Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae VF39

Abstract

Four mutants of Rhizobium leguminosarum biovar viciae VF39 altered in lipopolysaccharide (LPS) synthesis were isolated upon random TnS mutagenesis. These mutants produced matt colonies on TY medium and showed autoagglutination and loss of motility. On sodium dodecyl sulfate-polyacrylamide gels, they lacked a slow-migrating carbohydrate band, corresponding to the complete LPS (LPSI). All four mutants formed small white nodules on Vicia hirsuta. These nodules were infected but showed no nitrogen-fixing activity and senesced prematurely. Three of the mutants were complemented by a wild-type cosmid to synthesis of normal LPS and induction of nitrogen-fixing nodules. By hybridization and in vivo cloning experiments, the mutations were mapped within different EcoRI fragments which could be localized on the VF39 chromosome. Crosscomplementation analyses revealed that the three mutants were affected in different transcriptional units. The results indicate that a cluster of genes necessary for LPSI production and symbiotic efficiency is located within a defined region of 20 kilobases on the R. keguminosarum bv. viciae chromosome.The establishment of nitrogen-fixing nodules in the Rhizobium-legume symbiosis is a complex multistep interaction between microsymbiont and its specific host plant (for a review, see reference 2). Early events involve recognition and deformation or curling of root hair cells. Cortical cell division, leading to the formation of a unique organ, the nodule, is induced probably by diffusible bacterial substances. Invasion of the nodule by the bacteria is initiated by penetration of the root hair cell wall and the formation of an infection thread, in which the bacteria are carried towards the dividing root cortex cells. Bacteria are eventually released from the infection thread into the plant cells. They remain separated from the host cytoplasm by peribacteroid membranes and differentiate into bacteroids able to reduce atmospheric nitrogen.This complex developmental process obviously requires continuous signal exchange between plant and bacterial cells. Undoubtedly, bacterial cell surface components play an important role in this specific interaction; particularly extracellular polysaccharides (EPSs) and lipopolysaccharides (LPSs) have been hypothesized to be involved in the symbiotic process (for reviews see references 5 and 15).The LPS of gram-negative bacteria consists of lipid A, a core oligosaccharide and an 0-antigenic side chain. The Rhizobium LPSs are very heterogenous molecules and vary greatly among different species as among strains of a single species (6, 49). Therefore, rhizobial LPS has been suggested to be involved in specific recognition and attachment of bacteria to the root hair cells of compatible host plants, mediated by the binding of lectins (47). While recent work (12) supports an essential function of lectins in host-plant specificity, a role of LPS in host-specific nodulation remains unclear.Differences in LPS composition were described between nodulating and nonnodulating str...

The Rhizobium leguminosarum bv. viciae glnD gene, encoding a uridylyltransferase/uridylyl-removing enzyme, is expressed in the root nodule but is not essential for nitrogen fixation

“…The protocols for mating experiments, mobilization of plasmids between E. coli and Rhizobium strains as well as for Tn5 mutagenesis were as previously described (Simon, 1984 ;Priefer, 1989).…”

“…Nodulation and nitrogen fixation tests using Vicia hirsuta as a host plant and acetylene reduction assays for the determination of nitrogenase activity were as previously described (Priefer, 1989).…”

Cloning and analysis of a 35.3-kilobase DNA region involved in exopolysaccharide production by Xanthomonas campestris pv. campestris

“…Figure 1 shows the positions of all insertion mutations and the different phenotypes of the resulting clones. In cross-complementation experiments, cosmid pXCB 1002 containing the different TnS-lac insertions was transferred into the TnS-lac-induced mutants and tested for complementation (22). Selection for vector-encoded tetracycline resistance resulted in transconjugants carrying the cosmid integrated into the chromosome.…”

“…The possibility that the 3.8-kb fragment and neighboring DNA regions might be favored targets for insertion sequence element mutations is very interesting in view of the instability in EPS production which has been reported for several organisms, including Pseudomonas aeruginosa (9, 11), Pseudomonas atlantica (3, 4), Zoogloea ramigera (10), and X. campestris (17). To study the location and expression of EPS biosynthetic genes, we exposed pXCB1002 to transposon TnS-lac (TnS-B20; 25) mutagenesis in E. coli as described elsewhere (16,25 tris wild-type chromosome by marker exchange (22). The resulting transconjugants were tested for EPS production on TY agar supplemented with 2% sucrose.…”

“…Recombinant cosmids contained inserts ranging in size from 30 to 40 kilobases (kb) (EcoRI fragments). Bacterial matings on membrane filters (pore size, 0.2 ptm) and genetic complementation analyses were performed as described elsewhere (22,23). The cosmids were mobilized into B100-13, B100-22, and B100-152.…”