Sinorhizobium fredii and Sinorhizobium meliloti Produce Structurally Conserved Lipopolysaccharides and Strain-Specific K Antigens

1998 ◽  
Vol 64 (12) ◽  
pp. 4930-4938 ◽  
Author(s):  
Bradley L. Reuhs ◽  
Daniel P. Geller ◽  
John S. Kim ◽  
Jennifer E. Fox ◽  
V. S. Kumar Kolli ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Cultured Cells ◽  
Structural Information ◽  
Consistent Difference ◽  
Sinorhizobium Fredii ◽  
Anomeric Configuration ◽  
Produce Strain ◽  
Common Antigens ◽  
O Antigens ◽  
K Antigen

ABSTRACT Lipopolysaccharides (LPS) and capsular polysaccharides (K antigens) may influence the interaction of rhizobia with their specific hosts; therefore, we conducted a comparative analysis of Sinorhizobium fredii and Sinorhizobium meliloti, which are genetically related, yet symbiotically distinct, nitrogen-fixing microsymbionts of legumes. We found that both species typically produce strain-specific K antigens that consist of 3-deoxy-d-manno-2-octulosonic acid (Kdo), or other 1-carboxy-2-keto-3-deoxy sugars (such as sialic acid), and hexoses. The K antigens of each strain are distinguished by glycosyl composition, anomeric configuration, acetylation, and molecular weight distribution. One consistent difference between the K antigens ofS. fredii and those of S. meliloti is the presence of N-acetyl groups in the polysaccharides of the latter. In contrast to the K antigens, the LPS ofSinorhizobium spp. are major common antigens. Rough (R) LPS is the predominant form of LPS produced by cultured cells, and some strains release almost no detectable smooth (S) LPS upon extraction.Sinorhizobium spp. are delineated into two major RLPS core serogroups, which do not correspond to species (i.e., host range). The O antigens of the SLPS, when present, have similar degrees of polymerization and appear to be structurally conserved throughout the genus. Interestingly, one strain was found to be distinct from all others: S. fredii HH303 produces a unique K antigen, which contains galacturonic acid and rhamnose, and the RLPS did not fall into either of the RLPS core serogroups. The results of this study indicate that the conserved S- and RLPS of Sinorhizobiumspp. lack the structural information necessary to influence host specificity, whereas the variable K antigens may affect strain-cultivar interactions.

scholarly journals Epitope Identification for a Panel of Anti-Sinorhizobium meliloti Monoclonal Antibodies and Application to the Analysis of K Antigens and Lipopolysaccharides from Bacteroids

1999 ◽  
Vol 65 (11) ◽  
pp. 5186-5191 ◽  
Author(s):  
Bradley L. Reuhs ◽  
Samuel B. Stephens ◽  
Daniel P. Geller ◽  
John S. Kim ◽  
Joshua Glenn ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Sinorhizobium Meliloti ◽  
Cultured Cells ◽  
Root Nodules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoblot Analysis ◽  
Whole Cells ◽  
Bacterial Differentiation ◽  
Common Antigens ◽  
Phenol Water

ABSTRACT In two published reports using monoclonal antibodies (MAbs) generated against whole cells, Olsen et al. showed that strain-specific antigens on the surface of cultured cells of Sinorhizobium meliloti were diminished or absent in the endophytic cells (bacteroids) recovered from alfalfa nodules, whereas two common antigens were not affected by bacterial differentiation (P. Olsen, M. Collins, and W. Rice, Can. J. Microbiol. 38:506–509, 1992; P. Olsen, S. Wright, M. Collins, and W. Rice, Appl. Environ. Microbiol. 60:654–661, 1994). The nature of the antigens (i.e., the MAb epitopes), however, were not determined in those studies. For this report, the epitopes for five of the anti-S. meliloti MAbs were identified by polyacrylamide gel electrophoresis-immunoblot analyses of the polysaccharides extracted from S. melilotiand Sinorhizobium fredii. This showed that the strain-specific MAbs recognized K antigens, whereas the strain-cross-reactive MAbs recognized the lipopolysaccharide (LPS) core. The MAbs were then used in the analysis of the LPS and K antigens extracted from S. meliloti bacteroids, which had been recovered from the root nodules of alfalfa, and the results supported the findings of Olsen et al. The size range of the K antigens from bacteroids of S. meliloti NRG247 on polyacrylamide gels was altered, and the epitope was greatly diminished in abundance compared to those from the cultured cells, and no K antigens were detected in the S. meliloti NRG185 bacteroid extract. In contrast to the K antigens, the LPS core appeared to be similar in both cultured cells and bacteroids, although a higher proportion of the LPS fractionated into the organic phase during the phenol-water extraction of the bacteroid polysaccharides. Importantly, immunoblot analysis with an anti-LPS MAb showed that smooth LPS production was modified in the bacteroids.

scholarly journals Alfalfa Root Nodule Invasion Efficiency Is Dependent on Sinorhizobium melilotiPolysaccharides

2000 ◽  
Vol 182 (15) ◽  
pp. 4310-4318 ◽  
Author(s):  
Brett J. Pellock ◽  
Hai-Ping Cheng ◽  
Graham C. Walker
Keyword(s):  
Molecular Weight ◽  
Sinorhizobium Meliloti ◽  
Root Nodule ◽  
Fluorescent Protein ◽  
Infection Thread ◽  
Low Molecular Weight ◽  
Infection Threads ◽  
Green Fluorescent ◽  
K Antigen ◽  
Alfalfa Root

ABSTRACT The soil bacterium Sinorhizobium meliloti is capable of entering into a nitrogen-fixing symbiosis with Medicago sativa (alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by S. meliloti are crucial for establishing this symbiosis. Alfalfa nodule invasion by S. meliloti can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled S. meliloti cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how S. meliloti polysaccharides are functioning in the plant-bacterium interaction, and models are discussed.

scholarly journals Expression of a Serratia marcescens Chitinase Gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 Impedes Soybean and Alfalfa Nodulation

1999 ◽  
Vol 12 (8) ◽  
pp. 748-751 ◽  
Author(s):  
Hari B. Krishnan ◽  
Kil Yong Kim ◽  
Ammulu Hari Krishnan
Keyword(s):  
Serratia Marcescens ◽  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Nodule Formation ◽  
Broad Host Range ◽  
Sinorhizobium Fredii ◽  
Gene Encoding ◽  
Range Vector ◽  
Layer Chromatography ◽  
Hydrolysis Of

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.

scholarly journals Citrate Synthase Mutants of Sinorhizobium fredii USDA257 Form Ineffective Nodules with Aberrant Ultrastructure

2003 ◽  
Vol 69 (6) ◽  
pp. 3561-3568 ◽  
Author(s):  
Hari B. Krishnan ◽  
Won-Seok Kim ◽  
Jeong Sun-Hyung ◽  
Kil Yong Kim ◽  
Guoqiao Jiang
Keyword(s):  
Sinorhizobium Meliloti ◽  
Citrate Synthase ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Light And Electron Microscopy ◽  
Tca Cycle ◽  
Single Copy ◽  
Membrane Structures ◽  
Rhizobium Tropici ◽  
Sinorhizobium Fredii

ABSTRACT The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici. Southern blot analysis revealed that the fast-growing S. fredii strains and Rhizobium sp. strain NGR234 contained a single copy of the gene located in the bacterial chromosome. S. fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced. A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above. Our results demonstrate that a functional citrate synthase gene of S. fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.

Characterisation of dry and mucoid colonies isolated from Australian rhizobial inoculant strains for Medicago species

2005 ◽  
Vol 45 (3) ◽  
pp. 151 ◽  
Author(s):  
A. McInnes ◽  
P. Holford ◽  
J. E. Thies
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Dry Weight ◽  
Sequence Element ◽  
Shoot Dry Weight ◽  
Single Colony ◽  
Agar Media ◽  
Fixation Capacity ◽  
Medicago Species ◽  
K Antigen

The presence of dry and mucoid colonies in cultures of rhizobial strains used in the production of commercial Australian inoculants is of concern for quality assurance because of the possibility of altered capacity for nodulation and nitrogen fixation by the different colony types. In this study, single colony isolates obtained from dry and mucoid colonies present in commercial cultures of Sinorhizobium meliloti were investigated to identify stability in culture, genetic identity and changes in exopolysaccharide (EPS) production, nodulation and nitrogen fixation. The 2 strains studied were WSM688 and WSM826 (Australian inoculant strains for annual and perennial medics, respectively), both of which produced only mucoid colonies on agar media when originally isolated from nodules. Dry and mucoid single colony isolates from the ‘mother cultures’ of the 2 strains exhibited stable colony phenotypes during successive subculturing in our laboratory and were shown to be most closely related to S. meliloti using 16S rRNA partial sequencing. All isolates produced at least 1 of 3 exopolysaccharides (succinoglycan, EPS II and K antigen) that are required for successful nodulation of Medicago species by S. meliloti strains, as indicated by nodulation of host legumes. Strain WSM826 isolates probably produce succinoglycan, as shown by similarity to the succinoglycan-producing strain Rm1021 in a calcofluor binding assay. In contrast to published work, there was no evidence that loss of mucoidy in dry colony isolates of either strain was associated with the presence of an insertion sequence element in the expR gene that inhibits EPS II production. For strain WSM688, dry and mucoid isolates were identical by PCR fingerprinting and showed a similar capacity to nodulate and fix nitrogen with the target host legume M. truncatula in glasshouse tests. In contrast, strain WSM826 mucoid isolates produced PCR fingerprints that were different from each other and from the WSM826 dry colony isolates. Dry and mucoid colonies may have arisen from substantial genetic change or through contamination of cultures by other S. meliloti strains. One WSM826 mucoid isolate (826-3) produced significantly lower shoot dry weight when inoculated onto both the target host M. sativa and non-target host M. truncatula, even though the capacity to nodulate both hosts was retained. This suggests that this isolate was affected in its nitrogen fixation capacity. Further research is required to identify the origin and extent of colony variation in commercial S. meliloti cultures.

scholarly journals Deciphering the Symbiotic Significance of Quorum Sensing Systems of Sinorhizobium fredii HH103

2020 ◽  
Vol 8 (1) ◽  
pp. 68 ◽  
Author(s):  
Sebastián Acosta-Jurado ◽  
Cynthia Alías-Villegas ◽  
Andrés Almozara ◽  
M. Rosario Espuny ◽  
José-María Vinardell ◽  
...  
Keyword(s):  
Growth Rate ◽  
Quorum Sensing ◽  
Sinorhizobium Meliloti ◽  
Target Genes ◽  
Plasmid Transfer ◽  
Sinorhizobium Fredii ◽  
Independent Manner ◽  
Signaling Mechanism ◽  
Surface Polysaccharides ◽  
Carboxy Terminal

Quorum sensing (QS) is a bacterial cell-to-cell signaling mechanism that collectively regulates and synchronizes behaviors by means of small diffusible chemical molecules. In rhizobia, QS systems usually relies on the synthesis and detection of N-acyl-homoserine lactones (AHLs). In the model bacterium Sinorhizobium meliloti functions regulated by the QS systems TraI-TraR and SinI-SinR(-ExpR) include plasmid transfer, production of surface polysaccharides, motility, growth rate and nodulation. These systems are also present in other bacteria of the Sinorhizobium genus, with variations at the species and strain level. In Sinorhizobium fredii NGR234 phenotypes regulated by QS are plasmid transfer, growth rate, sedimentation, motility, biofilm formation, EPS production and copy number of the symbiotic plasmid (pSym). The analysis of the S. fredii HH103 genomes reveal also the presence of both QS systems. In this manuscript we characterized the QS systems of S. fredii HH103, determining that both TraI and SinI AHL-synthases proteins are responsible of the production of short- and long-chain AHLs, respectively, at very low and not physiological concentrations. Interestingly, the main HH103 luxR-type genes, expR and traR, are split into two ORFs, suggesting that in S. fredii HH103 the corresponding carboxy-terminal proteins, which contain the DNA-binding motives, may control target genes in an AHL-independent manner. The presence of a split traR gene is common in other S. fredii strains.

Antibody specificities of polyclonal rabbit and rainbow trout antisera against Vibrio ordalii and serotype 0:2 strains of Vibrio anguillarum

1993 ◽  
Vol 39 (5) ◽  
pp. 492-499 ◽  
Author(s):  
Lucy W. Mutharia ◽  
Bonnie T. Raymond ◽  
Teri R. Dekievit ◽  
Roselynn M. W. Stevenson
Keyword(s):  
Rainbow Trout ◽  
Vibrio Anguillarum ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Antigenic Determinants ◽  
Polyclonal Rabbit ◽  
Common Antigens ◽  
Antibody Specificities ◽  
O Antigens ◽  
Vibrio Ordalii

Polyclonal rabbit antisera raised against Vibrio ordalii and serotype 02 strains of Vibrio anguillarum showed extensive cross-reactivity with lipopolysaccharide from these bacterial pathogens of fish when tested in western immunoblot analysis. Results with absorbed polyclonal antisera indicated that lipopolysaccharide molecules from these strains had both common and strain-specific antigenic determinants, which allowed the antisera to be used to differentiate between V. ordalii and serotype 02 strains of V. anguillarum. Unlike rabbits, the immune response in rainbow trout to serotype 02 common antigenic epitopes was dependent on the source of the immunizing lipopolysaccharide antigens. Serum from fish immunized with V. ordalii antigens reacted more extensively with serotype 02 common antigens. In contrast, fish anti-V. anguillarum 02 serum did not interact with O antigens from the V. ordalii strains. Lipopolysaccharide from V. anguillarum serotype 02 and 02a strains showed identical antibody binding properties when interacted with rabbit or fish antiserum to either V. anguillarum 02 or V. ordalii. Lipopolysaccharide from V. anguillarum 02b strains did not interact with the tested rabbit or fish polyclonal sera. The results from this study suggest that fish and rabbits recognise different antigenic determinants in lipopolysaccharide from V. ordalii and serotpye 02 V. anguillarum strains; that V. ordalii and serotype 02 strains of V. anguillarum should be regarded as distinct serotype 02 subgroups based on the strain-specific antigenic determinants; and finally that the serological classification of V. anguillarum serotype 02b strains should be reexamined.Key words: antigenic heterogeneity, lipopolysaccharides, fish immune response.not available

scholarly journals Sinorhizobium fredii HH103 rkp-3 Genes Are Required for K-Antigen Polysaccharide Biosynthesis, Affect Lipopolysaccharide Structure and Are Essential for Infection of Legumes Forming Determinate Nodules

2012 ◽  
Vol 25 (6) ◽  
pp. 825-838 ◽  
Author(s):  
Isabel Margaret ◽  
Juan C. Crespo-Rivas ◽  
Sebastián Acosta-Jurado ◽  
Ana M. Buendía-Clavería ◽  
María T. Cubo ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Cajanus Cajan ◽  
Sinorhizobium Fredii ◽  
Polysaccharide Biosynthesis ◽  
Indeterminate Nodule ◽  
Indeterminate Nodules ◽  
Pseudaminic Acid ◽  
Determinate Nodule ◽  
Static Conditions ◽  
K Antigen

The Sinorhizobium fredii HH103 rkp-3 region has been isolated and sequenced. Based on the similarities between the S. fredii HH103 rkpL, rkpM, rkpN, rkpO, rkpP, and rkpQ genes and their corresponding orthologues in Helicobacter pylori, we propose a possible pathway for the biosynthesis of the S. fredii HH103 K-antigen polysaccharide (KPS) repeating unit. Three rkp-3 genes (rkpM, rkpP, and rkpQ) involved in the biosynthesis of the HH103 KPS repeating unit (a derivative of the pseudaminic acid) have been mutated and analyzed. All the rkp-3 mutants failed to produce KPS and their lipopolysaccharide (LPS) profiles were altered. These mutants showed reduced motility and auto-agglutinated when early-stationary cultures were further incubated under static conditions. Glycine max, Vigna unguiculata (determinate nodule–forming legumes), and Cajanus cajan (indeterminate nodules) plants inoculated with mutants in rkpM, rkpQ, or rkpP only formed pseudonodules that did not fix nitrogen and were devoid of bacteria. In contrast, another indeterminate nodule–forming legume, Glycyrrhiza uralensis, was still able to form some nitrogen-fixing nodules with the three S. fredii HH103 rifampicin-resistant rkp-3 mutants tested. Our results suggest that the severe symbiotic impairment of the S. fredii rkp-3 mutants with soybean, V. unguiculata, and C. cajan is mainly due to the LPS alterations rather than to the incapacity to produce KPS.

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