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.
