Organization of genes required for gellan polysaccharide biosynthesis in Sphingomonas elodea ATCC 31461

Abstract

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.

Proteins Encoded bySphingomonas elodeaATCC 31461rmlAandugpGGenes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities

“…We started the present study by sequencing the first gene (rmlA) of the putative rml operon, located within the gel cluster. More recently, the nucleotide sequence of this four-gene rml cluster was deposited in GenBank by Harding et al (15) and used in this work to deduce the amino acid sequences of the RmlB, RmlC, and RmlD proteins. These sequences were compared with the corresponding sequences from seven gram-negative and two gram-positive organisms ( Table 2).…”

“…Eighteen genes, organized in the gel cluster (15,34) and coding for enzymes presumably involved in the synthesis of dTDP-L-Rha, glucosyltransferases, and proteins required for gellan polymerization and export, were identified. The organization and the nucleotide sequence of the gel cluster are highly similar to those described for the gene cluster required for the synthesis of the sphingan S88 in Sphingomonas strain S88 (sps cluster) (15,45).…”

“…The rml four-gene cluster has been identified for different gram-negative and gram-positive bacterial species; these clusters exhibit a remarkable sequence similarity, although the gene order of the operon-like structures may vary from species to species (11,21,24,30). The two Sphingomonas strains S. elodea ATCC 31461 and Sphingomonas strain S88 exhibit a similar organization (ACBD3) and a high range of sequence conservation (87 to 96% amino acid identity) (15,34).…”

The Effect of Potassium Phosphate as a pH Stabilizer on the Production of Gellan by Sphingomonas paucibilis NK-2000

“…Gellan consists of linear repeating tetrasaccharides [→3)-β-D-Glc-(1→4)-β-D-GluA-(1→4)-β-D-Glc-(1→4)-α-L-Rha-(1→] composed of D-glucose (Glc), D-glucuronic acid (GlcA), and L-rhamnose residues (Rha) [12,28]. Due to the diversity of its structure and properties, gellan has a wide range of applications in the food, pharmaceutical and other industries as texturizing, stabilizing, thickening, emulsifying and gelling agents [9,25]. Gellan exhibits good stability, which is a distinct advantage in fruit-based products [18].…”

High Rate of N 2 Fixation by East Siberian Cryophilic Soil Bacteria as Determined by Measuring Acetylene Reduction in Nitrogen-Poor Medium Solidified with Gellan Gum

“…Hashidoko et al developed an improved soft gel medium for growth of N 2 -fixing bacteria in 2002 (15). In their study, 0.2% agar was replaced with 0.3% gellan gum, a bacterial extracellular polysaccharide (EPS) produced by Sphingomonas elodea (a synonym of Sphingomonas paucimobilis) ATCC 31461 (12,17,18). Initially, gellan gum was used for the purpose of preparing a highly transparent soft gel medium that was better for culturing microaerobic N 2 -fixing rhizobacteria.…”