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

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 by. trifolii mutant, 24AR, and the R.etli parent stra in, CE3, were isolated by hot phenol/water (phi /W), and phenol/EDTA/trieth ylamine (phi /EDTA/TEA) extraction. The LPS in various preparations was qua ntified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-P AGE), 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 C E3, The phi /EDTA/TEA, but not the phi /W, method extracts LPS I from mutan ts 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, whil e phi /W extraction gives very small amounts of LPS I. The LPS yield from a ll the strains using phi /EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi /W extraction are highly variable (850-fo ld range). The phi /EDTA/TEA method extracts LPS I and LPS II from mutant 2 4AR, but the phi /W method partitions LPS II exclusively into the phenol ph ase, making its recovery difficult. Overall, phi /EDTA/TEA extraction yield s more forms of LPS from the mutants and provides a simpler, faster, and le ss hazardous alternative to phi /W extraction. Nevertheless, it is conclude d that careful analysis of any LPS mutant requires the use of more than one extraction method.