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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.

The biological activity of reducing-end-modified oligogalacturonides was quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with the C-1 of their reducing end (a) covalently linked to a biotin hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to (a) alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by suspension-cultured cv Samsun cells, (c) elicit extracellular alkalinization by suspensioncultured N. tabacum cv Xanthi cells, and (d) elicit H 2 O 2 accumulation in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end is a key element for the recognition of oligogalacturonides in these systems. However, the degree of reduction in biological activity depends on the tissue culture system used and on the nature of the specific reducing-end modification. These results suggest that oligogalacturonides are perceived differently in each tissue culture system.Carbohydrates that act as signal molecules in plants (oligosaccharins) have been isolated from plant and fungal cell wall polysaccharides, from the cell walls of bacterial symbionts of plants, and from fungal glycopeptides (for review, see Ryan and Farmer, 1991;Darvill et al., 1992;Cô té and Hahn, 1994). We are interested in determining the mechanism by which one type of the plant cell wallderived oligosaccharins, the oligogalacturonides, elicit biological responses in plants.The biological effects elicited in plants by oligosaccharins are diverse (for review, see Ryan and Farmer, 1991;Darvill et al., 1992;Cô té and Hahn, 1994) but can generally be placed in two groups: delayed responses and rapid responses. Delayed responses are usually observed hours or days after oligosaccharin treatment and are often directly involved in adaptation to environmental conditions, whereas rapid responses generally occur at the plant cell surface and are observed within minutes after addition of oligosaccharins. The delayed responses elicited by oligogalacturonides can be broadly divided into those in which defense responses are induced and those in which growth and development are modified. The defense-related responses, depending on the plant species, include phytoalexin accumulation (Hahn et al., 1981), lignification of cell walls (Robertsen, 1986), and proteinase inhibitor accumulation (Bishop et al., 1984). The responses involving growth and development include induction of ethylene in tomato fruit (Brecht and Huber, 1988), inhibition of auxin-induced pea stem elongation (Branca et al., 1988), and regulation of tobacco (Nicotiana tabacum) TCL explant morphogenesis (Eberhard et al., 1989). The rapid responses induced by oligogalacturonides include enhanced protein phosp...

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Preparative gel electrophoresis was used to separate and purify extracellular, capsular and lipopolysaccharides (EPSs, CPSs, and LPSs, respectively) from crude bacterial extracts. The procedure effectively separates CPS from LPSs. In addition discreet size ranges of these various polysaccharides can be isolated. The 'rough' (R-type), 'smooth' (S-type), and 'semi-smooth' LPSs were separated from one another. In addition different size classes of 'semi-smooth', or S-type LPS, can be separated. This procedure was demonstrated for diverse bacterial species, including the soil bacteria Rhizobium fredii, and the enteric bacterial species, Salmonella enteritidis and Proteus mirabilis. In the latter case, it was also possible to separate capsular polysaccharide from its lipid-bound form.

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