Identification of a novel ABC transporter required for desiccation tolerance, and biofilm formation in Rhizobium leguminosarum bv. viciae 3841

Abstract

Rhizobium leguminosarum is a soil bacterium with the ability to form nitrogen-fixing nodules on the roots of leguminous plants. Soil-dwelling, free-living R. leguminosarum often encounters desiccation stress, which impacts its survival within the soil. The mechanisms by which soil bacteria resist the effects of desiccation stress have been described. However, the role of the cell envelope in the desiccation tolerance mechanisms of rhizobia is relatively uncharacterized. Using a transposon mutagenesis approach, a mutant of R. leguminosarum bv. viciae was isolated that was highly sensitive to desiccation. The mutation is located in the ATP-binding protein of an uncharacterized ATP-binding cassette transporter operon (RL2975-RL2977). Exopolysaccharide accumulation was significantly lower in the mutant and the decrease in desiccation tolerance was attributed to the decreased accumulation of exopolysaccharide. In addition to desiccation sensitivity, the mutant was severely impaired in biofilm formation, an important adaptation used by soil bacteria for survival. This work has identified a novel transporter required for physiological traits that are important for the survival of R. leguminosarum in the rhizosphere environment.

Fig. 1

Schematic of the RL2975—2977 ABC transporter operon. Small arrows indicate the positions of primers 17BF and 17BR (1 and 2) and 17B1F and 17B1R (3 and 4). Refer to Materials and methods for the primer sequences and amplification conditions used. The triangle indicates the transposon insertion site. The dashed line indicates the 17B probe fragment. Inset table summarizes the blastp analysis and predicted function of the three proteins encoded by the operon. *DUF990-domain of unknown function 990 is associated with ABC-2 transporters (Reizer et al., 1992). AA, amino acid; TM, transmembrane.

Fig. 2

Neighbor-joining tree of RL2976 and RL2977 with the transmembrane components of well-characterized ABC-2 transporters and DUF990 proteins. Bootstrap analysis was performed with 1000 replicates, and values (out of 1000) are as displayed on tree. RHE_CH01148, Rhizobium etli CFN 42 (YP_468682.1); RL2977, Rhizobium leguminosarum bv. viciae 3841 (YP_768561.1); DrrB, Mycobacterium avium ssp. paratuberculosis K-10 (NP_960171.1); NodJ, R. leguminosarum bv. viciae 3841 (YP_770467.1); CtrC, Neisseria meningitidis Z2491 (NP_283043.1); BexB, Haemophilus influenzae (P22235); KpsM, Escherichia coli (AAC38078.1); RHE_CH01149, R. etli CFN 42 (YP_468683.1); RL2976, R. leguminosarum bv. viciae 3841 (YP_768560.1); RkpT1, Sinorhizobium meliloti 1021 (NP_437116); RkpT2, S. meliloti 1021 (NP_437102); JDR-2, Paenibacillus sp. JDR-2 (ZP_02846839); H10, Clostridium cellulolyticum H10 (ZP_01574145.1); WSM1325C, R. leguminosarum bv. trifolii WSM1325 (ZP_02293733); DSM 13941, Roseiflexus castenholzii DSM 13941 (YP_001431939); DSM 8903, Caldicellulosiruptor saccharolyticus DSM 8903 (YP_001179931.1); WSM1325B, R. leguminosarum bv. trifolii WSM1325 (ZP_02293734); EryF, R. leguminosarum bv. viciae 3841 (YP_764713); GacX, Streptomyces glaucescens (CAL64857.1). *Represents proteins where experimental data exists on their cellular function.

Fig. 3

Compositional analysis of exopolysaccharides purified from Rhizobium leguminosarum 3841, wt (a) and 17B, mutant (b). Gas–liquid chromatography analysis of exopolysaccharide indicated a similar composition characteristics for HMW acidic exopolysaccharide. Abbreviations: GlcA, glucuronic acid; Gal, galactose; PvGal, 4,6-pyruvate acetal of galactose; Glc, glucose; Std, inositol used as a standard.

Fig. 4

A500MHz ¹H-NMR spectrometry comparative analysis of Rhizobium leguminosarum 3841, wild type and 17B, mutant exopolysaccharide.

Fig. 5

Capsule stain of RL2975-77 transporter mutants grown in VMM broth. Cells are shown at ×1000 magnification. (a) wild-type 3841; (b) 17B; (c) 38EV30.

Fig. 6

CLSM of biofilms produced by wild-type Rhizobium leguminosarum bv. viciae 3841 (a and c) and the 17B mutant (b and d). Biofilms of the wild type and mutant strains were stained with acridine orange (a and b) or using the Live/Dead™ cell viability kit (c and d).