B Hötte

The nucleotide sequence of a 3.4-kb EcoRI-PstI DNA fragment of Xanthomonas campestris pv. campestris revealed two open reading frames, which were designated xanA and xanB. The genes xanA and xanB encode proteins of 448 amino acids (molecular weight of 48,919) and 466 amino acids (molecular weight of 50,873), respectively. These genes were identified by analyzing insertion mutants which were known to be involved in xanthan production. Specific tests for the activities of enzymes involved in the biosynthesis of UDP-glucose and GDP-mannose indicated that the xanA gene product was involved in the biosynthesis of both glucose 1-phosphate and mannose 1-phosphate. The deduced amino acid sequence of xanB showed a significant degree of homology (59%) to the phosphomannose isomerase of Pseudomonas aeruginosa, a key enzyme in the biosynthesis of alginate. Moreover, biochemical analysis and complementation experiments with the Escherichia coli manA fragment revealed that xanB encoded a bifunctional enzyme, phosphomannose isomerase-GDP-mannose pyrophosphorylase.

On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliloti strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.

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.

Cosmid clones able to restore exopolysaccharide production in possibly insertion sequence element-induced surface mutants of Xanthomonas campestris pv. campestris were isolated. By fragment-specific Tn5-lac mutagenesis of one of the cosmids, pXCB1002, a new DNA region which is involved in exopolysaccharide biosynthesis and which is organized into at least 12 complementation groups was identified.