A symbiotic mutant of Sinorhizobium meliloti reveals a novel genetic pathway involving succinoglycan biosynthetic functions

Abstract

SummaryA large-scale screen for symbiotic mutants was carried out using the model root nodulating bacterium Sinorhizobium meliloti. Several mutations in the previously uncharacterized gene msbA2 were isolated. msbA2 encodes a member of the ATPbinding cassette exporter family. This protein family is known to export a wide variety of compounds from bacterial cells. S. meliloti MsbA2 is required for the invasion of nodule tissue, with msbA2 mutant cells stimulating nodule primordium morphogenesis, but failing to invade plant tissue beyond the epidermal cell layer. msbA2 mutants do not exhibit any of the free-living traits often found to correlate with symbiotic defects, suggesting that MsbA2 may take part in a specifically symbiotic function. In strains that overproduce the symbiotic signalling polysaccharide succinoglycan, loss of MsbA2 function is extremely deleterious. This synthetic lethal phenotype can be suppressed by disrupting the succinoglycan biosynthetic genes exoY or exoA. It can also be suppressed by disrupting putative glycosyltransferase-encoding genes found upstream of msbA2. Finally, the symbiotic phenotype of a msbA2 null mutant is suppressed by secondary mutations in these upstream transferase genes, indicating that the msbA2 mutant phenotype may be caused by an inhibitory accumulation of a novel polysaccharide that is synthesized from succinoglycan precursors.

Mutation of a Broadly Conserved Operon (RL3499-RL3502) from Rhizobium leguminosarum Biovar viciae Causes Defects in Cell Morphology and Envelope Integrity

“…R. leguminosarum, and rhizobia in general, has a complex cell envelope consisting of the cytoplasmic membrane, the peptidoglycan cell wall, the outer membrane (with several lipopolysaccharide [LPS] modifications), and a number of excreted surface polysaccharides (21,48). Numerous studies have investigated the importance of the cell envelope for survival of rhizobia both under free-living conditions and during symbiosis (11,13,24,58,61). For example, the outer membranes of Rhizobium spp.…”

“…Hyper-and hypo-osmotic stress tolerances have also been associated with cell envelope components. Periplasmic cyclic ␤-1,2-glucans and very-long-chain fatty acid (VLCFA)-modified lipid A are required for hypo-osmotic stress tolerance in R. leguminosarum and Sinorhizobium meliloti (11,24,58), while an intact outer membrane and exopolysaccharides are necessary for tolerance to hyperosmotic stress (61). In addition, several surface polysaccharides, LPS, and many membrane proteins have all been shown to play essential roles in the development of a successful symbiosis (7,16,25,41,48,59).…”

“…For example, the outer membranes of Rhizobium spp. are critical for resisting the harmful effects of detergents, hydrophobic antibiotics, and cationic peptides (11,24,58). In addition, loss of both the O antigen and exopolysaccharides in Rhizobium etli biovar phaseoli CE3 causes increased sensitivity to plant-derived toxic compounds such as coumestrol, suggesting that the cell envelope plays an important role in protecting rhizobia from toxins encountered in the rhizosphere (13).…”

The Sinorhizobium meliloti Essential Porin RopA1 Is a Target for Numerous Bacteriophages

“…pRK600 expresses trans-acting proteins required for mobilization of plasmids harboring the RK2 transfer origin (oriT). Tn5-110 minitransposon delivery and identification of transposon insertion sites by arbitrary PCR were described previously (8). Phagemediated transduction was also described previously (6,7).…”

Control of Gluconate Utilization in Sinorhizobium meliloti

“…In this series of mutants, the associated transposon insertions again mapped to SMa0246. To confirm that loss of SMa0246 function gives rise to the gluconate utilization phenotype, we created an in-frame deletion of SMa0246 using the same technique as described previously (4,5). The ⌬SMa0246 allele gave rise to a gluconate utilization phenotype identical to that observed for the spontaneous and transposon-induced alleles.…”