Several structurally related capsular polysaccharides that are secreted by members of the genus Sphingomonas are being developed as aqueous rheological control agents for diverse industrial and food applications. They include gellan (S-60), welan (S-130), rhamsan (S-194), S-657, S-88, S-198, S-7, and NW-11. We refer to these polysaccharides as sphingans, after the genus name. This paper characterizes the first gene cluster isolated from a Sphingomonas species (S88) that is required for capsule synthesis. Overlapping DNA segments which spanned about 50 kbp of S88 DNA restored the synthesis of sphingan S-88 in capsule-negative mutants. The mutations were mapped into functional complementation groups, and the contiguous nucleotide sequence for the 29-kbp cluster was determined. The genetic complementation map and the DNA sequences were interpreted as an extended multicistronic locus containing genes essential for the assembly and secretion of polysaccharide S-88. Many of the deduced amino acid sequences were similar to gene products from other polysaccharide-secreting bacteria such as Rhizobium meliloti (succinoglycan), Xanthomonas campestris (xanthan gum), and Salmonella enterica (O antigen). The S88 locus contained a four-gene operon for the biosynthesis of dTDP-L-rhamnose, an essential precursor for the sphingans. Unexpectedly, there were also two genes for secretion of a lytic or toxin-like protein nested within the polysaccharide cluster. The conservation and linkage of genes that code for a defensive capsule and genes for secretion of an offensive lysin or toxin suggest a heretofore unknown pathogenic life history for Sphingomonas strain S88.The ''sphingans'' are capsular polysaccharides that have similar but not identical structures and are secreted by members of the genus Sphingomonas (44). The group includes S-88, S-60 (gellan), S-130 (welan), S-194 (rhamsan), heteropolysaccharide 7,41,43). As diagrammed in Fig. 1, the sphingans have different side groups, and either L-rhamnose or L-mannose is found at one position in the backbone. L-Mannose itself is exceedingly rare in nature. Aqueous solutions of the polymers have unique and useful rheological properties (41). Gellan is currently produced by large-scale aerobic fermentation for use as a gelling agent in foods and microbiological culture media, and welan is marketed for control of aqueous viscosity. It is not clear how the structural variations in the polymers give rise to distinct rheological properties.Sphingomonas strains have been isolated from diverse environments and with a wide range of metabolic activities. The sphingan-secreting strains were isolated from plant tissue, water, and soil. Chemoheterotrophic strains with the ability to metabolize toluene, naphthalene, cresol, and other aromatic compounds were recently isolated from deep terrestrial subsurface sediments (12). And other strains have been studied for their ability to degrade lignin (38). Sphingomonas bacteria have also been isolated from human clinical specimens and from hospital wate...

Four representative species from three genera of gram-negative bacteria that secrete exopolysaccharides acquired resistance to the antibiotic bacitracin by stopping synthesis of the exopolysaccharide. Xanthomonas campestris, Sphingomonas strains S-88 and NW11, and Escherichia coli K-12 secrete xanthan gum, sphingans S-88 and NW11, and colanic acid, respectively. The gumD gene in X. campestris is required to attach glucose-P to C55-isoprenyl phosphate, the first step in the assembly of xanthan. A recombinant plasmid carrying the gumD gene of X. campestris restored polysaccharide synthesis to bacitracin-resistant exopolysaccharidenegative mutants ofX. campestris and Sphingomonas strains. Similarly, a newly cloned gene (spsB) from strain S-88 restored xanthan synthesis to the same X. campestris mutants. However, the intergeneric complementation did not extend to mutants of E. coli that were both resistant to bacitracin and nonproducers of colanic acid. The genetic results also suggest mechanisms for assembling the sphingans which have commercial potential as gelling and viscosifying agents.The bacitracins are a mixture of structurally related cyclic polypeptides with clinically useful antibiotic properties and are produced by certain species of Bacillus (13). Bacitracins interfere indirectly with the biosynthesis of bacterial cell walls by inhibiting the dephosphorylation of C55-isoprenyl pyrophosphate (IPP) (30). Bacitracin forms a complex with IPP and a divalent cation and prevents phosphatase from acting on IPP (32-34). Bacteria require the C.5-isoprenyl phosphates (IP) as carriers during synthesis of the repeat subunits of peptidoglycan. IPP is released when a repeat subunit, a disaccharidepentapeptide, is transferred to a growing peptidoglycan chain, but the IPP must be dephosphorylated before reuse. Although binding to IPP is thought to be the primary mode of action, bacitracin may also interfere with additional cellular processes: the actions of certain hydrolytic enzymes (19), the formation of ubiquinone precursors (29), the biosynthesis of membranederived oligosaccharides (41), and the involvement of membranes in cell division (26).One mechanism of resistance to bacitracin seems to result from increased de novo synthesis of IP. Cain et al. (3) have recently shown that increased intracellular levels of a lipid kinase, the product of the bacA gene of Escherichia coli, confers resistance to bacitracin. The kinase appears to increase the level of the carrier IP by phosphorylating isoprenyl alcohol, thereby circumventing the sequestration of IPP by bacitracin. Sutherland (35) had earlier suggested that resistance to bacitracin in Klebsiella species might be due to an abundance of isoprenoid lipids; however, actual quantitation of the lipids was incomplete. Fiedler and Rotering (7) described mutants of E. coli that display several phenotypes during growth under conditions of low osmolarity: partial resistance to bacitracin, failure to synthesize membrane-derived oligosaccharides, increased production of capsular...

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Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene ofSphingomonas strain S88 and the pssDE genes ofRhizobium leguminosarum were identified as encoding glucuronosyl-(β1→4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(β1→4)-glucuronosyl transferase inSphingomonas and that pssC codes for a glucuronosyl-(β1→4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.

By using temperature-sensitive (ts) and suppressor-sensitive (sus) mutants, 11 essential genes have been identified in phage 4105. The order of the genes has been established in two-and three-factor crosses. The genes can be arranged in a linear order; this order is identical in the vegetative phage and in the prophage. One gene essential for phage deoxyribonucleic acid (DNA) synthesis has been found. Marker rescue from prophage and mature DNA, taken up by competent bacteria, was studied by superinfection with phage carrying one sus and one ts mutation. In prophage DNA, all single markers studied are rescued at similar frequencies. The frequency of co-rescue of two markers is proportional to the recombinational distance between the markers. Thus, colinearity between the genetic map and the position on the DNA molecule of those mutations used to establish the map is demonstrated. The results indicate that the recombination frequencies observed in vegetative crosses are a relative measure of the physical distance between markers. All single markers are not rescued at equal frequencies from mature DNA. The frequency of co-rescue of two markers is related to the recombinational distance only over a distance about one-fourth or less of the genetic map. Markers separated by 10% recombination, or more, are co-rescued at 5 to 10% of the frequency of rescue of single markers. Shearing of mature DNA into half-sized molecules reduces the efficiency by which single markers are rescued by a factor of 5 to 10. The results of experiments on co-rescue of two markers from half-sized mature DNA indicate a preferred break-point near the middle of the genetic map; the results are compatible with a nonpermuted sequence in mature DNA. It is pointed out and discussed that mature DNA exhibits several anomalies in marker rescue experiments.