Presence of the oral bacterium Capnocytophaga canimorsus in the tooth plaque of canines.

Capnocytophaga canimorsus is a potentially pathogenic microorganism when transmitted to humans from the oral cavity of canines. Although there is some knowledge about the frequency of occurrence in canines, it is uncertain whether there is a correlation between its occurrence and lifestyle, health, or breed of dog. Samples of tooth plaque from a total of 131 canines were collected, cultured on selective media, and tested using physiological and molecular analyses to help discern the presence of C. canimorsus. Phylogenetic analyses determined that 49.2% of canines sampled carried a species of Capnocytophaga and 21.7% of the canines sampled in this study carried C. canimorsus. Statistical analyses found that male dogs and those that are neutered and spayed are more likely to host Capnocytophaga species. The data also suggested that breed was a statistically significant predictor of C. canimorsus, with the smaller breeds more likely to carry the potential pathogen. In addition, three "human" species of Capnocytophaga; C. ochracea, C. haemolytica, and one isolate of either C. gingivalis or C. granulosa were cultured from five canines. Sixteen canines sampled carried an unidentified Capnocytophaga species, with the sequences from all isolates forming a well-defined phylogenetic clade with 100% bootstrap support that may well represent a new species of Capnocytophaga.

Isolation and characterization of a Chryseobacterium strain from the gut of the American cockroach, Periplaneta americana.

A 16S rDNA sequence cloned directly from whole-gut microbiota of the American cockroach, Periplaneta americana, indicated the presence of a member of the Bacteroides/Flavobacterium group most closely related to the genus Flavobacterium. In an attempt to confirm this finding, we isolated a yellow-pigmented bacterium (strain FR2) from the hindgut of this insect. Strain FR2 was phylogentically and phenotypically most similar to species of Flavobacterium and related bacteria, namely Chryseobacterium indologenes. Fifty-four other yellow-pigmented bacteria isolated during a 1-year study shared the salient phenotypic characteristics of Chryseobacterium spp., and thus were considered the same phenotype. This phenotype's abundance was related to the fiber content of the insect diet, being consistently detected only in cockroaches fed a high-fiber diet (30% crude fiber by weight). The highest population density was in the hindgut, ranging from 2 x 10(6) to 1.2 x 10(7) colony forming units ml(-1) during a 1-year period. The nature of the symbiosis between the FR2 phenotype and P. americana is discussed.

Sulfonates as terminal electron acceptors for growth of sulfite-reducing bacteria (Desulfitobacterium spp.) and sulfate-reducing bacteria: effects of inhibitors of sulfidogenesis.

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)-Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135-149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.

Low-molecular-weight sulfonates, a major substrate for sulfate reducers in marine microbial mats.

Several low-molecular-weight sulfonates were added to microbial mat slurries to investigate their effects on sulfate reduction. Instantaneous production of sulfide occurred after taurine and cysteate were added to all of the microbial mats tested. The rates of production in the presence of taurine and cysteate were 35 and 24 microM HS(-) h(-1) in a stromatolite mat, 38 and 36 microM HS(-) h(-1) in a salt pond mat, and 27 and 18 microM HS(-) h(-1) in a salt marsh mat, respectively. The traditionally used substrates lactate and acetate stimulated the rate of sulfide production 3 to 10 times more than taurine and cysteate stimulated the rate of sulfide production in all mats, but when ethanol, glycolate, and glutamate were added to stromatolite mat slurries, the resulting increases were similar to the increases observed with taurine and cysteate. Isethionate, sulfosuccinate, and sulfobenzoate were tested only with the stromatolite mat slurry, and these compounds had much smaller effects on sulfide production. Addition of molybdate resulted in a greater inhibitory effect on acetate and lactate utilization than on sulfonate use, suggesting that different metabolic pathways were involved. In all of the mats tested taurine and cysteate were present in the pore water at nanomolar to micromolar concentrations. An enrichment culture from the stromatolite mat was obtained on cysteate in a medium lacking sulfate and incubated anaerobically. The rate of cysteate consumption by this enrichment culture was 1.6 pmol cell(-1) h(-1). Compared to the results of slurry studies, this rate suggests that organisms with properties similar to the properties of this enrichment culture are a major constituent of the sulfidogenic population. In addition, taurine was consumed at some of highest dilutions obtained from most-probable-number enrichment cultures obtained from stromatolite samples. Based on our comparison of the sulfide production rates found in various mats, low-molecular-weight sulfonates are important sources of C and S in these ecosystems.

Sulfidogenesis from 2-aminoethanesulfonate (taurine) fermentation by a morphologically unusual sulfate-reducing bacterium, Desulforhopalus singaporensis sp. nov.

A pure culture of an obligately anaerobic marine bacterium was obtained from an anaerobic enrichment culture in which taurine (2-aminoethanesulfonate) was the sole source of carbon, energy, and nitrogen. Taurine fermentation resulted in acetate, ammonia, and sulfide as end products. Other sulfonates, including 2-hydroxyethanesulfonate (isethionate) and cysteate (alanine-3-sulfonate), were not fermented. When malate was the sole source of carbon and energy, the bacterium reduced sulfate, sulfite, thiosulfate, or nitrate (reduced to ammonia) but did not use fumarate or dimethyl sulfoxide as a terminal electron acceptor for growth. Taurine-grown cells had significantly lower adenylylphosphosulfate reductase activities than sulfate-grown cells had, which was consistent with the notion that sulfate was not released as a result of oxidative C-S bond cleavage and then assimilated. The name Desulforhopalus singaporensis is proposed for this sulfate-reducing bacterium, which is morphologically unusual compared to the previously described sulfate-reducing bacteria by virtue of the spinae present on the rod-shaped, gram-negative, nonmotile cells; endospore formation was not discerned, nor was desulfoviridin detected. Granules of poly-beta-hydroxybutyrate were abundant in taurine-grown cells. This organism shares with the other member of the genus Desulforhopalus which has been described a unique 13-base deletion in the 16S ribosomal DNA. It differs in several ways from a recently described endospore-forming anaerobe (K. Denger, H. Laue, and A. M. Cook, Arch. Microbiol. 168:297-301, 1997) that reportedly produces thiosulfate but not sulfide from taurine fermentation. D. singaporensis thus appears to be the first example of an organism which exhibits sulfidogenesis during taurine fermentation. Implications for sulfonate sulfur in the sulfur cycle are discussed.

Taurine-sulfur assimilation and taurine-pyruvate aminotransferase activity in anaerobic bacteria.

We demonstrated the ability of strictly fermentative, as well as facultatively fermentative, bacteria to assimilate sulfonate sulfur for growth. Taurine (2-aminoethanesulfonate) can be utilized by Clostridium pasteurianum C1 but does not support fermentative growth of two Klebsiella spp. and two different Clostridium spp. However, the latter are able to assimilate the sulfur of a variety of other sulfonates (e.g., cysteate, 3-sulfopyruvate, and 3-sulfolactate) anaerobically. A novel taurine-pyruvate aminotransferase activity was detected in cell extracts of C. pasteurianum C1 grown with taurine as the sole sulfur source. This activity was not detected in extracts of other bacteria examined, in C. pasteurianum C1 grown with sulfate or sulfite as the sulfur source, or in a Klebsiella isolate assimilating taurine-sulfur by aerobic respiration. More common aminotransferase activities (e.g., with aspartate or glutamate as the amino donor and pyruvate, oxalacetate, or (alpha)-ketoglutarate as the amino acceptor) were present, no matter what sulfur source was used for growth. Partial characterization of the taurine-pyruvate aminotransferase revealed an optimal temperature of 37(deg)C and a broad optimal pH range of 7.5 to 9.5.

Sulfonates: novel electron acceptors in anaerobic respiration.

The enrichment and isolation in pure culture of a bacterium, identified as a strain of Desulfovibrio, able to release and reduce the sulfur of isethionate (2-hydroxyethanesulfonate) and other sulfonates to support anaerobic respiratory growth, is described. The sulfonate moiety was the source of sulfur that served as the terminal electron acceptor, while the carbon skeleton of isethionate functioned as an accessory electron donor for the reduction of sulfite. Cysteate (alanine-3-sulfonate) and sulfoacetaldehyde (acetaldehyde-2-sulfonate) could also be used for anaerobic respiration, but many other sulfonates could not. A survey of known sulfate-reducing bacteria revealed that some, but not all, strains tested could utilize the sulfur of some sulfonates as terminal electron acceptor. Isethionate-grown cells of Desulfovibrio strain IC1 reduced sulfonate-sulfur in preference to that of sulfate; however, sulfate-grown cells reduced sulfate-sulfur in preference to that of sulfonate.

Sulfonate-sulfur utilization involves a portion of the assimilatory sulfate reduction pathway in Escherichia coli.

Strains of Escherichia coli lacking serine transacetylase or a positive regulator (Cys B protein) of the assimilatory sulfate reduction (ASR) pathway were unable to assimilate sulfonate-S, while single mutants in O-acetyl-L-serine sulfhydrylase (either 'A' or 'B') were able to do so. Mutants unable to reduce sulfate to sulfite were nonetheless able to form and accumulate sulfide and then cysteine from sulfonates, while strains lacking sulfite reductase were not. Thus terminal portions of the ASR pathway are involved in reduction of sulfonate-S to that of cysteine. E. coli K-12 formed cysteine more slowly, and accumulated lesser amounts of it with sulfonate-sulfur than it did from either sulfate or sulfite. These observations are consistent with our earlier report that sulfate is the preferred sulfur source when present simultaneously with a sulfonate.

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12.

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g., sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.

Sulfonate-sulfur assimilation by yeasts resembles that of bacteria.

Three sulfonates were tested for their ability to serve as nutrients for Hansenula wingei, Rhodotorula glutinis, Trigonopsis variabilis and Saccharomyces cerevisiae. Cysteate, taurine and isethionate, under aerobic conditions, could be utilized as sources of sulfur, although in some instances final cell yields were less than those obtained with an equimolar amount of sulfate-sulfur. Sulfonate assimilation by S. cerevisiae resembled that of bacteria (reported earlier by us) in several aspects: first, sulfate-S was used in preference to that of sulfonate, when both were present; second, mutants unable to use sulfate as a source of sulfur because of deficiencies in ATP sulfurylase, adenylylsulfate kinase (APS kinase) or PAPS reductase were able to utilize sulfonates; and third, mutants deficient in sulfite reductase were unable to utilize sulfonates.

Sulphonate utilization by enteric bacteria.

A variety of sulphonates were tested for their ability to serve as nutrients for Escherichia coli, Enterobacter aerogenes and Serratia marcescens. Cysteate, taurine and isethionate could not serve as sole sources of carbon and energy but, under aerobic conditions, could be utilized as sources of sulphur. Both sulphate and sulphonate supported equivalent cell yields, but the generation times varied with the sulphonate being metabolized. The sulphonate-S of HEPES buffer, dodecane sulphonate and methane sulphonate was also utilized by some strains, whereas the sulphonate-S of taurocholate was not. None of the sulphonates tested served as a sulphur source for growth under anaerobic conditions. Sulphonate utilization appears to be a constitutive trait; surprisingly, however, cells of E. coli and Ent. aerogenes utilized sulphate-S in preference to that of sulphonate, when both were present. E. coli mutants unable to use sulphate as a source of sulphur because of deficiencies in sulphate permease, ATP sulphurylase, adenylylsulphate kinase (APS kinase) or glutaredoxin and thioredoxin were able to utilize sulphonates; hence sulphate is not an obligatory intermediate in sulphonate utilization. However, mutants deficient in sulphite reductase were unable to utilize sulphonates; therefore, this enzyme must be involved in sulphonate utilization, though it is not yet known whether it acts upon the sulphonates themselves or upon the inorganic sulphite derived from them.

Temporal sequence of the recovery of traits during phenotypic curing of a Cytophaga johnsonae motility mutant.

The lack of cell translocation and the resulting formation of nonspreading colonies of mutants of the gram-negative gliding bacterium Cytophaga johnsonae have been correlated with the loss of cell surface features of the organism. These cell surface traits include the ability to move polystyrene-latex beads over the cell surface and the ability to be infected by bacteriophages that infect the parent strain. In order to assess whether these traits reflect structures or functions that actually play a role in gliding, we studied a mutant (21A2I) selected for its inability to form spreading colonies; it is deficient in sulfonolipid, lacks bead movement ability, and is resistant to at least one bacteriophage. The provision of cysteate (a specific sulfonolipid precursor) restores lipid content and gliding to the mutant; hence, the lipids are necessary for motility. Growth with cysteate also restores bead movement and phage sensitivity. In order to determine the temporal relationship of these traits, we undertook a kinetic study of the appearance of them after addition of cysteate to the mutant. One predicts that appearance of a trait essential for cell translocation will either precede or accompany the appearance of this ability, while a nonessential trait need not do so. Sulfonolipid synthesis was the only trait that appeared before gliding; this is consistent with its established importance for motility. Bead movement and phage sensitivity first appeared only after gliding started, suggesting that the machinery involved in those processes is not necessary, at least for the initiation of gliding.

Defects in gliding motility in mutants of Cytophaga johnsonae lacking a high-molecular-weight cell surface polysaccharide.

We previously observed (W. Godchaux, L. Gorski, and E.R. Leadbetter, J. Bacteriol. 172:1250-1255, 1990) that two mutants (strains 21 and NS-1) of the gliding bacterium Cytophaga johnsonae that were totally deficient in motility-dependent colony spreading, movement of rafts (groups) of cells as observed with a microscope, and movement of polystyrene-latex spheres that attached to the cell surface (observed in wet mounts) were also deficient in a high-molecular-weight cell surface polysaccharide (HMPS) and suggested a role for that substance in gliding motility. Antisera have been prepared against the purified HMPS, and these were used to select mutants specifically and highly deficient in the polysaccharide. All five such mutants had rates of colony spreading and raft movement that were much lower than those of the parent strain, but the rate of increase in colony diameter was higher than that found for strains NS-1 and 21 (which do not undergo raft movement at all). Unlike these latter two strains, the HMPS mutants retained the ability to move polystyrene-latex spheres over their surfaces. Hence, HMPS deficiency results in defective motility but not nonmotility, and the HMPS deficiency cannot fully explain the phenotype of mutants 21 and NS-1; in these strains, gliding must be affected by additional biochemical lesions. The HMPS may, nonetheless, be advantageous in that it supports greater gliding speeds.

Outer membrane polysaccharide deficiency in two nongliding mutants of Cytophaga johnsonae.

Phenol-extractable polysaccharides firmly associated with the outer membrane of the gliding bacterium Cytophaga johnsonae could be resolved by gel filtration in sodium dodecyl sulfate (SDS) or by SDS-polyacrylamide gel electrophoresis into a high-molecular-weight (H) fraction (excluded by Sephadex G-200) and a low-molecular-weight (L) fraction. Fraction L was rich in components typical of lipid A and the core region of lipopolysaccharide (P, 3-hydroxy fatty acids, and 2-keto-3-deoxyoctonate) and evidently was a lipopolysaccharide with a limited number of distal, repeating polysaccharide units, as judged by SDS-polyacrylamide gel electrophoresis. In relation to total carbohydrate, the H fraction was rich in amino sugar but poor in (possibly devoid of) the lipid A and core components. Two nongliding mutants were highly deficient in the H fraction; one of these was deficient in sulfonolipid but could be cured by provision of a specific sulfonolipid precursor, a process that also resulted in the return of both the H fraction and gliding, as well as the ability to move polystyrene latex spheres over the cell surface. Hence, the polysaccharide may be the component that is directly involved in motility, and the presence of sulfonolipids in the outer membrane is necessary for the synthesis or accumulation of the polysaccharide. This conclusion was reinforced by the fact that the second nongliding, polysaccharide-deficient mutant had a normal sulfonolipid content.

Cysteine is not an obligatory intermediate in the biosynthesis of cysteate by Cytophaga johnsonae.

A cysteine auxotroph of Cytophaga johnsonae was able to incorporate sulfur from sulfate into cysteate, and thus into sulfonolipid, in the absence of cysteine synthesis. This indicates that cysteine is not an obligatory intermediate of the cysteate biosynthetic pathway even though cysteine sulfur can be utilized for cysteate synthesis.

Increase of ornithine amino lipid content in a sulfonolipid-deficient mutant of Cytophaga johnsonae.

The gram-negative gliding bacterium Cytophaga johnsonae contains not only large quantities of unusual sulfonolipids but also, as we report here, a second class of unusual lipids. These lipids were detected and quantified by two-dimensional thin-layer chromatography of lipids from cells grown in the presence of [14C]acetate and shown by chemical studies to be alpha-N-(3-fatty acyloxy fatty acyl)ornithines. Like the sulfonolipids, these ornithine lipids were localized in the outer membrane (whereas phosphatidylethanolamine was the predominant lipid of the inner membrane). In a sulfonolipid-deficient mutant, the missing lipid was replaced, specifically, by an increased amount of ornithine lipid. Cells grown in liquid media contained predominantly ornithine lipids with nonhydroxylated residues in the O-fatty acyl position. In contrast, surface-grown cells contained a high proportion of ornithine lipids in which the O-fatty acyl group was 3-hydroxylated. The sulfonolipids and ornithine lipids are apparently coregulated in the sense that, regardless of perturbations caused by mutation or growth conditions, their total amounts remain constant at 40% of total cell lipid.

Biosynthesis of a sulfonolipid in gliding bacteria.

Gliding bacteria of the genus Cytophaga synthesize sulfonolipids (1,2) that contain capnine (1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Studies of the incorporation of radiolabeled compounds by C. johnsonae show that cysteate is utilized preferentially to both cystine and inorganic sulfate as a precursor of capnine sulfur and to both cystine and serine as a precursor of carbons 1 and 2 of capnine. The results are consistent with a pathway in which capnine is formed by condensation of cysteate with a fatty acyl CoA. Cystine, added as the sole sulfur source in the presence of glucose, provides the sulfur but not the carbon for capnine. Hence, these cells form cysteate not by direct oxidation of cystine (or cysteine), but by transfer of its sulfur to a different carbon compound.

Sulfonolipids of gliding bacteria. Structure of the N-acylaminosulfonates.

Earlier (Godchaux, W., and Leadbetter, E. R. (1980) J. Bacteriol. 144, 592-602; (1983) J. Bacteriol. 153, 1238-1246) we demonstrated that an unusual class of sulfonolipids are major components of the cell envelope of gliding bacteria of the genus Cytophaga and of closely related genera. One of these lipids, to which we have assigned the trivial name capnine, was purified and was shown to be 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid (which might also be named as 1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Though capnine accumulates as such in the cells of some Capnocytophaga spp., most organisms of the Cytophaga-like genera contain, instead, sulfonolipids that are less polar than capnine. These less polar lipids have been purified from a Capnocytophaga sp., a marine Cytophaga sp., Cytophaga johnsonae, and a Flexibacter sp. Acid methanolysis of the lipids yielded both aminosulfonates and a collection of fatty acid methyl esters. The infrared absorption spectra of the lipids indicated that the fatty acids were in amide (and not ester) linkage to the aminosulfonates. In every instance, analysis by mass spectrometry and other methods revealed that most, if not all, of the aminosulfonates obtained by methanolysis were structurally identical to capnine (though small amounts of variants of that compound may be present in some cases). The less polar sulfonolipids are, therefore, predominantly N-fatty acyl capnines, 1-deoxy-1-sulfonic acid analogs of ceramides. The fatty acid methyl esters obtained from the lipids were heterogeneous, but in all cases were rich in hydroxylated fatty acyl groups, which constituted 66 to 95% of the total.

Unusual sulfonolipids are characteristic of the Cytophaga-Flexibacter group.

Capnocytophaga spp. contain a group of unusual sulfonolipids, called capnoids (W. Godchaux III and E. R. Leadbetter, J. Bacteriol. 144:592-602, 1980). One of these lipids, capnine, is 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid; the others are, apparently, N-acylated versions of capnine. The lipids were found, in amounts ranging from 2.5 to 16 mumol of capnoid sulfur per g of cells (wet weight), in two Cytophaga spp. and also in several closely related organisms: several Capnocytophaga spp., Sporocytophaga myxococcoides, two Flexibacter spp., and two Flavobacterium spp. With the exception of the flavobacteria, all of these bacteria have been shown to exhibit gliding motility. The two Flavobacterium spp. belong to a subset of that genus that shares many other characteristics with the cytophagas. Only the Capnocytophaga spp. contained large quantities of capnine as such; in all of the others, most (and possibly all) of the capnoids were present as N-acylcapnines. Capnoid-negative bacteria included some gliding organisms that may not be closely related to the cytophagas: two fruiting myxobacters, a gliding cyanobacterium (Plectonema sp.), Beggiatoa alba, Vitreoscilla stercoraria, Herpetosiphon aurantiacus, and Lysobacter enzymogenes. Nongliding bacteria representing nine genera were also tested, and all of these fell into the capnoid-negative group.

Capnocytophaga spp. contain sulfonolipids that are novel in procaryotes.

A group of unusual sulfonolipids was found in bacteria of the genus Capnocytophaga. One of these lipids, to which we have assigned the trivial name capnine, was isolated in 98% pure form and was identified, by infrared absorption spectrometry, high-resolution mass spectrometry, and other methods, as 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid. Another lipid appears to be an N-acylated version of capnine; after acid hydrolysis, its sulfur was recovered in a form chromatographically indistinguishable from that of capnine. The new lipids are related structurally to sphingosine and the ceramides, respectively, but differ markedly from those compounds in important respects, notably the presence of the sulfonate group. Some Capnocytophaga strains accumulated mostly capnine, whereas others accumulated mostly N-acylcapnine. All seven strains examined were found to contain the new lipids, in amounts ranging from 7 to 16 mumol/g of cells (wet weight). The lipids were found in isolated cell envelopes, where they were present in amounts ranging up to 400 mg/g of envelope protein; they are, accordingly, major cell components.