Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [-->4)-alpha- l-rhamnose-(1-->3)-beta- d-glucose-(1-->4)-beta- d-glucuronic acid-(1-->4)-beta- d-glucose-(1-->]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP- l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes ( gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified.