UNMARKED GENE INTEGRATION INTO THE CHROMOSOME OF MYCOBACTERIUM-SMEGMATIS VIA PRECISE REPLACEMENT OF THE PYRF GENE

After integration into the bacterial chromosome an exogenous gene may be stably expressed without continued selection for the recombinant lo cus. However, chromosomal integration events occur infrequently, requi ring the concomitant integration of a drug resistance marker in order to identify colonies of recombinant cells. The generation of a drug-re sistant recombinant strain can both reduce the in vivo applicability o f the strain and preclude the use of recombinant vectors which use the same drug resistance marker. We have constructed a plasmid, pINT-Delt a, which allows recombination of exogenous genes onto the Mycobacteriu m smegmatis chromosome. The exogenous gene completely replaces the pyr F gene and the resultant strain lacks any exogenous drug resistance ma rker. The methodologies described herein are general and applicable ev en to those bacteria for which extrachromosomal plasmids are not avail able. Using pINT-Delta we integrated the lacZ gene into the M. smegmat is chromosome via a precise exchange of lacZ and pyrF. The resultant s train was used to demonstrate that the expression of genes integrated at the pyrF locus is repressed twofold by inclusion of uracil in the g rowth medium. In addition, we used pINT-Delta to construct an M. smegm atis strain with a precise deletion of its pyrF locus. This strain, TS m-627, grows normally in rich medium but does not grow in medium lacki ng uracil. TSm-627 cells allow the pyrF gene to be used as a selectabl e marker for growth on medium lacking uracil. In TSm-627 cells, the py rF gene is also useful as a counterselectable marker on complete mediu m containing 5'-fluoroorotic acid and uracil. Two pyrF-containing plas mids, designed to exploit the new Delta pyrF strain, have been constru cted and their possible applications to problems in mycobacteriology a re discussed. (C) 1997 academic Press.