Isolation of specific receptors from Xanthomonas albilineans cell walls for lectins-like glycoproteins of sugarcane

Abstract

Two families of sugarcane glycoproteins differing in their molecular mass have been isolated from sugarcane stalks. These glycoproteins specifically bind to cell wall receptors of Xanthomonas albilineans, a sugarcane pathogen, producing bacterial agglutination. Bound glycoproteins can be desorbed from bacterial cell walls by galactitol, a component of the glycosidic moiety of the sugarcane protein. This indicates that sugarcane glycoproteins bind through their glycosidic rest to the peptide moiety of the bacterial receptor. Several cell wall receptors have been isolated by affinity chromatography and separated by capillary electrophoresis.     

Characterization of an Agrobacterium tumefaciens lectin.

An Agrobacterium tumefaciens suspension induces a strong agglutination of aldehyde-fixed pig erythrocytes at pH 5.0. The agglutination is inhibited by some polysaccharides, such as fucoidin, and also when the pH is raised to 7.0. Lectins (sugar-binding proteins) associated with the bacterial cell wall of A. tumefaciens strain 84.5 were directly evidenced by spectrofluorimetry using fluoresceinylated neoglycoproteins. The specific binding of the fluorescein-labelled neoglycoprotein bearing alpha-L-fucoside residues was also optimal at pH 5.0. A lectin was purified by affinity chromatography on agarose substituted with alpha-L-fucopyranoside. Furthermore, the haemagglutination activity of this lectin was inhibited by polysaccharides isolated from poplar leaves.     

Serological method for identification of Vibrio parahaemolyticus from marine samples

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

Serological method for identification of Vibrio parahaemolyticus from marine samples.

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

A New Method for Strain Identification of Rhizobium trifolii in Nodules

An indirect haemagglutination test has been developed for the detection of strains of Rhizobium trifolii in nodules of subterranean clover plants. Preserved sheep red blood cells, coated with isolated specific rhizobial lipopolysaccharide, were used as the indicator of agglutination; these cells were agglutinated by specific antilipopolysaccharide antibody. Detection of lipopolysaccharide antigen in a suspension of nodular tissue was carried out by reacting the suspension with antilipopolysaccharide antibody prior to the addition of coated red blood cells. The presence of antigen in the suspension was indicated by an inhibition of agglutination. The test was more sensitive than agglutination and immunodiffusion in the detection of rhizobial lipopolysaccharide antigens, and could be used for the rapid screening of large numbers of nodules.     

Isolation and characterization of IS 31831, a transposable element from Corynebacterium glutamicum

A transposable element from a coryneform bacterium, Corynebacterium glutamicum ATCC 31831 was isolated and characterized. The element IS 31831 is a 1453 bp insertion sequence with 24 bp imperfect terminal inverted repeats. It contains one open reading frame highly homologous at the amino acid level to the transposase of IS 1096 from Mycobacterium smeg-matis. Both IS 31831 and IS 1096 exhibit several common characteristics suggesting that they constitute a new family of insertion sequences. IS 31831 was isolated by taking advantage of the sucrose sensitivity of coryneform bacteria conferred by expression of the Bacillus subtilis sacB gene. An Escherichia coli/ Corynebacterium shuttle vector useful for the isolation of transposable elements from the coryneform group of bacteria was constructed.     

Antigenicity and immunogenicity of the polyagglutinable antigen of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa strains isolated from cystic fibrosis patients agglutinate in antisera against anti-polyagglutinable antigen (PA). Anti-PA antibodies were formed in rabbits when immunization was carried out with bacteria possessing core-bound PA, independently of whether the strains were of S or R phenotype. For bacterial agglutination with anti-PA antibodies two prerequisites are essential: the bacterial cell must be of R phenotype and must possess the core-linked PA. In contrast, the PA in the isolated LPS's can be demonstrated in passive haemagglutination for both (S or R) phenotypes, provided the PA is core-linked. Two PA forms have been recognized, one found only in P. aeruginosa species, both in free and bound form. The other one is shared by all members of Pseudomonas genus but is present only in a free, unbound form.     

Whole-Genome Sequence of Enterobacter sp. Strain SST3, an Endophyte Isolated from Jamaican Sugarcane (Saccharum sp.) Stalk Tissue

Enterobacter sp. strain SST3 is an endophytic bacterium isolated from Saccharum spp. Here we present its annotated draft genome that may shed light on its role as a bacterial endophyte of sugarcane. To our knowledge, this is the first genome announcement of a sugarcane-associated bacterium from the genus Enterobacter.     

Preparation and Characterization of Membranes from a Coryneform Isolated from Ratoon Stunted Sugarcane

Membranes from the coryneform were prepared by an optimized chemical procedure using EDTA and lysozyme and isolated upon equilibrium sucrose density gradient ultracentrifugation. Four bands, I to IV, of peak densities 1.15, 1.19, 1.21 and 1.245 g. cm^?3 respectively, were reproducibly obtained. Immunodiffusion of the isolated membrane bands against bacterial antiserum showed that the immunological integrity of the isolated membranes was retained. Membrane bands I and II had similar precipitin arcs having one major component. A shift towards greater immunological complexity in band IV was observed. Similarly, a change in polypeptide composition established by polyacrylamide gel electrophoresis was found. The major group of polypeptides inband I ranging in size from 39 to 67 kdal were successively reduced in bands II, III and IV whereas the polypeptide of 15.7 kdal and of those in the region of 140 kdal showed respective increases. Transmission electron microscopy revealed similar membrane, structures in all membrane bands with only minor differences between them.     

Antigenicity and immunogenicity of the polyagglutinable antigen of Pseudomonas aeruginosa

Pseudomonas aeruginosa strains isolated from cystic fibrosis patients agglutinate in antisera against anti-polyagglutinable antigen (PA). Anti-PA antibodies were formed in rabbits when immunization was carried out with bacteria possessing core-bound PA, independently of whether the strains were of S or R phenotype. For bacterial agglutination with anti-PA antibodies two prerequisites are essential: the bacterial cell must be of R phenotype and must possess the core-linked PA. In contrast, the PA in the isolated LPS's can be demonstrated in passive haemagglutination for both (S or R) phenotypes, provided the PA is core-linked. Two PA forms have been recognized, one found only in P. aeruginosa species, both in free and bound form. The other one is shared by all members of Pseudomonas genus but is present only in a free, unbound form.     

Lytic Enzyme toward Aniline-assimilating Rhodococcus erythropolis AN-13: Screening and Production

A bacterial strain, SH-548, that produces a lytic enzyme toward intact cells of aniline-assimilating Rhodococcus erythropolis AN-13, was isolated from soil. The isolated bacterium was identified as a Flavobacterium species. The growth conditions for the enzyme production by Flavobacterium sp. SH-548 were examined; organic nitrogen compounds, such as meat extract and Polypepton, were effective for its production. The lytic enzyme of this strain lysed intact cells of Rhodococcus, Bacillus, Nocardia, Corynebacterium, Brevibacterium, Streptococcus, Micrococcus, Cellulomonas and DAB (diaminobutyric acid)-type coryneform bacterial strains. However, it did not act on those of Staphylococcus aureus or gram-negative bacteria, Enterobacter, Escherichia, Klebsiella, Proteus or Pseudomonas strains. Bacterial strains having cell walls of the glycolyl type were readily lysed by this enzyme.     

Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.     

Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.     

Bacterial metabolism of 3-chloroacrylic acid.

Two bacterial strains were isolated with 3-chloroacrylic acid (CAA) as sole source of carbon and energy. Strain CAA1, a Pseudomonas cepacia sp., was capable of growth with only the cis-isomer of CAA. Strain CAA2, a coryneform bacterium, utilized both isomers of CAA as sole source of carbon and energy. Strain CAA1 contained cis-CAA hydratase and strain CAA2 contained two hydratases, one with cis-CAA hydratase activity and one with trans-CAA hydratase activity. The product of the hydratase activities with CAA was malonate semialdehyde. In both strains malonate semialdehyde was subsequently decarboxylated by a cofactor-independent decarboxylase yielding acetaldehyde and CO2.     

Enzyme-Linked Immunosorbent Assay (ELISA) of a Coryneform Isolated from Ratoon Stunted Sugarcane

An enzyme-linked immunosorbent assay (ELISA) was developed for the coryneform bacterium isolated from ratoon stunted surgarcane. Parameters governing the ELISA were optimized. These included antibody coating concentration, conjugate concentration and diluent, antigen diluent, the washing procedure, incubation temperature for sample and conjugate, pH and time. The level of detectability of the cultured bacterium by the ELISA was ≥ 1 × 10⁴ Zellen · ml^-1.     

Establishment of a functional genomics platform for Leifsonia xyli subsp. xyli

Leifsonia xyli subsp. xyli, the causal agent of ratoon stunting disease in sugarcane, is a xylem-limited, nutritionally fastidious, slow growing, gram-positive coryneform bacterium. Because of the difficulties in growing this bacterium in pure culture, little is known about the molecular mechanisms of pathogenesis. Currently, the genome sequence of L. xyli subsp. xyli is being completed by the Agronomical and Environmental Genomes group from the Organization for Nucleotide Sequencing and Analysis in Brazil. To complement this work, we produced 712 Lxx::Tn4431 transposon mutants and sequenced flanking regions from 383 of these, using a rapid polymerase chain reaction-based approach. Tn4431 insertions appeared to be widespread throughout the L. xyli subsp. xyli genome; however, there were regions that had significantly higher concentrations of insertions. The Tn4431 mutant library was screened for individuals unable to colonize sugarcane, and one noncolonizing mutant was found. The mutant contained a transposon insertion disrupting two open reading frames (ORF), one of which had homology to an integral membrane protein from Mycobacterium leprae. Sequencing of the surrounding regions revealed two operons, pro and cyd, both of which are believed to play roles in disease. Complementation studies were carried out using the noncolonizing Lxx::Tn4431 mutant. The noncolonizing mutant was transformed with a cosmid containing 40 kbp of wild-type sequence, which included the two ORF disrupted in the mutant, and several transformants were subsequently able to colonize sugarcane. However, analysis of each of these transformants, before and after colonization, suggests that they have all undergone various recombinant events, obscuring the roles of these ORF in L. xyli subsp. xyli pathogenesis.     

Aureobacterium resistens sp. nov., exhibiting vancomycin resistance and teicoplanin susceptibility

Two similar strains of a coryneform bacterium were isolated from human clinical material. Both strains were resistant to vancomycin but susceptible to teicoplanin. Detailed biochemical, chemotaxonomical, and molecular genetic investigations revealed that both isolates were members of a hitherto undescribed species of the genus Aureobacterium. The name Aureobacterium resistens sp. nov. is proposed for the new bacterium and the type strain is CCUG 38312.     

Novel cell-binding activity specific for N-acetyl-D-glucosamine in an Escherichia coli strain

Escherichia coli strains isolated from patients with different levels of urinary tract infection and from healthy persons were tested for their ability to haemagglutinate endo-beta-galactosidase-treated human erythrocytes. Among the 104 strains studied one revealed a strong agglutination reaction with the enzyme-treated erythrocytes. From the monosaccharides tested N-acetyl-D-glucosamine inhibited agglutination most effectively. Orosomucoid and asialo-orosomucoid had no effect on the haemagglutination whereas beta-galactosidase treated asialo-orosomucoid was inhibitory. These findings indicate that the E. coli strain studied contains a novel cell-binding activity with specificity for terminal N-acetyl-D-glucosamine residues.     

Antigenic and blood group activity of lamb gastric mucoproteins]

The antigenic properties of a lamb mucin and of a glycoprotein isolated from it are investigated. The proteins are studied by immunoelectrophoresis of the mucin and of the glycoprotein against either a sheep serum antiserum or an exclusively glycoprotein antiserum. No serum protein could be shown in the mucin. In addition, the glycoprotein gives a precipitin line only with the specific antiserum; its antigenic power seems to be concealed in the original mucin. The carbohydrates are studied through the investigation of the blood group activities by the hemagglutination inhibition technique. The mucin and the isolated glycoprotein show the same types of activities: they both prevent the agglutination of A1 and 0 red cells. The presence of two blood group activities in that glycoportein and its non-reactivity against A2 red cells corroborate the hypothesis that carbohydrate chains must be branched for the elicitation of these activities in a single molecule.     

The production and properties of an antiserum to potato (Solanum tuberosum) lectin.

Precipitation of potato (Solanum tuberosum) lectin by antisera was not affected by treatments that abolish lectin activity. An antiserum precipitated glycosylated derivatives of the lectin but not a deglycosylated peptide. The haemagglutination inhibition titre of this antiserum was not affected by removing anti-glycopeptide antibodies. This evidence suggests that the antiserum contains two populations of antibodies, specific for different domains of the lectin.