Glycobiology, 2000, Vol. 10, No. 10 1013-1023
© 2000 Oxford University Press
The type and yield of lipopolysaccharide from symbiotically deficient Rhizobium lipopolysaccharide mutants vary depending on the extraction method
Complex Carbohydrate Research Center, University of Georgia, 220 Riverbend Road, Athens, GA 3060 2, USA
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 (
/W), and phenol/EDTA/triethylamine (/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 /EDTA/TEA, but not the /W, method extracts LPS I from mutants CE358 and CE359. Conversely, the /W but not the /EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide. /EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while /W extraction gives very small amounts of LPS I. The LPS yield from all the strains using /EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from /W extraction are highly variable (850-fold range). The /EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the /W method partitions LPS II exclusively into the phenol phase, making its recovery difficult. Overall, /EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to /W extraction. Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method.
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