CLONING AND PARTIAL CHARACTERIZATION OF 2 CHROMOSOMAL LOCI FROM BACTEROIDES-OVATUS THAT CONTAIN GENES ESSENTIAL FOR GROWTH ON GUAR GUM

Previously, we isolated three transposon insertion mutants of Bacteroides ovatus (M-4, M-5, and M-7) that were unable to grow on the branched polysaccharide guar gum. In this study, we used a tetracycline resistance gene on the transposon to clone chromosomal DNA adjacent to the transposon insertions in each of the three mutants. Restriction analysis of the flanking chromosomal DNA in M-4 and M-7 revealed that the insertions in these two mutants were in the same location. The cloned DNA adjacent to the insertions in M-5 and M-7 was used as a hybridization probe to clone the wild-type loci. Two clones of about 10 kbp in size were obtained. Restriction analysis showed that these two clones did not overlap. The clone of the M-5 locus appeared to contain all of the genes affected by the M-5 insertion, but we were unable to demonstrate complementation of the M-5 mutation because of the instability of the clone in this background. Analysis of the clone of the M-7 locus showed that it contained a guar gum-regulated promoter, but the transcript originating from this promoter was not affected by the transposon insertion. Thus, the M-7 locus apparently contains at least two separate transcriptional units, the one defined by this promoter and the one interrupted by the transposon insertion. Insertion mutations downstream of the guar gum-regulated promoter demonstrated that there were essential guar gum utilization genes in this region. The M-7 mutant was eliminated by the wild type in the intestinal tracts of germfree mice. However, in this same model, the insertion mutation downstream of the guar gum-regulated promoter was less deleterious. Thus, the deleterious effect of the M-7 mutation is not simply due to an inability to grow on guar gum.