Separation and characterization of two chemically distinct lipopolysaccharides in two Pectinatus species

Abstract

Lipopolysaccharides (LPS) from the type strains of the anaerobic beer spoilage bacteria Pectinatus cerevisiiphilus and P. frisingensis were extracted with the 5:5:8 volume ratio modification of the phenolchloroform-petroleum ether method (H. Brade and C. Galanos, Eur. J. Biochem. 122:233-237, 1982). Sequential precipitations of LPS with water and acetone from the phenol phase yielded LPS which differed in that water-precipitable material (LPS-H20; 0.1 to 0.4% of the dry weight of the cells) was rough-type LPS, whereas acetone-precipitable material (LPS-Ac; 4.6 to 5.8% of the dry weight) contained both rough-type LPS and high-molecular-weight material resembling smooth LPS. The LPS were chemically characterized, and they contained D-glucosamine, 4-amino-4-deoxy-L-arabinose, 3-deoxy-D-manno-2-octulosonic acid, D-fucose, D-galactose, D-glucose, D-mannose, and phosphate. D-Fucose was present mostly in LPS-Ac, suggesting that it is a constituent of the 0 antigen. The major fatty acids were ester-and amide-linked (R)-3-hydroxytridecanoic and ester-linked undecanoic acids, with minor amounts of ester-linked tridecanoic and (R)-3-hydroxyundecanoic acids. The chemical compositions of LPS-H20 and LPS-Ac suggested that they differ not only in their smooth or rough nature but also in the structure of their core regions. This may explain their different precipitabilities from the extraction mixture. The extraction method was also shown to be applicable to the isolation of smooth-type LPS from Salmonella enterica serovar Typhimurium. Extraction of two Typhimurium strains carrying chemically different 0 antigens resulted in high yields (8% of the dry weight) of LPS. Strain SH2183, which contains the relatively hydrophobic 0-4,5,12 antigen yielded almost exclusively LPS-Ac, whereas the LPS of strain SH5770, which has a hydrophilic 0-6,7 antigen, was exclusively LPS-H20. No fractionation to smooth and rough LPS occurred with the Typhimurium strains.The gram-negative genus Pectinatus consists of obligately anaerobic rod-shaped bacteria which have been identified as spoilage-causing organisms in packaged beer (1,12,25,45). Since their initial description and establishment of the genus and the species Pectinatus cerevisiiphilus (25), another species, P. frisingensis, has been described (41).Lipopolysaccharides (LPS), also termed endotoxins, are structural components of the outer membrane of all gramnegative bacteria (for reviews, see references 27, 33, and 38). Since the chemical composition and structure of LPS show large variation between bacterial genera (27), the composition of LPS can serve as a taxonomic criterion. For this purpose, we previously performed a preliminary compositional analysis of the LPS of four isolates of Pectinatus, then classified as the species P. cerevisiiphilus (15). That study revealed exceptional properties in the LPS, such as the predominance of fatty acids with odd-numbered chain lengths of 11 and 13 carbons and the lack of heptose, a sugar which is commonly present in LPS. Because of the general in...

4‐O‐(2‐Amino‐2‐deoxy‐α‐d‐glucopyranosyl)‐3‐deoxy‐d‐manno‐2‐octulosonic acid, a constituent of lipopolysaccharides of the genus Pectinatus

“…This disaccharide is obviously a structural element of the core oligosaccharide, since it was detected in both R-type and S-type lipopolysaccharide obtained from both Pectinatus strains. The existence of phosphorylated GlcpN in the core explains our earlier finding that after strong acid hydrolysis of lipopolysaccharide as much as 40% of the GlcN was detected as GlcN phosphate [3]. Our results show that the presence of a-~-GlcpN-(1'-+4)-Kdo disaccharide is not the decisive factor in the previously reported 131 fractionation into S-type and R-type lipopolysaccharide.…”

“…These data suggest that in the intact lipopolysaccharide the disaccharide carried a hydrofluoric-acid-labile and alkali-labile substituent, probably a phosphate residue. It was obvious that the hexosamine component of the disaccharide was GlcN, since this was the only type of hexosamine detectable in the lipopolysaccharide [3]. The disaccharide was found in almost equal proportions in both R-type and S-type lipopolysaccharides of l?…”

“…frisingensis ATCC 33332T by the phenol/chloroform/petroleum-ether method (5 : 5 :8, by vol. modification [4]) as described previously [3]. By sequential precipitations with water and acetone, R-type and S-type lipopolysaccharides, respectively, were obtained from both species.…”

“…Both species are strict anaerobes, and they occur as contaminants and spoilage organisms in packaged beer [ 1, 21. The cell-surface lipopolysaccharides of the type strains of Pectinatus were recently reported to possess exceptional heterogeneity, and in fact two distinct lipopolysaccharide populations could be extracted from both species [3]. One of these lipopolysaccharides was of low-molecular-mass (rough, R) type, whereas the other contained additional sugar chains resembling smooth ( S ) lipopolysaccharide.…”

Cellular fatty acyl and alkenyl residues in Megasphaera and Pectinatus species: contrasting profiles and detection of beer spoilage

“…In this study we have analysed cellular fatty acids of M. cerevisiae, about which there is no previous information. Both species of the genus Pectinatus were also analysed, since there are contradictory data concerning the fatty acid composition; a recent report of the cellular fatty acids of Pectinatus (Moore et al, 1994) does not mention, for instance, the major 3-hydroxy fatty acid that we have described as a component of the lipopolysaccharide (LPS) of P. cerevisi$bilt/s and P. frisitzgensis (Helander et al, 1992(Helander et al, , 1994. …”