The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products

Abstract

Proteins directing the biosynthesis of galactoglucan (exopolysaccharide II) in Rhizobium melilotiThe soil bacterium Rhizobium meliloti is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. Bacterial nitrogen fixation takes place within root nodules resulting from a coordinated bacteria-plant interaction which requires the exchange of signals between both symbiotic partners (11, 29). R. meliloti is able to produce two acidic exopolysaccharides (EPSs), succinoglycan (EPS I) and galactoglucan (EPS II). At least one of these EPSs is required for invasion of Medicago sativa nodules by R. meliloti (3,32,36,61,86).EPS I is composed of repeating units containing one galactose and seven glucose molecules joined by ␤-1,4-, ␤-1,3-, and ␤-1,6-glycosidic linkages (63) and is decorated by acetate, pyruvate, and succinyl groups. EPS II consists of alternating glucose and galactose residues joined by ␣-1,3 and ␤-1,3 linkages (38). It is acetylated and pyruvylated. In the Rm1021 and Rm2011 strain backgrounds, the production of EPS II was observed only at low phosphate concentrations (87) or in the presence of a mutation in either the expR (32) or mucR (44, 86) gene. The corresponding gene products are thought to negatively regulate the expression of genes directing the biosynthesis of EPS II.The exo gene cluster, directing the biosynthesis of EPS I (50), and the exp gene cluster, responsible for the synthesis of EPS II (32), are separated by about 200 kb on megaplasmid 2 (15). Whereas the exo gene cluster was extensively studied and functions were assigned to most of the exo gene products (4-8, 12, 33, 34, 58, 64), little is known about the organization of the exp gene cluster and the functions of exp gene products.A 23-kb DNA region involved in EPS II biosynthesis was cloned and characterized by Tn5 mutagenesis (32). Six exp complementation groups required for EPS II biosynthesis in the expR101 mutant background were identified in this region. Here, we report on the 32-kb sequence of the R. meliloti Rm2011 exp gene cluster comprising 25 genes and on the inferred properties of the encoded exp gene products. MATERIALS AND METHODSBacterial strains and plasmids. Strains and plasmids used in this study are listed in Table 1.Media and growth conditions. Escherichia coli strains were grown in Penassay broth (Difco Laboratories) or in LB medium (65) at 37ЊC. R. meliloti strains were grown in TY medium (10), Vincent minimal medium (79), M9 medium (56), or LB medium (65) at 30ЊC.Antibiotics were supplemented as required at the following concentrations (micrograms per milliliter): for R.

The complete sequence of the 1,683-kb pSymB megaplasmid from the N 2 -fixing endosymbiont Sinorhizobium meliloti

“…Gram-negative bacteria exhibit complex sets of surface polysaccharides, including LPS (38), capsular polysaccharides (CPS) (39), EPS (40,41), and periplasmic glucans (42). Genes involved in the biosynthesis and export of cell surface carbohydrates are often clustered, and the exo/exs and exp gene clusters directing the synthesis of the EPS succinoglycan (EPS I) (43,44) and galactoglucan (EPS II) (13,14) were previously mapped on pSymB of S. meliloti. The production of surface polysaccharides is essential for successful nodule invasion by rhizobia.…”

“…Early studies focused on mutations that abolished synthesis of the succinoglycan EPS, EPS I, because these mutations resulted in a loss of the ability to form normal N 2 -fixing root nodules. This symbiotic defect was rescued by second-site mutations that increased the synthesis of a second galactoglucan EPS (EPS II), whose biosynthetic genes were also located on the pSymB megaplasmid (13,14). Other genes located on pSymB that are required for the formation of N 2 -fixing root nodules include the C 4 -dicarboxylate (dctA) and phosphate transport (phoCDET) genes and the bacA gene (15-18).…”

Rhizobial homologs of the fatty acid transporter FadL facilitate perception of long-chain acyl-homoserine lactone signals

“…In this setup, we determined activation of galactoglucan biosynthesis gene expression by judging culture morphology on tryptone-yeast (TY) agar and monitoring activity of the wgeA promoter. wgeA is the first gene of the wge operon of the galactoglucan biosynthesis gene cluster (21).…”

The complete cps gene cluster from Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of a new exopolysaccharide

“…CpsH displayed high similarity to a number of different glucosyltransferases (Table 2), therefore it seemed logical to predict that this represents the second of the three expected glucosyltransferases. CpsI had highest homology to galactosyltransferases ( it also contained an AX ( LDXD motif reported to be found in the β-glucosyltransferase and cellulose synthase superfamilies (Becker et al, 1997 ;Wiggins & Munro, 1998). This could imply that CpsI could be the third glucosyltransferase, thus a detailed biochemical analysis will be required to establish which sugar CpsI transfers.…”