J. Gen. App!. Microbiol., 39, 547-557 (1993) ISOLATION AND CHEMICAL CHARACTERIZATION OF LIPOPOLYSACCHARIDES FROM FOUR AQUASPIRILL UM SPECIES (A. ITERSONH SUBSP. NIPPONICUM IFO 13615, A. POLYMORPHUM IFO 13961, A. AQUA TICUM IFO 14918, A. METAMORPHUM IFO 13960 AND A. ME TAMORPHUM MUTANT STRAIN 12-3) HEIKE RAU, TAKESHI SAKANE,' AKIRA YOKOTA,' AND HUBERT MAYER* Max-Planck-Institut fur Immunbio!ogie, W-79108 Freiburg i. Br., Germany 'Institute for Fermentation , Osaka, Yodogawa-ku, Osaka 532, Japan (Received June 17, 1993) Lipopolysaccharides (LPSs), isolated from fourAquaspirillum species, i.e. the type strains of A. itersonii subsp. nipponicum, A. polymorphum, A. aquaticum and A. metamorphum together with A. metamorphum mutant strain 12-3 were characterized onto their chemical composition. A. itersonii subsp. nipponicum IFO 13615 and A. polymorphum IFO 13961, both belonging to the a-1 subgroup of Proteobacteria, possess L-glycero-D- manno-heptose, glucuronic acid and galacturonic acid in their LPS and both have 3-hydroxy-tetradecanoic acid as hydroxylated fatty acid. A. itersonii subsp. nipponicum has additionally 3-hydroxy-hexadecanoic acid and a small amount of 3-hydroxy-octadecanoic acid. In the LPSs of A. aquaticum IFO 14918 and both A. metamorphum IFO 13960 strains, which belong to the /3-1 subgroup of Proteobacteria, neither heptoses nor uronic acids could be detected. The main sugar components were rhamnose and glucose and both possess the same three major fatty acid constituents dodecanoic acid, tetradecanoic acid and 3-hydroxydecanoic acid. From the DOC-PAGE pattern it was evident that A. itersonii subsp. nipponicum and A. polymorphum have a similar LPS-type as revealed for two Rhodospirillum strains, R. fulvum DSM 117 and R. molischianum NTHC 131. These strains being members of the a-1 group of Proteobac- teria possess LPSs which show distinct gaps between the band of R-type LPS and other slower-moving bands. In the case of A. metamorphum * Address reprint requests to: Dr . Hubert Mayer, Max-Planck-Institut fur Immunbiologie, Stubeweg 51, W-79108 Freiburg i. Br., Germany. 547 548 RAU et al. VOL. 39 such gap was also visible. The LPS of A. aquaticum showed R-type character on DOC-PAGE. Lipopolysaccharides (LPSs), main components of the Gram-negative cell wall, are amphiphilic glycoconjugates, consisting of a lipid component, lipid A, and a hydrophilic polysaccharide part, which can be subdivided into the 0-specific chain and the core oligosaccharide (23). Lipid A, as structural unit of LPS, is responsible for the manifold endotoxic properties and the other biological activities, such as lethal toxicity, pyrogenicity, local Shwartzman-reactivity, reactivity in the Limulus- test system. It induces TNF-activity, B-cell-mitogenicity, and synthesis of pro- staglandin in macrophages (6). Although lipid A is the phylogenetically most conserved part of lipopolysaccharide molecule, a lot of structural variants were found (16,17). In many cases LPS and its lipid A part can be used as markers with regard to possible phylogenetical relations existing between bacterial species. The genus Spirillum, containing different aerobic and microaerophilic spirilla, was previously divided into 3 genera named Aquaspirillum, Oceanospirillum and Spirillum (8). The members of the genus Aquaspirillum are aerobic fresh water spirilla, which cannot grow even under rather moderate halophilic conditions (3% NaCI). The cells are helical with 0.2 to 1.5,um in diameter. They move by means of fascicles of flagella at one or both poles. These Gram-negative microorganisms grow chemoorganotroph and aerob, having a strictly respiratory metabolism with oxygen as the terminal electron acceptor (8). The G + C content of DNA is 49-65 mol% (11,12). The lipopolysaccharide of five different Aquaspirillum strains Aquaspirillum itersonii subsp. nipponicum IFO 13615, Aquaspirillum polymorphum IFO 13961, Aquaspirillum aquaticum IFO 14918, Aquaspirillum metamorphum IFO 13960 and its R-mutant (strain 12-3), all obtained from the strain collection of Institute for Fermentation, Osaka-were investigated. A. itersonii subsp. nipponicum IFO 13615 and A. polymorphum IFO 13961 belong together with the Rhodospirillum species, Rhodopseudomonas globiformis and Azospirillum brasilense to the a-1 group of the Proteobacteria basing on 16S-rRNA-catalog data (31). The two other Aquaspiril- lum strains, A. aquaticum IFO 14918 and A. metamorphum IFO 13960, belong to the $-1 subgroup of Proteobacteria, which has been designated as rRNA- superfamily III (29, 30, 32). The name Aquaspirillum aquaticum will not anymore be used. Aquaspirillum aquaticum has recently been renamed as Comamonas terrigena and forms together with Comamonas acidovorans and Comamonas testost- eroni the genus Comamonas, which represents the Acidovorans-Cluster or /3-1 group of Proteobacteria (21, 29, 30, 32). Seven Aquaspirillum strains (A. metamorphum, A. anulus, A. delicatum, A. giesbergeri, A. gracile, A. psychrophilum and A. sinu- osum) belong to the Comamonadaceae. They have not yet been renamed, since phenotypic data are not yet available. Data on their quinone composition support the phylogenetical data. The two members of the a-1 group have ubiquinone-10; 1993 Lipopolysaccharides from Aquaspirillum Species 549 the two Aquaspirillum strains belonging to the /3-1 group have ubiquinone-8 (Sakane and Yokota, in preparation). The present investigation will provide further arguments based on LPS composition for the classification of four Aquaspi- rillum strains in two different subdivisions of the Proteobacteria. MATERIALS AND METHODS Bacterial strains and growth conditions. Aquaspirillum aquaticum IF014918T ( = ATCC 11330T) (T, type strain), Aquaspirillum metamorphum IFO 13960T ( =ATCC 15280T), Aquaspirillum itersonii subsp. nipponicum IFO 13615T ( =ATCC 11332T) were obtained from the Culture Collection of the Institute for Fermentation, Osaka (Osaka, Japan). A strain 12-3 was obtained as a spontaneous mutant of A. metamorphum IFO 13960T, which is deficient in S-layer protein (Sakane and Yokota, unpublished). Aquaspirillum strains were cultured aero- bically at 28°C in a medium containing: peptone 0.5%, yeast extract 0.1 %, sodium succinate 0.2%, pH 7.0. Preparation of LPS. LPS was by the phenol-water extracted of Westphal and Jann (28) and followed by repeated centrifugation at 105,000 X g for 4 h. In the case of R-type LPSs (A. metamorphum mutant strain 12-3), the combined phenol- water/phenol-chloroform-petroleum ether (PCP) extraction procedure according to Galanos et al. ( 7) was used to obtained pure materials. Sodium deoxycholatepolyacrylamide gel electrophoresis (DOC-PAGE). DOC- PAGE of isolated lipopolysaccharide was carried out according to the method of Komuro and Galanos (10). The gels were silver-stained after oxidation with periodic acid (27) or stained with periodic acid/Schiffs reagent (PAS) (5,25). SDSpolyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE of whole cells was carried out according to the method of Laemmli (13). The gel was stained with Coomassie blue. Analytical procedures. Fatty acids, liberated by 1 M McOH/HC1 (85°C, 16h) as their methylester derivatives, were quantified by combined gas liquid chromatog- raphy-mass spectrometry (GC-MS) on a fused silica DB WAX (Fissons, Mainz- Kastell, Germany) capillary column (0.25 mm I.D. X 15 m). For quantitative determination of neutral sugars, amino sugars, hexuronic acids, 2-keto-3-deoxy-D- manno-octonic acid (KDO) by combined GC-MS on a fused silica DB 5.MS (Fissons, Mainz-Kastell, Germany) capillary column (0.25 mm I. D. X 25 m) sample was treated according to the method of Russa et al. (24). Briefly, the sample was hydrolyzed with 1% acetic acid (100°C, 1.5 h), reduced with NaB2H4, methanolysed (0.5 M methanolic HCI, 85°C, 16 h), carboxy-reduced with NaB2H4 (4°C, 48 h), hydrolyzed with 1 M TFA (120°C, 2 h), reduced with NaB2H4 and peracetylated with pyridine/acetic anhydride (1:1, v/v) (100°C, 1 h). Amino sugars were released with 4 M HCl (100°C, 18 h) and identified by high voltage paper electrophoresis in a pyridine-formic acid-acetic acid-water (1:1.5 :10 : 90, v/v/v/v; pH 2.8) buffer system (9). 550 RAU et al. VOL. 39 RESULTS The two LPSs of A. itersonii subsp. nipponicum IFO 13615 and A. poly- morphum IFO 13961 contain L-glycero-D-manno-heptose (L,D-heptose) in compa- rable amounts (Table 1). In the case of A. itersonii subsp. nipponicum the major sugar components after carboxy-reduction with NaB2H4 and formation of alditol acetates were glucose and glucuronic acid, L,D-heptose and KDO. L,D-Heptose and KDO were found in molar ratios of 1.7:1, whereas the ratio of glucose to glucuronic acid was 2.5: 1. With regard to galactose and galacturonic acid a ratio of 1: 5 could be calculated based on fragments of the 2H-reduced galacturonic acid by mass spectrometry. The major neutral and acidic sugars present in A. poly- morphum-LPS were mannose, glucose and glucuronic acid, galactose and galactu- ronic acid, L,D-heptose and KDO. Mannose, L,D-heptose and KDO were found in molar ratios of 1.8 : 2.0: 1. In case of glucose and glucuronic acid a ratio of 1.5: 1 could be calculated based on fragments of the 2H-reduced glucuronic acid by mass spectrometry; in case of galactose and galacturonic acid a ratio of 1: 3.6 could be calculated. In both lipopolysaccharides, glucosamine was detected as amino sugar. Quantification (Table 1) was done by GC-MS of the alditol acetates using xylose as internal standard. The LPS of A, itersonii subsp. nipponicum IFO 13615 was found to be rich in Table 1. Chemical analysis of the LPSs of the two members of a-group. 1993 Lipopolysaccharides from Aquaspirillum Species 551 hydroxy fatty acids: 3-hydroxy-tetradecanoic acid (3-OH-14:0) and 3-hydroxy- hexadecanoic acid (3-OH-16:0) were present in large amounts (molar ratio 1.0: 1.1), whereas another 3-hydroxy fatty acid, 3-hydroxy-octadecanoic acid (3- OH-18 : 0), was found only in small amounts.