Fimbriae and Haemagglutinins in Erwinias of the Carotovora Group

1. Thirteen Erwinia carotovora strains produced mannose-sensitive haemagglutinins associated with the presence of type-1 fimbriae.
2. Three fimbriate Er. rhapontici strains produced haemagglutinins of the mannose-resistant and eluting (MRE) type.
3. There were 12 Er. carotovora , 12 Er. atroseptica and 2 Er. chrysanthemi strains that produced no detectable haemagglutinins and were non-fimbriate.     

Type 3 fimbriae of Klebsiella sp.: molecular characterization and role in bacterial adhesion to plant roots.

Type 3 fimbriae of Klebsiella were purified and characterized. The fimbriae were 4 to 5 nm in diameter and 0.5 to 2 microns long. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the fimbrillin had an apparent molecular weight of 23,500, and it differed from enterobacterial type 1 fimbrillins in its amino acid composition. Hydrophobic amino acids comprised 33.6% of all amino acids in the fimbrillin, which lacked cystine, phenylalanine, and arginine. Serologically, the type 3 fimbriae were also distinct from the type 1 fimbriae. Purified type 3 fimbriae agglutinated tannin-treated human blood group O erythrocytes; this confirms the role of type 3 fimbriae as hemagglutinins. Purified 125I-labeled type 3 fimbriae bound to the roots of Poa pratensis, and this binding could be inhibited by Fab fragments to the purified fimbriae. Anti-type 3 fimbriae Fab fragments also inhibited bacterial adhesion to plant roots. These results demonstrate that type 3 fimbriae mediate adhesion of klebsiellas to plant roots. Eight nitrogen-fixing strains of Klebsiella also produced type 3 fimbriae when grown under anaerobic nitrogen fixation conditions. It is proposed that type 3 fimbriae are involved in the establishment of the plant-bacterium association concerning nitrogen-fixing Klebsiella strains.     

Role of haemagglutinins in adhesion of Erwinia carotovora to potato tissue

The adhesion of two strains each of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica to potato tuber discs, leaflets and tuber cell cultures was examined and found to occur independently of the presence of either mannose-sensitive (MSHA) or mannose-resistant (MRHA) haemagglutinins. Adhesion was generally greater when bacteria were grown in nutrient broth than on phosphate-buffered agar. The specific MSHA inhibitor α-methyl mannoside reduced the adhesion of two strains to tuber discs and leaflets and the specific MRHA inhibitor, asialofetuin inhibited strains only on leaflets. A reduction in adhesion of a MSHA-producing strain by α-methyl mannoside was observed by scanning electron microscopy which found that adhesion was localized at intercellular junctions.     

Purification and Characterization of Type 1 Fimbriae of Salmonella typhi

Type 1 fimbriae have been purified from a Salmonella typhi strain of clinical origin. Purified fimbriae retained their ability to bind to erythrocytes in a mannose-inhibitable fashion and, in doing so, behaved preferentially as a monovalent adhesin. SDS-PAGE analysis of the fimbrial preparation showed the presence of a 20-kDa major polypeptide component (fimbrillin) and of additional larger polypeptides present in smaller amounts. The amino-terminal sequence of fimbrillin was determined and turned out to be very similar but not identical to that of type 1 fimbrillins of other Salmonella serovars. A Western blot analysis of the purified fimbrial preparation using an antiserum raised against native fimbriae suggested that fimbrial proteins did not carry any major sequential epitope and that, in native fimbriae, conformational epitopes, possibly generated between different subunits, might provide for the major immunogenic epitopes. Analysis of different S. typhi clinical isolates using the anti-fimbrial antiserum showed an overall immunological similarity of these structures within this serovar.     

Heterogeneous post-translational modification of Actinobacillus actinomycetemcomitans fimbrillin

Fresh isolates of Actinobacillus actinomycetemcomitans produce bundle-forming fimbriae. The exact molecular mass of A. actinomycetemcomitans fimbrillin, a structural subunit of fimbriae, was determined by liquid chromatography-electrospray ionization mass spectrometry. Three major molecular species with 6,226.0, 6,366.0, and 6,513.0 Da were detected in a purified fimbrial fraction from the strain 310-a. These molecular masses were significantly higher than the molecular weight (5,118 Da) calculated from nucleotide sequence data of the fimbrillin gene, flp, suggesting that the fimbrial peptides were post-translationally modified. Modification of the fimbrial peptides was also suggested by an N-terminal amino acid sequence analysis of fimbrillin peptic fragments, with the modified amino acids being due to seven serine or asparagine residues located in the C-terminal region. A periodate oxidation/biotin-hydrazide labeling assay of fimbrillin suggested that it might be glycosylated.     

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.     

Molecular cloning and characterization of F9 fimbriae from a uropathogenic Escherichia coli

Abstract The genes responsible for the formation of F9 fimbriae of the uropathogenic Escherichia coli strain C1018 were cloned by a cosmid cloning procedure. A positive clone was further subcloned by removing two Bam HI fragments and the remaining plasmid pPIL288-10 had a size of 25 kb. This clone still produced fimbriae as judged by electron microscopy and mannose-resistant haemagglutination (MRHA). Antisera were raised against the clone and against fimbriae purified from the clone. The first antiserum was used in a Western blot to prove the purity of the F9 fimbriae. The antiserum raised against purified fimbriae was used in inhibition tests of MRHA and adherence of cloned bacteria to human uroepithelial cells.     

Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis.

Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain.     

Relationships among Broad-Spectrum and Narrow-Spectrum Mannose-Resistant Fimbrial Hemagglutinins in Different Yersinia Species

When 117 strains of Yersinia were grown in serial broth culture at 22 or 30 C, 44 (38%) formed a broad-spectrum, mannose-resistant hemagglutinin (MR/Y-HA) associated with thick (8 nm) channelled (MR/Y) fimbriae; eight other strains (7%) formed a narrow-spectrum, mannose-resistant hemagglutinin (MR/K-like HA) associated with thin (4 nm) non-channelled (type 3-like) fimbriae. The distribution of these two fimbrial hemagglutinins in different Yersinia species is discussed and their properties are compared with those of other reported mannose-resistant hemagglutinins. The thick fimbriae of four Yersinia species were antigenically similar as judged by immunoelectronmicroscopy. Likewise, the thin fimbriae of three Yersinia species were antigenically similar, though different from the thick fimbriae.     

Serological and biochemical variation among potato strains of Erwinia carotovora subsp. atroseptica and their taxonomic relationship to other E. carotovora strains

The serological and biochemical characteristics of 32 Erwinia carotovora subsp. atroseptica strains from potato were compared with 48 other pectolytic Erwinia strains. Biochemical characteristics were examined by the API 20E and API 50CHE systems. Numerical analysis using the Euclidean distance coefficients and clustering by the unweighted average pair group method indicated that these E. carotovora subsp. atroseptica strains formed a distinct cluster (subphenon A₁) that could be differentiated from other E. carotovora strains. Three non-potato strains also belonged to this group; two of these were from tomato and the other from Chinese cabbage. Named E. carotovora subsp. atroseptica strains from other hosts clustered into other phenons. Sixty-three per cent of subphenon A₁ strains tested in this study typed into serogroup I. One potato strain in another phenon also typed into this serogroup. The subphenon A₁ strains that did not type into serogroup I typed into serogroups XVIII, XX, or XXII. Many of these strains, however, expressed several different O antigens which were also expressed by E. carotovora strains in other phenons.     

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.     

PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato diseases.

Using a sequenced pectate lyase-encoding gene (pel gene), we developed a PCR test for Erwinia carotovora. A set of primers allowed the amplification of a 434-bp fragment in E. carotovora strains. Among the 89 E. carotovora strains tested, only the Erwinia carotovora subsp. betavasculorum strains were not detected. A restriction fragment length polymorphism (RFLP) study was undertaken on the amplified fragment with seven endonucleases. The Sau3AI digestion pattern specifically identified the Erwinia carotovora subsp. atroseptica strains, and the whole set of data identified the Erwinia carotovora subsp. wasabiae strains. However, Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. odorifera could not be separated. Phenetic and phylogenic analyses of RFLP results showed E. carotovora subsp. atroseptica as a homogeneous group while E. carotovora subsp. carotovora and E. carotovora subsp. odorifera strains exhibited a genetic diversity that may result from a nonmonophyletic origin. The use of RFLP on amplified fragments in epidemiology and for diagnosis is discussed.     

Development of hyperfimbriated strains of Vibrio cholerae O1

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR vector. These clones were sequenced. The fimA sequences were found to be identical between V cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.     

魔芋内生拮抗细菌的分离及其抗菌物质特性研究

从魔芋的内生菌中筛选到能抑制魔芋软腐病病原菌生长、产芽胞的杆状细菌,16SrDNA序列分析表明该菌是一株枯草芽胞杆菌,命名为BSn5。BSn5的胞外蛋白提取液有抗菌活性,并具有对热不稳定,对蛋白酶K敏感,对胰蛋白酶不敏感的特性,SDS-PAGE检测显示该蛋白提取液仅由分子量为31.6kDa的蛋白质组成。通过非变性聚丙烯酰胺凝胶电泳纯化该蛋白,纯化的蛋白能够抑制软腐病病原菌的生长,进一步表明该31.6kDa蛋白即为该菌的抗菌活性物质。该蛋白与目前所知的枯草芽胞杆菌产生的抗菌物质均不同,可能是一种新的抗菌蛋白。     

Frequency of Erwinia carotovora in the Alyth Burn in eastern Scotland and the sources of the bacterium

Contamination of the Alyth Burn by Erwinia carotovora was monitored monthly over 2 years at nine sites spread over a distance of ca 20 km. The bacterium was detected only once in the upper reaches of the river where it flows in uninhabited moorland but frequency of detection and contamination level tended to increase progressively as the river flowed through the middle reaches mostly in grassland to the lower reaches in arable land where the bacterium was almost always present. Erwinia populations rose from < 10² cells/1 before May to frequently > 10³ but < 10⁴ cells/l thereafter at sites in the arable land zone. A similar pattern was found in the grassland zone except that erwinia numbers were lower. Erwinia numbers at one site in the arable land zone were positively and negatively correlated with the river water temperature and flow rate respectively when there was a 1 month lag between the environmental data and the population recorded. More than 80% of isolates tested were E. carotovora subsp. carotovora.
Water from field drains in arable fields, especially those recently planted with potatoes, was frequently contaminated by E.c. carotovora , with numbers and a temporal pattern similar to those of the Alyth Burn. Drainage water from non-arable fields was rarely contaminated. Infected and rotting potatoes deposited in rivers temporarily contaminated the water. Survival of E. carotovora in dilute phosphate buffer was greater at pH 5˙7 than at pH 7˙7 and they survived for at least 10 d in river water.     

Fimbrial haemagglutinins in Enterobacter species

Fifty-two strains from seven species of Enterobacter, grown under a variety of conditions, were examined in rocked-tile tests for production of haemagglutinins and with the electron microscope for fimbriae. Thirteen non-haemagglutinating strains were non-fimbriate. Most (33) of the 39 haemagglutinating strains produced only one kind of haemagglutinin, either the mannose-sensitive haemagglutinin associated with type -1 Fimbriae or, the mannose-resistant, Klebsiella-like haemagglutinin associated with type-3 fimbriae. Multiply haemagglutinating strains were most common in E. aerogenes, in which species a third kind of haemagglutinin, also mannose-resistant, was found. The findings are discussed briefly in the light of the current taxonomy of Enterobacter.     

Serogroups of potato pathogenic Erwinia carotovora strains: identification by lipopolysaccharide electrophoretic patterns

Lipopolysaccharides were purified from 51 strains of Erwinia carotovora subsp. carotovora, atroseptica and betavasculorum representing 12 different serogroups. Analysis of the lipopolysaccharides by SDS-PAGE showed that, irrespective of the strain or serogroup from which they were extracted, the lipopolysaccharides were composed of up to 30 components. The mobility of the components decreased in a regular fashion giving a ladder-like appearance on the gel. The relative mobilities of components of lipopolysaccharide from each serogroup was constant so that each serogroup gave an easily identifiable ladder profile. The potato pathogenic serogroups I, XVIII, XX and XXII were examined in detail. Of 20 strains received as serogroup I, 18 gave a pattern identical to an authentic serogroup I strain. The two strains which did not give the same pattern were shown by immunological tests not to be serogroup I. Five atroseptica strains of serogroup XXII gave a distinct pattern characteristic of the serogroup while atroseptica strains of serogroups XVIII (four strains) and XX (five strains) gave patterns that could not be distinguished from each other. Analysis of lipopolysaccharides by SDS-PAGE has been shown to be an alternative to immunological tests to identify serogroups of Erwinia carotovora associated with blackleg. It is also capable of differentiating these serogroups from erwinia serogroups not normally regarded as causing blackleg. The analysis has the advantage that, irrespective of serogroup, a positive result is always obtained.     

Salicylic acid and the plant pathogen Erwinia carotovora induce defense genes via antagonistic pathways

Infection of tobacco plants with the plant pathogenic bacterium Erwinia carotovora subsp. carotovora or treatment of plants with Erwinia -derived elicitor preparations leads to the induction of a number of genes thought to play a role in plant defense response to pathogens. In order to determine the role of salicylic acid (SA) in the induction of the Erwinia responsive genes, the accumulation of mRNAs for these and other genes encoding pathogenesis-related proteins (PR genes) in response to both Erwinia elicitors and SA was determined. PR genes were identified which were preferentially induced by Erwinia elicitor preparations, one gene was induced by SA but not by Erwinia , and another gene was induced by both type of treatments. The differential expression of these genes and the timing of induction suggest that SA is not the signal molecule leading to the early response of plants to Erwinia . This was demonstrated by experiments using transgenic NahG plants that overproduce a salicylate hydroxylase inactivating SA. The elicitation of PR genes by Erwinia was similar in NahG and wild-type plants. Therefore, induction of plant defense genes by Erwinia and SA seems to be by two distinct pathways leading to expression of separate sets of genes. Furthermore, we could demonstrate that Erwinia elicitors antagonize the SA-mediated induction of PR genes. Similarly, SA appeared to inhibit the induction of PR genes elicited by Erwinia . The observed antagonism between the two signal transduction pathways indicates the presence of a common regulatory element in both pathways that acts downstream of SA in the SA-mediated response.     

Immunogenicity of Vibrio cholerae O1 Fimbriae in Animal and Human Cholera

Parenteral immunization with either formalin-fixed whole cells of the fimbriate Bgd17 strain or purified fimbriae protected against Vibrio cholerae O1 infection in rabbits, independent of biotype and serotype. Parenteral immunization of adult rabbits with purified fimbriae prior to V. cholerae O1 challenge resulted in a reduction of 2 to 3 orders of magnitude in the number of bacteria recovered from the small intestines of immunized rabbits in comparison to non-immunized controls. IgG and IgA antibodies against fimbrillin of V. cholerae O1 were detected in the convalescent sera of patients with cholera; however, little fimbrial antigen was detected in the commercially available cholera vaccines when examined by polyclonal and monoclonal antibodies against fimbriae. These data suggest that fimbrial hemagglutinin is a major adhesin of V. cholerae O1 and that parenteral immunization with fimbriae generates a specific immune response in the gut that may serve as one means of mitigating subsequent V. cholerae O1 gut infection.