Mesorhizobium lotiProducesnodPQ-Dependent Sulfated Cell Surface Polysaccharides

Abstract

Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a ␤-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.Most soils are limited in reduced forms of nitrogen. Thus, plants undergo symbioses with microorganisms that provide the plants with reduced nitrogen. The best studied of these symbioses occur between leguminous plants and a family of gram-negative bacteria known as rhizobia. The symbiosis culminates in the formation of novel structures on the plant root called nodules. To enter and colonize the nodules, the bacteria elicit a series of morphological changes in specialized epidermal cells called root hairs. The bacteria induce curling of the root hairs, trapping bacterial microcolonies in the center of the structure. The bacteria then elicit the formation of a plantderived tubular structure that emanates from the center of the curl, extending through the root hair and ultimately penetrating through the epidermis into the cortical layers of the root. This structure, known as an infection thread, is occupied by the bacteria and allows their entry into the interior of the root. The bacteria are released from the infection thread into the cytoplasm of plant cells within the nodule, where they differentiate into intracellular forms called bacteroids, which then reduce molecular dinitrogen for use...

Identification of Mesorhizobium loti Genes Relevant to Symbiosis by Using Signature-Tagged Mutants

“…The nodPQ genes (mlr7575 and mlr7576) are required for the biosynthesis of 3'-phosphoadenosine-5'-phosphosulfate, which is used for the sulfation of cellsurface polysaccharides, but not of Nod factor, in M. loti. The mutant in nodPQ elicited effective nodules with reduced nodulation kinetics (43). In addition, we confirmed that strain 06T02h06 (mutant in msl0067) was a purine auxotroph and strain 10T01g06 (mutant in mll5681) was a pyrimidine auxotroph (data not shown).…”

Sinorhizobium meliloti SyrA Mediates the Transcriptional Regulation of Genes Involved in Lipopolysaccharide Sulfation and Exopolysaccharide Biosynthesis

“…PAPS was analyzed as described previously (4), as modified by Townsend et al (62). Briefly, wild-type strains harboring vector control, multicopy syrA, or multicopy nodPQ, were cultured in 1 ml of TY medium with Na 2 35 SO 4 to saturation.…”

“…Al-though common in mammalian cells, sulfated carbohydrates appear to be rare in bacteria, having only been reported in S. meliloti (9), Mycobacterium (45,55), Mesorhizobium loti (62), and Pseudoalteromonas (56) to date. The physiological function of these sulfated molecules remains obscure, although mutants of S. meliloti and M. loti with decreased polysaccharide sulfation exhibit alterations in symbiosis (11,62; D. H. Keating, G. R. O. Campbell, and G. C. Walker, submitted for publication). A recent publication reported the identification of a gene, lpsS, which encodes an LPS sulfotransferase activity in S. meliloti (11).…”

The rkp-1 Cluster Is Required for Secretion of Kdo Homopolymeric Capsular Polysaccharide in Sinorhizobium meliloti Strain Rm1021

“…The method we employed for polysaccharide preparation involved the treatment of the cells with EDTA-TEA, which is expected primarily to extract macromolecules present in the outer membrane (39,61,73). We therefore utilized an alternative method, phenol-water extraction (71), to isolate total radiolabeled cellular polysaccharides, which then were fractionated on Tris-Tricine SDS-PAGE and visualized using phosphorimaging (Fig. 3A).…”

“…The fractions obtained from size-exclusion chromatography were dialyzed to remove detergent (57) and then lyophilized. Aliquots (100 to 200 g) were subjected to glycosyl composition analysis by preparing the trimethylsilyl methyl glycoside derivatives (70), and gas chromatography-mass spectrometry was performed as described previously (71).…”