The type and yield of lipopolysaccharide from symbiotically deficient Rhizobium lipopolysaccharide mutants vary depending on the extraction method

Abstract

At least 18 lipopolysaccharide (LPS) extraction methods are available, and no single method is universally applicable. Here, the LPSs from four R.etli, one R.leguminosarum bv. trifolii mutant, 24AR, and the R.etli parent strain, CE3, were isolated by hot phenol/water (phi;/W), and phenol/EDTA/triethylamine (phi/EDTA/TEA) extraction. The LPS in various preparations was quantified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE), and by immunoblotting. These rhizobia normally have two prominent LPS forms: LPS I, which has O-polysaccharide, and LPS II, which has none. The LPS forms obtained depend on the method of extraction and vary depending on the mutant that is extracted. Both methods extract LPS I and LPS II from CE3. The phi/EDTA/TEA, but not the phi/W, method extracts LPS I from mutants CE358 and CE359. Conversely, the phi;/W but not the phi;/EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide. phi/EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while phi/W extraction gives very small amounts of LPS I. The LPS yield from all the strains using phi/EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi/W extraction are highly variable (850-fold range). The phi/EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the phi/W method partitions LPS II exclusively into the phenol phase, making its recovery difficult. Overall, phi/EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to phi/W extraction. Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method.

Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria

“…The LPS extracted into the phenol phase was treated as described by Carrion et al (23). The LPS was purified from these crude preparations with affinity chromatography using polymyxin B-Sepharose (Pierce) (24,25). Briefly, the crude LPS was dissolved in 50 mM NH 4 CO 3 and applied to the column.…”

ARhizobium leguminosarumAcpXL Mutant Produces Lipopolysaccharide Lacking 27-Hydroxyoctacosanoic Acid

“…LPS extraction and purification. The wild-type and mutant LPSs were first extracted by the TEA/EDTA/ procedure as previously described (43). For each strain, the bacterial pellet (approximately 10 g wet weight) from 8 liters of culture was extracted with 40 ml of TEA/EDTA/ (0.25 M EDTA containing 5% phenol and titrated to pH 6.9 with triethylamine [TEA]) with constant stirring at 37°C for 1 h. The extract was then centrifuged at 10,000 rpm for 1 h, and the supernatant was collected and dialyzed (2,000 molecular weight cutoff; Spectrapor) against deionized water.…”

“…For each strain, the bacterial pellet (approximately 10 g wet weight) from 8 liters of culture was extracted with 40 ml of TEA/EDTA/ (0.25 M EDTA containing 5% phenol and titrated to pH 6.9 with triethylamine [TEA]) with constant stirring at 37°C for 1 h. The extract was then centrifuged at 10,000 rpm for 1 h, and the supernatant was collected and dialyzed (2,000 molecular weight cutoff; Spectrapor) against deionized water. This material was further purified by polymyxin affinity chromatography with polymyxin B-Sepharose (Pierce Chemical Company) as previously described (20,43,46). Briefly, after sample application in 50 mM NH 4 HCO 3 , the column (10-ml bed volume) was sequentially eluted with 30 ml of a solution of 0.3 M TEA adjusted to pH 6.4 with acetic acid plus 10% ethylene glycol, followed by 30 ml of a solution of 2.0 M urea in 0.1 M NH 4 HCO 3 to elute the non-LPS components.…”

The Sinorhizobium meliloti MsbA2 protein is essential for the legume symbiosis

“…A previous study has shown that the ability to visualize polysaccharide alterations in bacterial mutants is often dependent upon the method used for extraction and analysis (Ridley et al, 2000). The polysaccharides isolated into the aqueous and the phenol phases were then resolved by DOC-PAGE and treated with either periodate or alcian blue followed by silver staining.…”