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

Four mutants of Rhizobium leguminosarum biovar viciae VF39 altered in lipopolysaccharide (LPS) synthesis were isolated upon random Tn5 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. Cross-complementation 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. leguminosarum bv. viciae chromosome

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue

Patschkowski T, Schlüter A, Priefer UB. Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue. Mol Microbiol. 1996;21(2):267-280.Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK-fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R, leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr-like gene, designated fixK, whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix+ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN-deletion mutant, whereas no nitrogen-fixation activity was detectable for a flxK/fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified down-stream of fixK-fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation

Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions.

The broad-host-range vectors pSUP104, pSUP106, pSUP204, pSUP304, and pSUP404 are based on conventional Escherichia coli vectors (such as pBR325 and pACYC184) which have been modified to include the mobilization and broad-host-range replication functions of the IncQ plasmid RSF1010. These vector plasmids now can be maintained in a wide range of bacterial genera including Rhizobium, Agrobacterium, and Pseudomonas. They are efficiently mobilized by RP4 and thus are of particular interest for bacteria refractory to transformation. They offer the selection markers and cloning sites characteristic of the basic E. coli vectors. Therefore, they can be applied and adapted to a variety of cloning strategies. However, the cloning of very large fragments (e.g., in cosmid hybrids of pSUP106) was found to affect the stability of the recombinant molecules in a Rec+ background. This instability was not observed with smaller inserts of about 5 kilobases