Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens.

Products of the virB operon are proposed components of a membrane-associated T-DNA transport apparatus in Agrobacterium tumefaciens. Here we identified the virB10 gene product and raised specific antiserum to the protein. While the virB10 reading frame contains two potential ATG translation start sites located 32 codons apart, we found that only the downstream ATG was required for efficient VirB10 synthesis. Cellular localization studies and analysis of translational fusions with the Escherichia coli alkaline phosphatase gene (phoA) indicated that VirB10 was anchored in the inner membrane and contained a periplasmic domain. This work also demonstrated the utility of alkaline phosphatase as a reporter for secreted proteins in A. tumefaciens. Several high-molecular-weight forms of VirB10 were observed after treatment of A. tumefaciens whole cells or inner membranes with protein cross-linking agents, suggesting that VirB10 exists as a native oligomer or forms an aggregate with other membrane proteins. These results provide the first biochemical evidence that a VirB protein complex is membrane associated in A. tumefaciens.

Identification of bacterial periplasmic glycine betaine-binding protein after electrophoresis and affinity labeling.

Antibodies were elicited in rabbits against periplasmic proteins obtained by cold osmotic shock from the Gram-negative eubacterium Rhizobium meliloti. When analyzed by crossed immunoelectrophoresis (CIE), the periplasmic proteins gave rise to 20 distinct immunoprecipitates corresponding to the same number of bands in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions and in SDS-PAGE. The periplasmic glycine betaine-binding protein (GB-BP) was identified by autoradiography after affinity labeling with [14C]glycine betaine in PAGE and in CIE gels. The binding proved to be quite specific to glycine betaine, since the GB-BP was not labeled by choline (a metabolic precursor of glycine betaine in Escherichia coli and Rhizobium meliloti) and 15 distinct L-amino acids, including L-proline which, like glycine betaine is also an osmoprotectant. Affinity labeling of the GB-BP with [14C]glycine betaine after protein separation by PAGE or CIE is a simple and sensitive technique permitting the GB-BP to the unambiguously detected and identified in samples of complex protein mixtures containing down to 2 micrograms of GB-BP in PAGE and only 0.2 micrograms in CIE.

Immunochemical analysis of lipopolysaccharides from free-living and endosymbiotic forms of Rhizobium leguminosarum.

Rhizobium leguminosarum B556 and 8002 differ only with respect to carrying symbiotic plasmids with specificity for Pisum or Phaseolus hosts, respectively. Protease-treated samples derived from free-living cultures of both strains revealed a ladder of lipopolysaccharide (LPS-1) bands after periodate-silver staining of sodium dodecyl sulfate-polyacrylamide gels. These bands were arranged as doublets. After Western (immuno-) blotting, all LPS-1 bands reacted with monoclonal antibody JIM 21, whereas monoclonal antibody MAC 57 reacted only with the upper (slower-migrating) band and monoclonal antibody MAC 114 reacted only with the lower band of each doublet pair. Preparations obtained from bacteroids of Pisum or Phaseolus nodules showed significant differences in the size distribution and antigenicity of LPS. In bacteroids from Phaseolus sp., JIM 21 and MAC 57 each stained a ladder of LPS-1 bands on sodium dodecyl sulfate-polyacrylamide gels which corresponded in mobility to the upper band of each doublet pair seen in free-living cultures. MAC 114 did not react with the LPS from Phaseolus sp.-derived bacteroids. In bacteroids from Pisum sp., only fast-migrating (lower-molecular-weight) forms of LPS-1 could be visualized on gels, but both upper and lower bands of each doublet were still present and could be stained by the appropriate monoclonal antibody, MAC 57 or MAC 114, respectively. Similarly, bacteroids from R. leguminosarum 3841, which nodulates Pisum species, differed with respect to the structure and antigenicity of their LPS-1 from bacteroids of a related strain, B625, which nodulates Phaseolus species. Physiological factors were investigated that could account for these differences between the structures of LPS-1 from free-living cultures of B556 and 8002 and that from bacteroids. The following modifications in growth conditions each tended to reduce the expression of MAC 114 antigen and enhance the expression of MAC 57 antigen: succinate rather than glucose as the carbon source; microaerobic (2.5%, vol/vol) oxygen concentrations; and acidic (pH 5 to 6) culture medium. When all three of these conditions were combined, the LPS-1 that resulted was very similar to that in bacteroids from Pisum nodules. However, it was not possible to reproduce the LPS-1 pattern observed for bacteroids from Phaseolus nodules, which maintained a ladder of LPS bands reacting with MAC 57 antibody.

Cell-associated oligosaccharides of Bradyrhizobium spp.

We report the initial characterization of the cell-associated oligosaccharides produced by four Bradyrhizobium strains: Bradyrhizobium japonicum USDA 110, USDA 94, and ATCC 10324 and Bradyrhizobium sp. strain 32H1. The cell-associated oligosaccharides of these strains were found to be composed solely of glucose and were predominantly smaller than the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species. Linkage studies and nuclear magnetic resonance analyses demonstrated that the bradyrhizobial glucans are linked primarily by beta-1,6 and beta-1,3 glycosidic bonds. Thus, the bradyrhizobia appear to synthesize cell-associated oligosaccharides of structural character substantially different from that of the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species.

Galactosyl residue in exopolysaccharide from Rhizobium meliloti JJ-1 exposed to manganese in furanoid.

Exopolysaccharide (EPS) elaborated by Rhizobium meliloti JJ-1 in a manganese supplemented medium was isolated. Periodate oxidation, reduction with sodium borohydride, followed by hydrolysis and subsequent capillary gas liquid chromatography of the derived alditol acetates revealed that D-galactose in this complex biopolymer is in furanoid form. This observation was further confirmed by 13carbon nuclear magnetic resonance (13C NMR).

Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein.

The nucleotide sequence of the nodE gene of Rhizobium trifolii strain ANU843 was determined. Like the nodE gene of R. leguminosarum strain 248 it encodes a protein with a predicted mol. wt of 42.0 kd. The predicted NodE proteins of R.trifolii and R.leguminosarum have a homology of 78%. Using antibodies raised against the NodE protein of R.trifolii it was shown that the NodE products of R.leguminosarum and R.trifolii are localized in the cytoplasmic membrane. Furthermore, these NodE proteins are predicted to contain at least two non-polar transbilayer alpha-helices. The nodE genes of R.trifolii and R.leguminosarum were separated from the nodF genes that precede them in the respective nodFE operons. Using the resulting clones, in which NodE was produced under control of the flavonoid-inducible nodABCIJ promoter of R.leguminosarum, it was shown that the NodE product is the main factor that distinguishes the host range of nodulation of R.trifolii and R.leguminosarum. Hybrid nodE genes, which consist of a 5' part of the R.leguminosarum nodE gene and a 3' part of the R.trifolii gene, were constructed. From the properties of these hybrid genes it was concluded that a central region of 185 amino acids at the most, containing only 44 non-identical amino acids, determines the difference in the host-specific characteristics of the two NodE proteins.

Critical spacing between two essential cysteine residues in the interdomain linker of the Bradyrhizobium japonicum NifA protein.

A special sequence motif in the Bradyrhizobium japonicum NifA protein, consisting of two functionally essential cysteines separated by four other amino acids (Cys-aa4-Cys), has been proposed to be part of a potential metal-binding site [(1988) Nucleic Acids Res. 16, 2207-2224]. Using the techniques of oligonucleotide-directed mutagenesis, we report here that several of the four intervening amino acids can be replaced by others without loss of NifA function. The deletion of one amino acid to give a Cys-aa3-Cys motif renders the protein inactive whereas the creation of a Cys-aa5-Cys motif (one amino acid inserted) still leads to a partially active NifA protein.

Isolation and characterization of the unusual lipopolysaccharide component, 2-amino-2-deoxy-2-N-(27-hydroxyoctacosanoyl)-3-O-(3-hydroxy- tetradecanoyl)-gluco-hexuronic acid, and its de-O-acylation product from the free lipid A of Rhizobium trifolii ANU843.

Two new, unusual lipid A components have been isolated and characterized from the free lipid A of Rhizobium trifolii ANU843. 2-Amino-2-deoxy-2-N-(27-hydroxyoctacosanoyl)-3-O-(3-hydroxy- tetradecanoyl)-gluco-hexuronic acid and its de-O-acylation product were purified from the chloroform/methanol extract of a mild acid hydrolysate of the lipopolysaccharide by chromatography on C18 reverse-phase columns and layers. The compositions of the two compounds were determined by releasing the acyl components by exhaustive acid-catalyzed methanolysis and identifying them as their methyl esters by gas chromatography and gas chromatography/mass spectrometry. The sugar component was identified by converting it to the alditol acetate derivative of glucosamine in a two-step reduction and identifying it as such by gas chromatography/mass spectrometry. The linkages of the fatty acyl components to the sugar residue and the configuration of the sugar component was confirmed by 1H and 13C NMR spectroscopy. The complete structures of the two compounds were further confirmed by fast atom bombardment mass spectrometry. It is still unsure whether the de-O-acylated derivative was formed from the di-acyl compound by de-O-acylation during acid hydrolysis. These structures represent the first report of 2-amino-2-deoxy-gluco-hexuronic acid in the free lipid A of a Gram-negative bacterium and confirms our earlier contention (Hollingsworth, R.I., and Carlson, R. W. (1989) J. Biol. Chem. 264, 9000-9303) of the involvement of 27-hydroxyoctacosanoic acid in the structure of the lipopolysaccharide of Rhizobium trifolii ANU843.

Evidence for divalent cation (Ca2+)-stabilized oligomeric proteins and covalently bound protein-peptidoglycan complexes in the outer membrane of Rhizobium leguminosarum.

Two unusual characteristics of some outer membrane proteins of Rhizobium leguminosarum are described. First, most of the major outer membrane proteins could only be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after lysozyme treatment of the isolated cell envelopes, suggesting a very strong, possibly covalent, interaction of these proteins with the peptidoglycan. These peptidoglycan-associated outer membrane proteins belonged to two distinct groups of immunologically related proteins, groups II and III, as defined by typing with monoclonal antibodies. As members of both groups of proteins could be radioactively labeled by growing cells in the presence of N-[3H]acetylglucosamine, we propose that variation in the apparent molecular weight of the antigens within each group is caused by varying numbers of peptidoglycan subunit residues on only two or three different outer membrane proteins. Second, group III outer membrane proteins, with masses of 35 to 46 kilodaltons, formed oligomers stabilized by divalent cations which resisted complete denaturation in 2% sodium dodecyl sulfate at 100 degrees C. Reconstitution experiments showed that of the divalent cations tested, Ca2+ and, to a lesser extent, Mn2+ and Sr2+ were the best stabilizers.

Application of gas-liquid chromatography to the routine identification of nonfermenting gram-negative bacteria in clinical specimens.

A total of 430 strains of glucose-nonfermenting gram-negative bacteria representing 35 species were analyzed for their cellular fatty acid composition by gas-liquid chromatography (GLC). On the basis of qualitative differences in their cellular fatty acid composition, these bacteria could be divided into 19 distinct chromatographic groups. Eight Pseudomonas species, Achromobacter xylosoxidans, group Vd, and Agrobacterium radiobacter were identified from their fatty acid compositions alone. The other glucose-nonfermenting gram-negative bacterial species studied here, classified within nine distinct GLC groups, were easily recognized by using the GLC fatty acid analysis supplemented with a limited number of conventional biochemical tests. The results support the hypothesis that bacterial fatty acid composition is rather specific and that qualitative GLC fatty acid analysis can be adapted in the clinical laboratory either to provide additional criteria for differentiation of closely related groups or to serve as a rapid and highly reproducible method for their routine identification.

A new core tetrasaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU 843.

A second core oligosaccharide fragment has been isolated and characterized from the lipopolysaccharide (LPS) of Rhizobium trifolii ANU 843. The oligosaccharide is a tetrasaccharide composed of galactose, galacturonic acid, mannose, and 3-deoxy-D-manno-2-octulosonic acid. The mannose residue is alpha-linked to the 4-position of 3-deoxy-D-manno-2-octulosonic acid and the galacturonic acid residue is alpha-linked to the 6-position of mannose. The galactose residue, which is acetylated at the 4-position, is attached to the 4-position of mannose by an alpha-linkage. All of the aldoses are in the pyranose form. The composition of the tetrasaccharide was determined by gas-liquid chromatography of the alditol acetate derivatives of the component monosaccharides. The configuration of anomeric linkages was determined by 1H NMR spectroscopy. Fast atom bombardment-mass spectrometry (FAB-MS) was performed on acetylated, per(trideutero)acetylated and underivatized tetrasaccharide giving sequence information in addition to information on the residue which was acetylated. Similar studies were performed on the oligosaccharide after reduction with sodium cyanoborohydride and peracetylation with labeled and unlabeled acetic anhydride as before. Further linkage and sequence analysis was obtained from methylation analysis, and from electron impact mass spectrometry of the per(trideutero)acetylated oligosaccharide and from collision-induced dissociation fast atom bombardment tandem mass spectrometry using linked scans at constant B/E on the cyanoborohydride-reduced, per (trideutero)acetylated oligosaccharide. The exact location of the acetyl group was deduced from 1H NMR double resonance experiments in conjunction with mass spectrometric data.

27-Hydroxyoctacosanoic acid is a major structural fatty acyl component of the lipopolysaccharide of Rhizobium trifolii ANU 843.

A new hydroxylated, very long-chain fatty acid has been isolated and characterized from the lipopolysaccharide (LPS) of Rhizobium trifolii ANU 843. The lipid A of the organism was degraded by mild alkali and borohydride and the products methylated, peracetylated, and fractionated on a C18 reverse-phase column. The major lipid fraction was reduced with lithium triethylborohydride, methylated, peracetylated, and subjected to thin layer chromatography. The methylated peracetylated acid and the reduced diacetylated diol (1,27-dihydroxyoctacosane diacetate) were isolated and characterized by mass spectrometry and 1H NMR spectroscopy using homonuclear decoupling. The identity and linkage of the new fatty acid in the lipopolysaccharide was confirmed by 1H NMR spectroscopy of purified lipid A fractions and similar NMR studies of lipid A after acylation by phenylisocyanate. In the native LPS, the 27-hydroxy C-28 fatty acid is acylated at the 27-hydroxy position by other 3-hydroxy fatty acids. About 50% of the total fatty acid content of the LPS of R. trifolii ANU 843 is 27-hydroxyoctacosanoic acid. This oxyacyloxy structure involving 27-hydroxyoctacosanoic appears to be the major structural feature of the lipid A of this organism.

Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies.

Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.

Lipid A with 2,3-diamino-2,3-dideoxy-glucose in lipopolysaccharides from slow-growing members of Rhizobiaceae and from "Pseudomonas carboxydovorans".

Lipid A's from two Bradyrhizobium species and from the phylogenetically closely related species "Pseudomonas carboxydovorans" were found to contain 2,3-diamino-2,3-dideoxy-glucose as lipid A backbone sugar. In contrast, three representatives of the genus Rhizobium, as well as the phylogenetically related species Agrobacterium tumefaciens, contain solely glucosamine as lipid A backbone sugar. These findings support independent studies on the phylogenetical relatedness based on 16S rRNA-data of the genus Bradyrhizobium with "Pseudomonas carboxydovorans" and Rhodopseudomonas palustris, which form a tight phylogenetical cluster and which all contain the 2,3-diamino-2,3-dideoxy-glucose-containing lipid A. The relatedness of these species to the glucosamine-containing species of the genus Rhizobium and to Agrobacterium tumefaciens is rather distant as documented by 16S rRNA studies.

Host-range related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum.

Proton nuclear magnetic resonance (1H NMR) and fast atom bombardment mass spectrometric analyses were performed on enzymatically derived oligosaccharides from the acidic excreted polysaccharides (EPS) from representative bacterial strains of the pea-nodulating symbiont, Rhizobium leguminosarum (128C53, 128C63, and 300) and the clover-nodulating symbiont, Rhizobium trifolii (NA-30, ANU843, 0403, TA-1, LPR5035, USDA20.102, and 4S). The results revealed structural similarities and differences between EPS of these two species. Octasaccharide units containing galactose, glucuronic acid, alpha-L-threo-hex-4-enopyranosyluronic acid, and glucose in a molar ratio of 1:1:1:5 were obtained from the EPS of the three R. leguminosarum strains and had the same primary glycosyl sequence and location of pyruvate, acetate, and 3-hydroxybutyrate substituents. About 80% of the galactose residues were acylated with 3-hydroxybutyrate, and there were two acetyl groups per repeating unit distributed between the 2 glucose residues of the main chain-derived sequence of the octasaccharides. In contrast, the R. trifolii strains had varied EPS structures, each of which differed from the common R. leguminosarum EPS structure. The EPS from one group of R. trifolii strains (0403 and LPR5035) most closely resembled the R. leguminosarum EPS but differed in that a lower number of galactose and glucose residues were substituted by 3-hydroxybutyryl and acetyl groups, respectively. The EPS from a second group of R. trifolii strains (ANU843, TA-1, and NA-30) was even more different than the R. leguminosarum EPS. These R. trifolii octasaccharides bore a single acetyl group on O-3 of the glucuronic acid residue. In addition, the level of acylation by 3-hydroxybutyryl groups was 50% of that present in the R. leguminosarum EPS. The remaining two strains of R. trifolii (USDA20.102 and 4S) had very different patterns of acylation to each other and to all of the other strains. The EPS from strain USDA20.102 practically lacked 3-hydroxybutyryl groups and had a unique degree and pattern of acetylation. The oligomers from the EPS of R. trifolii strain 4S completely lacked 3-hydroxybutyryl groups and galactose. The latter EPS contained only one O-1-carboxyethylidene group and had a different degree and pattern of acetylation. Interestingly, these two latter strains differ from the other R. trifolii strains in nodulation rates on rare clover species in the Trifolium cross-inoculation group. Thus, we define several groups of R. trifolii based upon their EPS structures and establish their similarities and distinct differences with the EPS of R. leguminosarum.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection and subcellular localization of two Sym plasmid-dependent proteins of Rhizobium leguminosarum biovar viciae.

The previously described Sym plasmid-dependent 24-kilodalton rhi protein of Rhizobium leguminosarum biovar viciae was localized in the cytosol fraction. Another Sym plasmid-dependent protein of 50 kilodaltons is secreted into the growth medium, and its expression is dependent on both the nodD gene and a nod gene inducer.

Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules.

Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules. In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain. The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants. We also assayed for high- and low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH completely lacked both forms. For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium. The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies. The implications of these observations with regard to nodule entry are discussed.

A core oligosaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU843.

The major oligosaccharide from the core region of the lipopolysaccharide from R. trifolii ANU843 was isolated and its structure determined. It is a trisaccharide consisting of two galacturonic acid residues and one 3-deoxy-D-manno-2-octulosonic acid (KDO) residue. The two galacturonic acid residues are terminally linked alpha to the C-4 and C-7 atoms of KDO. This structure was determined through use of 1H- and 13C-n.m.r. spectroscopy, f.a.b.-m.s., and g.l.c.-m.s. techniques. This oligosaccharide had not previously been reported to be present in the lipopolysaccharides from Gram-negative bacteria.