reactions. Cluster 1A strains also had a characteristic cellular fatty acid profile containing cyclo-(11,12)-nonadecanoic acid (C19:0 Cyclo C11-12) and missing tridecanoic acid (C13:0), heptadecanoic acid (C17:0), and cis-9-heptadecenoic acid (C17:1 CIS 9), which separated them from other pectolytic erwinias. Collectively, these data indicate that the members of cluster 1A are members of a new species, which we name Erwinia cacticida. Three cactus strains in cluster 1B appear to represent a second new species that is closely related to E. cacticida; these strains are designated E. cacticida-like pending the availability of additional strains for testing. The remaining cactus strains (in cluster 4) have the physiological, DNA, and fatty acid profiles of Erwinia carotovora.

Identification of carotenoids in Erwinia herbicola and in a transformed Escherichia coli strain.

The yellow pigments of Erwinia herbicola Eho 10 and of a transformed Escherichia coli LE392 pPL376 have been identified as carotenoids. HPLC separation, spectra and in some cases mass spectroscopy demonstrated the presence of phytoene (15-cis isomer), beta-carotene (all-trans, 9-cis and 15-cis), beta-cryptoxanthin ( = 3-hydroxy beta-carotene), zeaxanthin (3,3'-dihydroxy beta-carotene) and corresponding carotene glycosides. In addition, lycopene and gamma-carotene accumulated in the presence of the inhibitor 2-(4-chlorophenylthio)-triethylamine.HCl. Carotenoid content in the transformed E. coli was two-fold higher than in E. herbicola. The pattern of the carotenoids was similar in the two organisms. Inactivation of the katF gene in E. coli resulted in an 85% lowering of carotenoid formation, as did the addition of 0.5% glucose to the medium. Suppression of carotenoid formation by inactivation of the katF gene lowered, but did not abolish, the protection offered by carotenoids against inactivation by alpha-terthienyl plus near-ultraviolet light (320-400 nm).

[Polyamines in representative taxa of coryneform and other bacteria]. / Poliaminy predstavitelei nekotorykh taksonov korinepodobnykh i drugikh bakterii.

The composition of polyamines is studied for the first time in representatives of the genus Micrococcus and taxon "conglomeratus", strains Staphylococcus aureus CCM 209, Deinococcus erythromyxa CCM 706 as well as of Erwinia carotovora ATCC 15713 polyamines, which are not extracted by perchloric acid. Considerable amounts of spermine and rarely of spermidine are revealed in cells of Gram positive microorganisms, that differs them from Gram negative bacteria possessing high concentrations of putrescine, spermidine and their derivatives. A procedure is developed for detection of polyamines in cells of Gram positive microorganisms. It is recommended to use the hydrolysis of their cells by 6N HCl for 4 at 120 degrees C or for 8-10h at 100 degrees C with the subsequent electrophoretic separation. Putrescine, as well as comparable with it amount of agmatine and spermidine traces are found in Erwinia carotovora ATCC 15713 cell hydrolyzates, whereas putrescine and agmatine traces are found in perchloric extracts of intact cells. Spermine is not observed in the cells. The binding of polyamines with biopolymers of cells of Gram positive bacteria and their difference by the given character from the Gram negative procaryotes are under discussion.

Isolation, characterization, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi.

A catechcol-type siderophore, assigned the trivial name chrysobactin, was isolated from the phytopathogenic bacterium Erwinia chrysanthemi and characterized by degradation and spectroscopic techniques as N-[N2-(2,3-dihydroxybenzoyl)-D-lysyl]-L-serine. Chrysobactin, which was also obtained by chemical synthesis, was shown to be active in supplying iron to a group of mutants of E. chrysanthemi defective in biosynthesis of the siderophore.

Bacteriophage T4 resistant mutants of the plant pathogen Erwinia carotovora.

Bacterial lipopolysaccharide (LPS) has been implied in a variety of pathogenic and symbiotic plant-bacterium interactions. In order to study the role of LPS in pathogenicity of Erwinia carotovora, a broad host range phytopathogen, we isolated LPS defective mutants of two subspecies of Erwinia carotovora, subsp. carotovora (Ecc) and subsp. astroseptica (Eca). This was accomplished by using the Escherichia coli phage T4 as a selective agent. Screening of Erwinia isolates revealed that some of them were sensitive to T4 and thus T4 could be employed in mutant isolation. Fully T4 resistant mutants were all shown to be defective in their LPS structure. Preliminary pathogenicity tests on tobacco did not, however, reveal any decrease in the virulence of the LPS defective strains.

Immunological characterization of ice nucleation proteins from Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola.

Antibodies were raised against the InaW protein, the product of the ice nucleation gene of Pseudomonas fluorescens MS1650, after protein isolation from an Escherichia coli clone. On Western blots (immunoblots), these antibodies recognized InaW protein and InaZ protein (the ice nucleation gene product of Pseudomonas syringae S203), produced by both E. coli clones and the source organisms. The InaZ protein appeared in P. syringae S203 during stationary phase; its appearance was correlated with the appearance of the ice nucleation-active phenotype. In contrast, the InaW protein occurred at relatively constant levels throughout the growth phases of P. fluorescens MS1650; the ice nucleation activity was also constant. Western analyses of membrane preparations of P. syringae PS31 and Erwinia herbicola MS3000 with this antibody revealed proteins which were synthesized with development of the nucleating phenotype. In these species the presence or absence of the nucleating phenotype was controlled by manipulation of culture conditions. In all nucleation-positive cultures examined, cross-reacting low-molecular-weight bands were observed; these bands appeared to be products of proteolytic degradation of ice nucleation proteins. The proteolysis pattern of InaZ protein seen on Western blots showed a periodic pattern of fragment sizes, suggesting a highly repetitive site for protease action. A periodic primary structure is predicted by the DNA sequence of the inaZ gene.

Characterization and virulence properties of Erwinia chrysanthemi lipopolysaccharide-defective, phi EC2-resistant mutants.

Outer membrane alterations were characterized in spontaneous mutants of the Erwinia chrysanthemi 3937jRH, which were selected for resistance to bacteriophage phi EC2. All but one of the mutants analyzed were affected in their lipopolysaccharide (LPS) structure, lacking the entire heterogeneous region of apparent high molecular weight present in the wild-type E. chrysanthemi LPS. At least two 3937jRH mutants, one selected as phi EC2 resistant (RH6065) and the other previously selected (D. Expert and A. Toussaint, J. Bacteriol. 163:221-227, 1985) as bacteriocin resistant (R1456), were cross-resistant to bacteriophage Mu and had rough LPSs with an altered core structure. Two phi EC2r mutants (RH6053 and RH6065) were most severely affected in their outer membrane integrity and also lost their virulence on saintpaulia plants, although they still possessed normal extracellular levels of pectinolytic and cellulolytic activities. The two Mur mutants RH6065 and R1456 were also able to induce systemic resistance in the challenged plant. All the other phi EC2r mutants retained the virulence of 393jRH.

Characterization of type 1 and mannose-resistant fimbriae of Erwinia spp.

Type 1 fimbriae from Erwinia carotovora subsp. carotovora and mannose-resistant fimbriae from Erwinia rhapontici were purified and characterized. The type 1 fimbrillin had an apparent molecular weight of 16,500; that of the mannose-resistant fimbrillin was 18,000. The amino-terminal amino acid sequences of the two fimbrillins were related, but tryptic peptide maps showed significant differences between the proteins. No serological cross-reaction was found between the two fimbrial filaments, nor did they cross-react with type 1 or type 3 fimbriae purified from other enterobacterial species. Immunofluorescent staining of bacterial populations revealed that they were heterogeneous with respect to fimbriation.

Structure of the core oligosaccharide from lipopolysaccharide of Erwinia carotovora.

The lipopolysaccharide of Erwinia carotovora was analyzed by quantitative sugar analysis, methylation analysis, and chromic oxide oxidation. This led to the following structure of the core oligosaccharide: (Formula; see text).

Fructan from Erwinia herbicola.

Levan production by strains of Erwinia herbicola is common, and this property has some taxonomic significance for species differentiation within the "herbicola" group. The extracellular polysaccharide elaborated by strain 403 was characterized by nuclear magnetic resonance spectroscopy and methylation analysis. Results showed it to be a typical bacterial levan.

Characterization of plasmids in Erwinia stewartii.

Plasmids in 39 strains of Erwinia stewartii were examined by agarose gel electrophoresis. Most virulent strains had from 11 to 13 plasmids ranging in molecular mass from 2.8 to 210 megadaltons and contained plasmids of 210, 70, 49, 43, 29.5, 16.8, 8.8, and 2.8 megadaltons. Plasmids in strains SW2 and SS104 were characterized by both electron microscopy and agarose gel electrophoresis and may be useful as convenient references for sizing plasmids by electrophoresis. Specific size classes of plasmids could not be associated with antibiotic and heavy metal resistance, carbohydrate utilization, bacteriocin production, or pathogenicity to corn. However, avirulent strains tended to have fewer plasmids than virulent strains.

[Some properties of bacteriocins from Erwinia]. / Nekotorye svoistva bakteriotsinov Erwinia.

The following properties of eight bacteriocins produced by Erwinia strains were investigated: thermal stability, sensitivity to proteolytc enzymes and enzymes involved in nucleic acid catabolism, dialyzability through a cellophane membrane. Their molecular weight was also measured. The bacteriocins proved to be protein substances with a molecular weight of 17,000--33,000 that differed in their thermal stability and sensitivity to proteolytic enzymes.

Herbicolins--New peptide antibiotics from Erwinia herbicola.

Erwinia herbicols (strain A 111) produces two acylated peptide antibiotics herbicolins A and B. Isolation of herbicolins was performed by adsorption on a polystryrol adsorbent followed by elution with methanol. Further purification was achieved by gel filtration on Sephadex LH-20, counter-current distribution or by TLC. Herbicolin A was chemically characterized, containing 2 glycines, 1 L-threonine, 1 D-allo-threonine, 1 D-glutamic acid, 1 D-leucine, 1 L-agrinine and beta-hydroxy myristic acid. Herbicolins A and B are inactive against bacteria, but highly active against yeasts and filamentous fungi.