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 , 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 , 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 . 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. Dodecanoic acid (12:0) was the main nonhydroxylated fatty acid (129.5 nmol/mg). In the lipopolysaccharide of A. polymorphum IFO 13961, the predominant fatty acids were tetradecanoic acid (14:0) and 3-OH-14 : 0 (molar ratio 1.4: 1), the latter being the only hydroxylated fatty acid in this fraction. Identification and quantification were done by GC-MS of the fatty acid methyl esters using heptadecanoic acid (17 :0) as internal stan- dard. Rhamnose and glucose were the main sugar constituents found in the LPS of A. aquaticum IFO 14918 (Table 2). Glucosamine was detected as the sole amino sugar and uronic acids could not be detected. In the case of A. metamorphum IFO 13960 the predominant sugar component of LPS was rhamnose (3504.9 nmol/mg), which was accompanied by a small amount of 3-0-methyl rhamnose (acofriose). Glucose was also present in considerable amounts. The LPS of the mutant strain 12-3 had glucose and KDO as predominant sugar components (molar ratio of 1.5: 1), but only small amounts of rhamnose and no 3-0-methyl rhamnose. As in the case of A. aquaticum IFO 14918, in both A, metamorphum strains no uronic acid could be detected and glucosamine was found as the only amino sugar (Table 2).

Table 2. Chemical analysis of the LPSs of the two members of $-group. 552 RAU et al. VOL. 39

The main components of the fatty acid fraction in the LPS of A. aquaticum IFO 14918 were 12:0, 14:0 and 3-hydroxy-decanoic acid (3-OH-10 :0) in ratios of 1:1.2 : 3.2. Another hydroxylated fatty acid, namely 3-hydroxy-undecanoic acid (3-OH-11:0), could be detected, but only in small amounts. The LPS of A. metamorphum IFO 13960 possessed the same three major fatty acid constituents (12 :0, 14:0 and 3-OH-10 :0), but in molar ratios of 1:1.4: 1.5. In addition to 3- OH-10 :0, two other hydroxylated fatty acids were present, 3-OH-14 : 0 and 3-OH- 16:0, but only in small amounts. From the DOC-PAGE pattern of LPSs it is evident that A. itersonii subsp. nipponicum IFO 13615 and A. polymorphum IFO 13961 belong to a similar LPS-type. A strong band of R-type LPS is visible in both organisms and in case of A. itersonii subsp. nipponicum IFO 13615 one additional band is demonstrable which is a bit slower-moving than Salmonella-LPS of SR-type (Fig. 1, lane 3). Between these two bands a prominent gap is demonstrated. The LPS of A. polymorphum IFO 13961 shows besides this R-type band three bands: two single bands and one double band in between (Fig. 1, lane 2). Gaps are present, too, indicating that lipopolysaccharides of only defined lengths are synthesized. The LPS of A. aquaticum IFO 14918 on DOC-PAGE shows R-type character (Fig. 1, lane 5). From the DOC-PAGE pattern of LPS of A. metamorphum IFO 13960 it is evident that this bacterium synthesizes lipopolysaccharide chains of essentially a

Fig. 1. DOC-PAGE patterns of LPSs from (1) Salmonella montevideo SH 94, (2) Aquaspirillum polymorphum IFO 13961, (3) Aquaspirillum itersonii subsp. nipponicum IFO 13615, (4) Aquaspirillum metamorphum IFO 13960, (5) Aquaspirillum aquaticum IFO 14918, (6) Rhodospirillum fulvum DSM 117, (7) Rhodospirillum molischianum NTHC 131, (8) Salmonella typhimurium SH 777; 5 ,ug each. 1993 Lipopolysaccharides from Aquaspirillum Species 553 single defined chain length, besides one band of an R-type LPS (Fig. 2, lane 2). The mutant strain 12-3 shows only one fast-moving band of an R-type LPS (Fig. 2, lane 3). In A, metamorphum IFO 13960 a S-layer protein was observed, which was stained with Coomassie staining as well as with PAS-staining, indicating its glyco-

Fig. 2. DOC-PAGE patterns of LPSs from (1) Salmonella montevideo SH94, (2) Aquaspirillum metamorphum IFO 13960, (3) Aquaspirillum metamorphum IFO 13960 mutant strain 12-3, (4) Salmonella typhi his386, (5) Salmonella minnesota R595, (6) Salmonella montevideo SH94; 3 ug each.

Fig. 3. Coomassie blue-stained SDS-PAGE (A) and PAS-stained DOC-PAGE (B) of whole cells of Aquaspirillum metamorphum IFO 13960 (wild type) and 12-3 (surface layer protein-less mutant). Lanes: 1, molecular size markers (A), and Salmonella minnesota LPS (B); 2, IFO 13960; and 3, strain 12-3. 554 RAU et al. VOL. 39 protein character. In A. metamorphum mutant 12-3, no S-layer protein is visible (Fig. 3).

DISCUSSION

Lipid A and the deep R-core region were previously shown to be phylogeneti- cally rather preserved structures and are in many instances also reflecting the phylogenetical relatedness of Gram-negative . Thus, the majority of species of the a-3 subgroup of Proteobacteria share as highly characteristic structural element the rare amide-linked 3-oxo-myristic acid (18). In the a-2 subgroup representatives of n-2 hydroxylated long-chain fatty acids, such as 27-OH-28 : 0 and 29-OH-30 : 0, which are so far not or extremely rarely encountered (3 ) in natural products, are highly characteristic ester-linked constituents in the lipid A moiety of lipopolysaccharides in this phylogenetic cluster (2). So far, however, characteristic and widespread constituents in LPS-specimens of members of the a-1 group have not been encountered. Here we report on the composition of two Aquaspirillum species of the a-1 group and compare their DOC-PAGE profiles with those previously established for a number of Rhodospirillum species of the same subgroup of Proteobacteria (22). Perusal of Fig. 1 shows similar irregular banding profile for LPSs of Aquaspirillum polymorphum, Rhodospirillum fulvum and Rhodospirillum molischianum, which indicate absence of block polymerization in these species. They also differ considerably from those obtained with two other LPSs derived from Aquaspirillum strains of the $ subgroup of Proteobacteria, namely Aquaspiril- lum metamorphum and Aquaspirillum aquaticum. A. metamorphum IFO 13960, as well as the R-mutant 12-3 derived therefrom, offered an interesting possibility to investigate whether a loss of 0-chains concomitantly leads to a loss of the pro- teinaceous S-layer, previously described to occur in the wild-type strain of A. metamorphum IFO 13960 (1,19). And in fact as shown in Fig. 3, by Coomassie, as well as by PAS-staining (5,25), the R-mutant does not contain the S-layer protein which is supposed to be glycosylated judged from its easy staining with PAS (Sakane and Yokota, unpublished). A corresponding finding, i.e. an LPS having a homogeneous 0-chain length, and thus serving as an anchor for a crystalline S-layer protein, which is lost when only a truncated 0-chain remained in the mutant, was reported by Dooley et al. (4) with a strain of Aeromonas hydrophila. Whether, however, also the smaller gaps observed in the LPS-profile with Aquaspirillum itersonii subsp. nipponicum and Rhodospirillum fulvum are indicative for the presence of S-layers has not yet been investigated. Several Rhodospirillum species, however, such as R. rubrum, R. molischianum and R. salexigens, have been described as possessing crystalline cell-surface layers (19). Concerning the sugar composition, it is noteworthy that LPSs from A. itersonii subsp. nipponicum and A. polymorphum both contain glucuronic and galacturonic acids in their LPSs, similar to findings with Rhodospirillum salinarum and to Rhodospirillum fulvum (23). Heptoses were identified in A. itersonii subsp. nippo- 1993 Lipopolysaccharides from Aquaspirillum Species 555

nicum and in A, polymorphum, as well as in R. salinarum and in R. fulvum (23), where, however, the latter species contains both L-glycero-D-manno- and D-glycero- D-manno-heptose (D,D-heptose). It is obvious that neither in the sugar nor in the fatty acid composition more than casual similarities are existing between these two Aquaspirillum and two Rhodospirillum species (a-1 group strains) and the two other investigated Aquaspirillum species, namely A, aquaticum and A. metamor- phum, which were recognized as members of the /3-branch of the phylogenetical tree. The latter two species share not only the same main fatty acids in their lipid A's, namely the rare amide-linked 3-OH-10 : 0 and the ester-linked 12 : 0 and 14:0, but also the same sugar components. The finding of large quantities of L-rhamnose in A. metamorphum IFO 13960 LPS, accompanied by a small amount of 3- 0-methyl-rhamnose, is reminiscent to previous findings with the Rhodobacter capsulatus 37b4 LPS and a number of other lipopolysaccharides with chains composed of rhamnose (or D-mannose), where repeatedly small amounts of 3- 0-methyl-rhamnose (respectively, 3-0-methyl-mannose) were identified in non- reducing terminal positions, and which may act as start or rather stop signal in the 0-chain biosynthesis (26). The R-mutant of A. metamorphum 12-3 has only tiny amounts of rhamnose left but glucose and KDO in addition to glucosamine, the latter two sugars in increased amounts, indicating their origin as R-core constitu- ents. Similarly, also the LPS of A. aquaticum showing an R- or SR-type in DOC-PAGE shares the same (R-core) sugars and has additionally a small amount of rhamnose. These data then document that the phylogenetic relationship existing between A. aquaticum and A. metamorphum is clearly reflected in their LPS composition, which shows similarity to each other but also to the LPSs of Rhodo- cyclus gelatinosus and Sphaerotilus natans, which are other members of the $-1 subgroup (32) and which were so far the only examples possessing the rare amide-linked 3-OH-10 : 0 (14,15). The data document also the phylogenetic distance to Aquaspirillum of the a-1 group, namely to A. itersonii subsp. nipponicum and to A. polymorphum. These Aquaspirillum species show compositional similar- ities to a few other members of the a-1 subgroup, namely to Rhodospirillum fulvum, Rhodospirillum salinarum and, to a lesser extent, also to Rhodospirillum rubrum (20). Further studies with additional strains of the genus Aquaspirillum are needed to generalize the present findings.

The authors thank E. Metz and C. Warth for technical assistance and D. Borowiak for GC-MS analysis.

REFERENCES

1) Beveridge, T. J. and Murray, R. G. E., Surface arrays on the cell wall of Spirillum metamorphum. J. Bacteriol., 124, 1529-1544 (1975). 2) Bhat, U.R., Carlson, R. W., Busch, M., and Mayer, H., Distribution and phylogenetic significance of 27-hydroxy-octacosanoic acid in the lipopolysaccharides from bacteria belonging to the a-2 subgroup of Proteobacteria. Int. J. Syst. Bacteriol., 41, 213-217 (1991). 556 RAU et al. VOL. 39

3) Bowman, J. P., Skerratt, J. H., Nichols, P. D., and Sly, L. I., Phospholipid fatty acid and lipopolysaccharide fatty acid signature lipids in methane-utilizing bacteria. FEMS Microbiol. Ecol., 85, 15-22 (1991). 4) Dooley, J. S. G., Lallier, R., Shaw, D. H., and Trust, T. J., Electrophoretic and immunochemical analysis of the lipopolysaccharides from various strains of Aeromonas hydrophila. J. Bacteriol., 164, 263-269 (1985). 5) Dubray, G. and Bezard, G., A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal. Biochem., 119, 325-329 (1982). 6) Galanos, C., Luderitz, 0., Rietschel, E. T., Westphal, 0., Brade, L., Freudenberg, M., Schade, U., Imoto, M., Yoshimura, H., Kusumoto, S., and Shiba, T., Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur. J. Biochem., 148, 1-5 (1985). 7) Galanos, C., Luderitz, 0., and Westphal, 0., Preparation and properties of a standardized lipopolysaccharide from Salmonella abortus equi (novo-pyrexal). Zentralbl. Bakteriol. Hyg., I Abt Orig. A, 243, 226-244 (1979). 8) Hylemon, P. B., Wells, J. S., Krieg, N. R., and Jannasch, H. W., The genus Spirillum: A taxonomic study. Int. J. Syst. Bacteriol., 23, 340-380 (1973). 9) Kickhofen, B. and Warth, R., Eine Trennkammer fur die Hochspannungselektrophorese nach dem Michl'schen Prinzip. J. Chromatogr., 33, 558-560 (1968). 10) Komuro, T. and Galanos, C., Analysis of Salmonella lipopolysaccharide by sodium deoxycholate- polyacrylamide gel electrophoresis. J. Chromatogr., 450, 381-387 (1988). 11) Krieg, N. R., Aerobic/ microaerophilic, motile, helical/ vibroid Gram-negative bacteria. In Bergey's Manual of Systematic Bacteriology, Vol. 1, ed. by Krieg, N. R. and Holt, J. G., Williams and Wilkins, Baltimore, London (1984), p. 71-124. 12) Krieg, N. R. and Hylemon, P. B., The of the chemoheterotrophic spirilla. Ann. Rev. Microbiol., 30, 303-325 (1976). 13) Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, 227, 680-685 (1970). 14) Masoud, H., Lindner, B., Weckesser, J., and Mayer, H., The structure of the lipid A component of Rhodocyclus gelatinosus Dr2 lipopolysaccharide. Syst. Appl. Microbiol., 13, 227-233 (1990). 15) Masoud, H., Urbanik-Sypniewska, T., Lindner, B., Weckesser, J., and Mayer, H., The structure of the lipid A component of Sphaerotilus natans. Arch. Microbiol., 156, 167-175 (1991). 16) Mayer, H., Campos-Portuguez, S. A., Busch, M., Urbanik-Sypniewska, T., and Bhat, U. R., Lipid A variants- or how constant are the constant regions in lipopolysaccharide? In Cellular and Molecular Aspects of Endotoxin Reactions, ed. by Nowotny, A., Spitzer, J. J., and Ziegler, E. J., Excerpta Medica, Amsterdam/New York/Oxford (1990), p. 111-120. 17) Mayer, H., Krauss, J. H., Yokota, A., and Weckesser, J., Natural variants of lipid A. In Endotoxin, ed. by Friedman, H., Klein, T. W., Nakano, M., and Nowotny, Plenum Publ. Corp., New York (1990), p. 45-70. 18) Mayer, H., Masoud, H., Urbanik-Sypniewska, T., and Weckesser, J., Lipid A composition and phylogeny of Gram-negative bacteria. Bull. JFCC, 5, 19-25 (1989). 19) Messner, P. and Sleytr, U. B., Crystalline bacterial cell surface layers. Adv. Microbiol. Physiol., 33, 213-275 (1992). 20) Pietsch, K., Weckesser, J., Fischer, U., and Mayer, H., The lipopolysaccharides of Rhodospirillum rubrum, Rhodospirillum molischianum, and Rhodopila globiformis. Arch. Microbiol., 154, 433-437 (1990). 21) Pot, B., Willems, A., Gillis, M., and De Ley, J., Intra- and intergeneric relationship of the genus Aquaspirillum: Prolinoborus, a new genus for Aquaspirillum fasciculus, with the species Prolinobo- rus fasciculus comb. nov. Int. J. Syst. Bacteriol., 42, 44-57 (1992). 22) Rau, H., Thesis, Freiburg i. Br., Germany (1993). 23) Rietschel, E. T., Brade, L., Lindner, B., and Zahringer, U., Biochemistry of lipopolysaccharides. In Bacterial Endotoxic Lipopolysaccharides, Vol. I, Molecular Biochemistry and Cellular Biology, 1993 Lipopolysaccharides from Aquaspirillum Species 557

ed. by Morrison, D. C., and Ryan, J. L,, CRC Press, Boca Raton/Ann Arbor/London/Tokyo (1992), p. 3-41. 24) Russa, R., Urbanik-Sypniewska, T., Choma, A., and Mayer, H., Identification of 3-deoxy-lyxo-2- heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae. FEMS Microbiol. Lett., 84, 337-344 (1991). 25) Sakane, T. and Yokota, A., Rapid method for LPS analysis with DOC-PAGE and PAS stain. IFO Res. Commun., 15, 42-51 (1991). 26) Tharanathan, R. N., Mayer, H., and Weckesser, J., Location of 0-methyl sugars in antigenic (lipo-)polysaccharides of photosynthetic bacteria and cyanobacteria. Biochem. J., 171, 403-408 (1978). 27) Tsai, C. M. and Frasch, C. E., A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem., 119, 115-119 (1982). 28) Westphal, 0. and Jann, K., Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem., V, 83-91 (1965). 29) Willems, A., De Ley, J., Gillis, M., and Kersters, K., Comamonadaceae, a new family encompass- ing the Acidovorans RNA complex, including Variovorax paradoxus gen. nov. comb. nov., for Alcaligenes paradoxus (Davis 1969). Int. J. Syst. Bacteriol., 41, 445-450 (1991). 30) Willems, A., Pot, B., Falsen, E., Vandamme, P., Gillis, M., Kersters, K., and De Ley, J., Polyphasic taxonomic study of the emended genus Comamonas: Relationship to Aquaspirillum aquaticum, E. Falsen Group 10, and other clinical isolates. Int. J. Syst. Bacteriol., 41, 427-444 (1991). 31) Woese, C. R., Stackebrandt, E., Weisburg, W. G., Paster, B. J., Madigan, M. T., Fowler, V. J., Hahn, C. M., Blanz, P., Gupta, R., Nealson, K. H., and Fox, G. E., The phylogeny of purple bacteria: The a-subdivision. Syst. Appl. Microbiol., 5, 315-326 (1984). 32) Woese, C. R., Weisburg, W. G., Paster, B. J., Hahn, C. M., Tanner, R. S., Krieg, N. R., Koops, H.-P., Harms, H., and Stackebrandt, E., The phylogeny of purple bacteria: The $-subdivision. Syst. Appl. Microbiol., 5, 327-336 (1984).