Striking Complexity of Lipopolysaccharide Defects in a Collection of Sinorhizobium meliloti Mutants

Abstract

Although the role that lipopolysaccharide (LPS) plays in the symbiosis between Sinorhizobium meliloti and alfalfa has been studied for over a decade, its function in this process remains controversial and poorly understood. This is largely due to a lack of mutants affected by its synthesis. In one of the definitive studies concerning this issue, Clover et al. (R. H. Clover, J. Kieber, and E. R. Signer, J. Bacteriol. 171:3961-3967, 1989) identified a series of mutants with putative LPS defects, judged them to be symbiotically proficient on Medicago sativa, and concluded that LPS might not have a symbiotic function in S. meliloti. The mutations in these strains were never characterized at the molecular level nor was the LPS from most of them analyzed. We have transduced these mutations from the Rm2011 background from which they were originally isolated into the sequenced strain Rm1021 and have characterized the resulting strains in greater detail. We found the LPS from these mutants to display a striking complexity of phenotypes on polyacrylamide electrophoresis gels, including additional rough LPS bands and alterations in the molecular weight distribution of the smooth LPS. We found that some of the mutants contain insertions in genes that are predicted to be involved in the synthesis of carbohydrate components of LPS, including ddhB, lpsB, lpsC, and lpsE. The majority, however, code for proteins predicted to be involved in a wide variety of functions not previously recognized to play a role in LPS synthesis, including a possible transcription elongation factor (GreA), a possible queuine synthesis protein, and a possible chemotaxis protein. Furthermore, using more extensive assays, we have found that most of these strains have symbiotic deficiencies. These results support more recent findings that alterations in LPS structure can affect the ability of S. meliloti to form an effective symbiosis.The bacterium Sinorhizobium meliloti is capable of forming a symbiosis with alfalfa, in which it reduces nitrogen from the atmosphere into ammonia for the plant in exchange for other nutrients. Establishing a symbiosis requires close association between the plant and bacterium. The bacteria live intracellularly within the plant in an organ called the root nodule. From the time at which the bacteria colonize the roots of the plant to the development of nitrogen-fixing nodules, the two organisms are in intimate contact. It is therefore not surprising that this developmental process involves many components of the bacterial cell envelope. Various cell surface polysaccharides have proven to be involved, including lipopolysaccharide (LPS), K antigen, exopolysaccharides, and cyclic ␤-(1,2)-glucans (36).LPS comprises a large percentage of the bacterial outer envelope and consists of three components: lipid A, containing both sugar and fatty acid molecules, which serves to anchor the LPS to the cell envelope; the core, a nonrepeating oligosaccharide; and the O antigen, consisting of repeating oligosaccharide subunits. In the case o...

Sinorhizobium meliloti lsrB is involved in alfalfa root nodule development and nitrogen-fixing bacteroid differentiation

“…Interestingly, lpsCDE genes are divergently localized with lsrB on the S. meliloti genome. S. meliloti lpsCDE genes encode glycotrasferases involved in biosynthesis of LPS core, which is required for efficient nodulation on alfalfa [8,20]. Therefore, we determined the transcripts of theses genes by RT-PCR, cloned the predicted promoters of lpsCDE, and fused them with the GUS reporter gene.…”

Transcriptional Regulator LsrB of Sinorhizobium meliloti Positively Regulates the Expression of Genes Involved in Lipopolysaccharide Biosynthesis

“…The lpsL gene encodes a UDP-glucuronate 5=-epimerase, rkpK encodes a UDP-glucose 6-dehydrogenase, and ddhB encodes a CDP-glucose 4,6-dehydratase to catalyze the synthesis of CDP-4-keto-6-deoxy-D-glucose from CDP-D-glucose, which is associated with O-antigen biosynthesis. Genetic evidence demonstrates that null mutants of lpsB, lpsC, rkpK, and lpsL in the S. meliloti 1021 background induce defective nitrogen fixation nodules on some ecotypes of Medicago sativa or Medicago truncatula A17 (11,13). Root nodules hosting lpsB::TnphoA mutants from M. sativa cv.…”

“…In S. meliloti, several LPS biosynthesis genes have been identified, including lpsB, lpsCDE, lpsL, rkpK, and ddhB (10)(11)(12)(13)(14). The lpsB, lpsC, and lpsDE genes encode a type I glycosyltransferase, ␤-1,4-glycosyltransferase, and RfaG glycosyltransferases, respectively, which are involved in the biosynthesis of the core oligosaccharides.…”

Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9

“…The LPS was extracted from early-stationaryphase cultures (1.5 ml) by SDS lysis and resolved by SDS-polyacrylamide gel electrophoresis exactly as described previously (6). To visualize the different LPS species, the gels were fixed overnight (40% [vol/vol] ethanol, 5% [vol/vol] glacial acetic acid) and then treated with sodium meta-periodate (0.7% [wt/vol] in fixative solution) for 5 min.…”

“…To visualize the different LPS species, the gels were fixed overnight (40% [vol/vol] ethanol, 5% [vol/vol] glacial acetic acid) and then treated with sodium meta-periodate (0.7% [wt/vol] in fixative solution) for 5 min. The gels were then stained with silver nitrate exactly as described previously (6).…”