Cloning and partial characterization of two chromosomal loci from Bacteroides ovatus that contain genes essential for growth on guar gum

Abstract

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.