Partial Characterization of LPSs from Rhizobium fredii USDA 205 Before and After NOD Gene Induction

The lipopolysaccharides (LPS) from Rhizobium fredii USDA 205 and its nod- mutant, HC 205, which has been cured of the 192-MDa symbiotic plasmid, were isolated by the phenol-water extraction method. The LPS from the wild-type strain which was grown in association with host-plant root extract or a flavone analog to a constituent of the root extract, which is required for the induction of at least a portion of the nodulation genes, was also isolated. The conditions required for the induction of the nod genes were determined by work with a nod- mutant of R. fredii USDA 201 which contains a Mu-lac insertion under the control of a nodulation gene promoter. The isolated LPSs were purified by column chromatography and partially analyzed by proton-NMR, gas chromatography (GC), colormetric assays, and polyacrylamide gel electrophoresis (PAGE) employing either sodium dodecyl sulfate (SDS) or 7-deoxycholic acid (DOC) as a detergent. Significant differences were seen in the DOC-PAGE banding patterns of the induced LPS samples when compared to the non-induced LPSs. The most important distinction being a relative reduction in overall LPS molecular weight by discreet increments for the induced LPS from USDA 205. This was accompanied by small molecular weight differences in the predominant bands of similar molecular weight. GC and colormetric analysis indicate that the polysaccharide portion of the LPSs of all samples is composed predominantly of galactose and 2-keto-3-deoxyoctonic acid (KDO), a common constituent of gram-negative bacterial LPSs. There were some compositional differences between the induced and non-induced LPSs but the significance of these differences remains unclear at this time

Lipopolysaccharide Core Structures in \u3cem\u3eRhizobium etli\u3c/em\u3e and Mutants Deficient in \u3cem\u3eO\u3c/em\u3e-Antigen

Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane, and for Rhizobium spp. has been shown to play a critical role in the establishment of an effective nitrogen-fixing symbiosis with a legume host. Many genes required for O-chain polysaccharide synthesis are in the lps α region of the CE3 genome; this region may also carry lps genes required for core oligosaccharide synthesis. The LPSs from several strains mutated in the α region were isolated, and their mild acid released oligosaccharides, purified by high performance anion-exchange chromatography, were characterized by electrospray- and fast atom bombardment-mass spectrometry, NMR, and methylation analysis. The LPSs from several mutants contained truncated O-chains, and the core region consisted of a (3-deoxy-D-manno-2-octulosomic acid) (Kdo)-(26)-α-Galp-(16)-[α-GalpA-(14)]-α-Manp-(15)-Kdop (3-deoxy-D-manno-2-octulosomic acid) (Kdo)pentasaccharide and a α-GalpA-(14)-[α-GalpA-(15)]-Kdop trisaccharide. The pentasaccharide was altered in two mutants in that it was missing either the terminal Kdo or the GalA residue. These results indicate that the lps α region, in addition to having the genes for O-chain synthesis, contains genes required for the transfer of these 2 residues to the core region. Also, the results show that an LPS with a complete core but lacking an O-chain polysaccharide is not sufficient for an effective symbiosis

Enzyme-Synthesized Highly Branched Maltodextrins Have Slow Glucose Generation at the Mucosal α-Glucosidase Level and Are Slowly Digestible In Vivo.

For digestion of starch in humans, α-amylase first hydrolyzes starch molecules to produce α-limit dextrins, followed by complete hydrolysis to glucose by the mucosal α-glucosidases in the small intestine. It is known that α-1,6 linkages in starch are hydrolyzed at a lower rate than are α-1,4 linkages. Here, to create designed slowly digestible carbohydrates, the structure of waxy corn starch (WCS) was modified using a known branching enzyme alone (BE) and an in combination with β-amylase (BA) to increase further the α-1,6 branching ratio. The digestibility of the enzymatically synthesized products was investigated using α-amylase and four recombinant mammalian mucosal α-glucosidases. Enzyme-modified products (BE-WCS and BEBA-WCS) had increased percentage of α-1,6 linkages (WCS: 5.3%, BE-WCS: 7.1%, and BEBA-WCS: 12.9%), decreased weight-average molecular weight (WCS: 1.73×108 Da, BE-WCS: 2.76×105 Da, and BEBA-WCS 1.62×105 Da), and changes in linear chain distributions (WCS: 21.6, BE-WCS: 16.9, BEBA-WCS: 12.2 DPw). Hydrolysis by human pancreatic α-amylase resulted in an increase in the amount of branched α-limit dextrin from 26.8% (WCS) to 56.8% (BEBA-WCS). The α-amylolyzed samples were hydrolyzed by the individual α-glucosidases (100 U) and glucogenesis decreased with all as the branching ratio increased. This is the first report showing that hydrolysis rate of the mammalian mucosal α-glucosidases is limited by the amount of branched α-limit dextrin. When enzyme-treated materials were gavaged to rats, the level of postprandial blood glucose at 60 min from BEBA-WCS was significantly higher than for WCS or BE-WCS. Thus, highly branched glucan structures modified by BE and BA had a comparably slow digesting property both in vitro and in vivo. Such highly branched α-glucans show promise as a food ingredient to control postprandial glucose levels and to attain extended glucose release

Partial Characterization of LPSs from Rhizobium fredii USDA 205 Before and After NOD Gene Induction

The lipopolysaccharides (LPS) from Rhizobium fredii USDA 205 and its nod- mutant, HC 205, which has been cured of the 192-MDa symbiotic plasmid, were isolated by the phenol-water extraction method. The LPS from the wild-type strain which was grown in association with host-plant root extract or a flavone analog to a constituent of the root extract, which is required for the induction of at least a portion of the nodulation genes, was also isolated. The conditions required for the induction of the nod genes were determined by work with a nod- mutant of R. fredii USDA 201 which contains a Mu-lac insertion under the control of a nodulation gene promoter. The isolated LPSs were purified by column chromatography and partially analyzed by proton-NMR, gas chromatography (GC), colormetric assays, and polyacrylamide gel electrophoresis (PAGE) employing either sodium dodecyl sulfate (SDS) or 7-deoxycholic acid (DOC) as a detergent. Significant differences were seen in the DOC-PAGE banding patterns of the induced LPS samples when compared to the non-induced LPSs. The most important distinction being a relative reduction in overall LPS molecular weight by discreet increments for the induced LPS from USDA 205. This was accompanied by small molecular weight differences in the predominant bands of similar molecular weight. GC and colormetric analysis indicate that the polysaccharide portion of the LPSs of all samples is composed predominantly of galactose and 2-keto-3-deoxyoctonic acid (KDO), a common constituent of gram-negative bacterial LPSs. There were some compositional differences between the induced and non-induced LPSs but the significance of these differences remains unclear at this time

Chemical and rheological properties of bacterial succinoglycan with distinct structural characteristics

Succinoglycans are bacterial exopolysaccharides with an octasaccharide repeating unit, composed of glucose and galactose in a 7:1 molar ratio of, and non-carbohydrate substituents, including pyruvate, succinate and acetate. The succinoglycans produced by four different strains of Sinorhizobium meliloti, gram-negative soil bacteria, were analyzed for their molecular weight distribution and degree of non-carbohydrate substitution, as well as their chemical properties were related to their rheological properties. These results showed that the ratio of high molecular weight to low molecular weight succinoglycan was varied from 0.50 to 2.36. Degree of succinylation among the bacterial strains was in the range of 0.30-1.90. Therefore, we concluded that each strain produced succinoglycans with different average degrees of polymerization and succinylation: and that these characteristics were correlated to the theological properties of the solutions. The effect of molecular weight on the theological properties appeared to be less than that of the succinyl group abundance. Published by Elsevier Ltd

Chronic intracellular infection of alfalfa nodules by Sinorhizobium meliloti requires correct lipopolysaccharide core

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

Identification of a Plasmid-Borne Locus in Rhizobium etli KIM5s Involved in Lipopolysaccharide O-Chain Biosynthesis and Nodulation of Phaseolus vulgaris

Screening of derivatives of Rhizobium etli KIM5s randomly mutagenized with mTn5SSgusA30 resulted in the identification of strain KIM-G1. Its rough colony appearance, flocculation in liquid culture, and Ndv(−) Fix(−) phenotype were indicative of a lipopolysaccharide (LPS) defect. Electrophoretic analysis of cell-associated polysaccharides showed that KIM-G1 produces only rough LPS. Composition analysis of purified LPS oligosaccharides from KIM-G1 indicated that it produces an intact LPS core trisaccharide (α-d-GalA-1→4[α-d-GalA-1→5]-Kdo) and tetrasaccharide (α-d-Gal-1→6[α-d-GalA-1→4]-α-d-Man-1→5Kdo), strongly suggesting that the transposon insertion disrupted a locus involved in O-antigen biosynthesis. Five monosaccharides (Glc, Man, GalA, 3-O-Me-6-deoxytalose, and Kdo) were identified as the components of the repeating O unit of the smooth parent strain, KIM5s. Strain KIM-G1 was complemented with a 7.2-kb DNA fragment from KIM5s that, when provided in trans on a broad-host-range vector, restored the smooth LPS and the full capacity of nodulation and fixation on its host Phaseolus vulgaris. The mTn5 insertion in KIM-G1 was located at the N terminus of a putative α-glycosyltransferase, which most likely had a polar effect on a putative β-glycosyltransferase located downstream. A third open reading frame with strong homology to sugar epimerases and dehydratases was located upstream of the insertion site. The two glycosyltransferases are strain specific, as suggested by Southern hybridization analysis, and are involved in the synthesis of the variable portion of the LPS, i.e., the O antigen. This newly identified LPS locus was mapped to a 680-kb plasmid and is linked to the lpsβ2 gene recently reported for R. etli CFN42

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGRΩfixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed in nodule cells infected by the fixF mutant. LPSs were purified from free-living NGR234 cultured in the presence of apigenin. Structural analyses showed that the polysaccharide portions of these LPSs are specialized, rhamnose-containing O antigens attached to a modified core-lipid A carrier. The primary sequence of the O antigen is [-3)-α-l-Rhap-(1,3)-α-l-Rhap-(1,2)-α-l-Rhap-(1-](n), and the LPS core region lacks the acidic sugars commonly associated with the antigenic outer core of LPS from noninduced cells. This rhamnan O antigen, which is absent from noninduced cells, has the same primary sequence as the A-band O antigen of Pseudomonas aeruginosa, except that it is composed of l-rhamnose rather than the d-rhamnose characteristic of the latter. It is noteworthy that A-band LPS is selectively maintained on the P. aeruginosa cell surface during chronic cystic fibrosis lung infection, where it is associated with an increased duration of infection