| Abstract: | We have previously described a system of experimental evolution in which many of the mutants of Klebsiella aerogenes selected for faster growth on xylitol ('evolvants') synthesized elevated levels of ribitol dehydrogenase and have presented genetic evidence implicating gene duplication in the enzyme superproduction in some of the evolvants. Here we describe a physical approach to the screening for gene duplications and subsequent structure determination. Nick-translated, cloned ribitol operon (rbt) DNA was used as a hybridization probe to identify fragments containing rbt operon sequences in restriction digests of total bacterial DNA. Whilst several of the evolvants probably harbour duplications spanning the entire rbt operon, one of the spontaneously arising evolvants (strain A3) was shown to harbour a small (5.8 kilobase pairs) direct DNA repeat which encodes the dehydrogenase (but not the kinase) of the closely linked D-arabitol operon as well as the dehydrogenase (but not the kinase) of the rbt operon. The hybridization data suggest that there are 4 to 5 copies of the repeat arranged contiguously on the chromosome. The genetic instability of strain A3, the rbt fragment hybridization pattern of an A3 segregant and the activities of the pentitol catabolic enzymes in A3 are all consistent with the proposed gene duplication structure. |
