The polysaccharide components from cultured cells of Rhizobium fredii USDA205 and Rhizobium meliloti AK631 were extracted with hot phenol-water and separated by repetitive gel filtration chromatography. Polyacrylamide gel electrophoresis, nuclear magnetic resonance spectrometry, and gas chromatography analyses showed that both of these bacterial species produce unique polysaccharides that contain a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). These polysaccharides, which constituted a major portion of the extracted carbohydrate, are not excreted into the growth media (i.e., they are not extracellular polysaccharides) and are structurally distinct from the lipopolysaccharides. The primary structure of the preponderant polysaccharide from R. fredii USDA205 was determined by high-performance anion-exchange liquid chromatography, nuclear magnetic resonance spectrometry, fast atom bombardment-mass spectrometry, and gas chromatography-mass spectrometry; it consists of repeating units of [-- The gram-negative bacteria of the Rhizobiaceae family include the nitrogen-fixing symbionts of legumes (Rhizobium,Azorhizobium, and Bradyrhizobium spp.) and the pathogenic Agrobacterium genus. These species produce various types of extracellular polysaccharides (EPSs) and cell surface polysaccharides that appear to be important factors in symbiotic and pathogenic effectiveness.Lipopolysaccharides (LPSs), unique to gram-negative bacteria, are outer membrane inclusions that consist of (i) a glycolipid membrane anchor, which contains characteristic Il-hydroxy fatty acids on a glucosamine backbone (lipid A);(ii) a core oligosaccharide that is linked to the lipid A via 3-deoxy-D-manno-2-octulosonic acid (Kdo) and is highly conserved within a species; and (iii) a highly variable 0 antigen. Polyacrylamide gel electrophoresis (PAGE)-silver stain analyses of rhizobial LPSs frequently result in banding regions (5, 9), indicating that the 0 antigens of these LPSs may commonly consist of oligosaccharides of specific lengths.Rhizobia also produce acidic EPSs and cyclic P-glucans.EPSs are secreted compounds and are isolated from the growth media of cultured cells. The EPSs produced by rhizobia are high-molecular-weight heteropolysaccharides composed of small repeating units that contain either uronic acids or noncarbohydrate substituents, such as succinate and pyruvate, as the acidic functions (4, 22, 29). The cyclic 3-glucans are commonly harbored within the periplasmic space but may also be secreted; consequently, they are found in both the growth media and the bacterial pellet of cultured cells (42). The role of the 13-glucans in symbiosis remains a moot subject. * Corresponding author.Numerous studies have shown that bacterial LPS and EPS are important in the infection and nodulation processes; however, the symbiotic phenotype of LPS-and EPS-defective microsymbionts appears to depend on the type of nodule formed by the host.(i) Inoculation of determinate nodule-forming host plants with rhizobial LPS mutants that are affected ...

The hypothesis of increasing the branch density of starch to reduce its digestion rate through partial shortening of amylopectin exterior chains and the length of amylose was investigated. Starch products prepared using -amylase, -amylase and transglucosidase, maltogenic R-amylase, and maltogenic R-amylase and transglucosidase showed significant reduction of rapidly digested starch by 14.5%, 29.0%, 19.8%, and 31.0% with a concomitant increase of slowly digested starch by 9.0%, 19.7%, 5.7%, and 11.0%, respectively. The resistant starch content increased from 5.1% to 13.5% in treated starches. The total contents of the prebiotics isomaltose, isomaltotriose, and panose (Isomaltooligosaccharides) were 2.3% and 5.5%, respectively, for -amylase/transglucosidase-and maltogenic R-amylase/transglucosidase-treated starches. The molecular weight distribution of enzyme-treated starches and their debranched chain length distributions, analyzed using high-performance sizeexclusion chromatography with multiangle laser light scattering and refractive index detection (HPSEC-MALLS-RI) and HPSEC-RI, showed distinctly different patterns among starches with different enzyme treatments. A larger proportion of low molecular weight fractions appeared in starches treated additionally with transglucosidase. All enzyme-treated starches showed a mixture of B-and V-type X-ray diffraction patterns, and 1 H NMR spectra showed a significant increase of R-1,6 linkages. Both the increase of the starch branch density and the crystalline structure in the treated starches likely contribute to their slow digestion property.

No abstract

Effective invasion of alfalfa by Rhizobium meliloti RmlO21 normally requires the presence of succinoglycan, an exopolysaccharide (EPS) produced by the bacterium. However, RmlO21 has the ability to produce a second EPS (EPS II) that can suppress the symbiotic defects of succinoglycan-deficient strains. EPS II is a polymer of modified glucose-(f8-1,3)-galactose subunits and is produced by RmlO21 derivatives carrying either an expRiO0 or mucR mutation. If the ability to synthesize succinoglycan is blocked genetically, expRiOl derivatives of Rm1O21 are nodulationproficient, whereas mucR derivatives of RmlO21 are not. The difference in nodulation proficiency between these two classes of EPS 11-producing strains is due to the specific production of a low molecular weight form of EPS H by expR101 strains. A low molecular weight EPS II fraction consisting of 15-20 EPS II disaccharide subunits efficiently allows nodule invasion by noninfective strains when present in amounts as low as 7 pmol per plant, suggesting that low molecular weight EPS II may act as a symbiotic signal during infection.The soil bacterium Rhizobium meliloti fixes nitrogen in symbiotic association with the leguminous plant Medicago sativa (alfalfa). Molecular analyses have revealed that early steps in establishment of this symbiosis depend upon an exchange of small signaling molecules between the two partners (1, 2). Bacteria invade root nodules via a tube of plant origin called the infection thread. This process depends upon the production of exopolysaccharides (EPSs) by K meliloti (3), but the role of EPSs in invasion is not yet clear.Studies focusing on the acidic Calcofluor-binding EPS (succinoglycan) of R. meliloti strain RmlO21 have clearly shown that the EPS is normally required for effective nodulation (4). Succinoglycan is a polymer of octasaccharide repeating units, each consisting of a backbone of three glucoses and one galactose, a side chain of four glucoses, and succinyl, acetyl, and 1-carboxyethylidene (pyruvyl) modifications in a ratio of approximately 1:1:1 (5-7). R meliloti RmlO21 mutants that fail to synthesize succinoglycan form empty nodules that are devoid of bacteria and bacteroids and are unable to fix nitrogen refs. 3 and 8). Genetic analyses led to the identification of a 25-kb cluster of exo genes (9-13) on the second symbiotic megaplasmid that are required for the production of succinoglycan (3,14,15), and biosynthetic roles have been assigned to most of these exo gene products (16). It has been reported that low molecular weight succinoglycan can partially rescue the symbiotic deficiencies of exo mutants (17,18). The active species was tentatively assigned as a tetramer of the octasaccharide subunit, but the exact structure is not known (17). Glazebrook and Walker (19) reported that R. meliloti RmlO21 derivatives carrying the expRi01 mutation synthesize a second EPS (EPS II) that is composed of disaccharide repeats of glucose and galactose carrying acetyl and pyruvyl modifications on the glucose and galactose, r...

Our analyses of lipopolysaccharide mutants of Sinorhizobium meliloti offer insights into how this bacterium establishes the chronic intracellular infection of plant cells that is necessary for its nitrogen-fixing symbiosis with alfalfa. Derivatives of S. meliloti strain Rm1021 carrying an lpsB mutation are capable of colonizing curled root hairs and forming infection threads in alfalfa in a manner similar to a wild-type strain. However, developmental abnormalities occur in the bacterium and the plant at the stage when the bacteria invade the plant nodule cells. Loss-of-function lpsB mutations, which eliminate a protein of the glycosyltransferase I family, cause striking changes in the carbohydrate core of the lipopolysaccharide, including the absence of uronic acids and a 40-fold relative increase in xylose. We also found that lpsB mutants were sensitive to the cationic peptides melittin, polymyxin B, and poly-L-lysine, in a manner that paralleled that of Brucella abortus lipopolysaccharide mutants. Sensitivity to components of the plant's innate immune system may be part of the reason that this mutant is unable to properly sustain a chronic infection within the cells of its host-plant alfalfa.

Formation of nitrogen-fixing nodules on legume roots by Rhizobium sp. NGR234 requires an array of bacterial factors, including nodulation outer proteins (Nops) secreted through a type III secretion system (TTSS). Secretion of Nops is abolished upon inactivation of ttsI (formerly y4xI), a protein with characteristics of two-component response regulators that was predicted to activate transcription of TTSS-related genes. During the symbiotic interaction, the phenotype of NGR omega ttsI differs from that of a mutant with a nonfunctional secretion machine, however. This indicated that TtsI regulates the synthesis of other symbiotic factors as well. Conserved sequences, called tts boxes, proposed to act as binding sites for TtsI, were identified not only within the TTSS cluster but also in the promoter regions of i) genes predicted to encode homologs of virulence factors secreted by pathogenic bacteria, ii) loci involved in the synthesis of a rhamnose-rich component (rhamnan) of the lipopolysaccharides (LPS), and iii) open reading frames that play roles in plasmid partitioning. Transcription studies showed that TtsI and tts boxes are required for the activation of TTSS-related genes and those involved in rhamnose synthesis. Furthermore, extraction of polysaccharides revealed that inactivation of ttsI abolishes the synthesis of the rhamnan component of the LPS. The phenotypes of mutants impaired in TTSS-dependent protein secretion, rhamnan synthesis, or in both functions were compared to assess the roles of some of the TtsI-controlled factors during symbiosis.

When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron, we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs) were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome.

The rhizobial production of extracellular polysaccharide (EPS) is generally required for the symbiotic infection of host plants that form nodules with an apical meristem (indeterminate nodules). One exception isRhizobium meliloti AK631, an exoB mutant of Rm41, which is deficient in EPS production yet infects and fixes nitrogen (i.e., is Fix ؉ ) on alfalfa, an indeterminate nodule-forming plant. A mutation of lpsZ in AK631 results in a Fix ؊ strain with altered phage sensitivity, suggesting that a cell surface factor may substitute for EPS in the alfalfa-AK631 symbiosis. Biochemical analyses of the cell-associated polysaccharides of AK631 and Rm5830 (AK631 lpsZ) demonstrated that the lpsZ mutation affected the expression of a surface polysaccharide that is analogous to the group II K polysaccharides of Escherichia coli; the polysaccharide contains 3-deoxy-D-manno-2-octulosonic acid or a derivative thereof in each repeating unit. Rm5830 produced a polysaccharide with altered chromatographic and electrophoretic properties, indicating a difference in the molecular weight range. Similar results were obtained in a study of Rm1021, a wild-type isolate that lacks the lpsZ gene: the introduction of lpsZ into Rm1021 exoB (Rm6903) both suppresses the Fix ؊ phenotype and results in a modified expression of the K polysaccharide. Chromatography and electrophoresis analysis showed that the polysaccharide extracted from Rm6903 lpsZ ؉ differed from that of Rm6903 in molecular weight range. Importantly, the effect of LpsZ is not structurally specific, as the introduction of lpsZ ؉ into Rhizobium fredii USDA257 also resulted in a molecular weight range change in the structurally distinct K polysaccharide produced by that strain. This evidence suggests that LpsZ has a general effect on the size-specific expression of rhizobial K polysaccharides.Bradyrhizobium and Rhizobium spp. (rhizobia) are gramnegative bacteria that participate in a mutualistic association with leguminous plants. The establishment of this association involves a molecular interaction between the host plant and the microsymbiont: flavonoid molecules that are secreted by the host plant elicit specific responses in the microsymbiont, including the activation of the nodulation (nod) genes. The nod gene products then synthesize structurally specific lipo-oligosaccharide molecules (Nod factors), first described LeRouge et al. (14), which induce root hair deformation and cortical cell division in the region of nodule development (5,6,10). In addition to Nod factor activity, numerous studies (reviewed in references 8, 11, and 16) have established correlations between effective infection and the specific production of bacterial lipopolysaccharides (LPSs) and extracellular polysaccharides (EPSs). A distinguishing feature of indeterminate nodulation, in which a nodule-specific apical meristem is formed, is a general requirement for EPS production by the microsymbiont; EPS Ϫ mutants do not infect indeterminate nodule-forming host plants but effectively nodulate determinate ...