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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.

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.

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.

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.

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.

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.

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.