In situ transposon replacement and isolation of a spontaneous tandem genetic duplication.

Abstract

Using a specialized transducing P1 phage carrying an insertion of Tn5-132, an insertion of Tn5-wt in the chromosome of Myxococcus xanthus, which codes for resistance to kanamycin, can be replaced with one of Tn5-132, which codes for resistance to tetracycline. That Tn5-132 in the daughter is inserted at the same location in the chromosome as Tn5-wt was in the parent was shown by a variety of physical and genetic tests. Southern blot hybridizations of restriction digests of daughter and parent DNAs probed for sequences homologous to Tn5 show that the physical location is the same. When KmR was transduced from the parent to the TcR daughter by the generalized transducing myxophage Mx4 or Mx8, all the transductants were TcS. Likewise, when the daughter was used as donor, TcR transductants of its KmR parent were KmS. Flanking markers that were linked to KmR in the parent were linked to TcR in the daughter. Spontaneous tandem genetic duplications of portions of bacterial chromosomes can be trapped by transducing a selectable marker from a donor to a recipient that has a different selectable marker at the same genetic location and selecting transductants with both markers. Using Tc-replacement, this technique can be applied to any region of the chromosome. We used it to isolate a spontaneous tandem duplication of part of the M. xanthus chromosome. The duplication was characterized by Southern blot hybridizations probed for Tn5-homologous DNA. It was also shown to be unstable by quantitation of loss of drug resistance. Transduction of the novel joint led to reconstruction of the duplication in the recipient strain. All these tests gave results consistent with the proposed structure. The methods described here are applicable to any bacterium into which transposons can be introduced, and for which some means of genetic exchange is available.