U. B. Priefer

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.

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.

By mutational analysis it was found that a 3.9-kb SmaI-XhoII DNA fragment of Xanthomonas campestris pv. campestris is involved in lipopolysaccharide (LPS) biosynthesis. LPS samples isolated from different mutants carrying mutations in the 3.9-kb SmaI-XhoII DNA fragment exhibited banding patterns in silver-stained sodium dodecyl sulfate-polyacrylamide gels markedly different from that of the wild-type LPS. Moreover, comparison of the monosaccharide composition obtained by high-performance anion-exchange chromatography with pulsed amperometric detection of LPS purified from wild-type Xanthomonas campestris pv. campestris B100 and from mutants with mutations in the 3.9-kb SmaI-XhoII DNA fragment revealed a lack of rhamnose moieties in the mutant LPS. Sequence analysis of this DNA fragment revealed four open reading frames (ORFs), designated ORF302, ORF183, ORF295, and ORF351. The deduced amino acid sequences of these ORFs showed a high degree of homology to the deduced amino acid sequences of the rfbC, rfbD, rfbA, and rfbB genes of Salmonella typhimurium LT2, which have been shown to encode a set of enzymes responsible for conversion of glucose 1-phosphate to dTDP-rhamnose.