A vector for systematic gene inactivation in Bacillus subtilis

Genetique Microbienne, lnstitut National de la Recherche Ag ronom ique, Domaine de Vilvefl, 78352 Jouy-en-Josas cedex, France 2 Author for correspondence: Valirie Vagner. Tel: +33 1 34 65 25 34. Fax: +33 1 34 65 25 21.e-mail: vagner@ biotec.jouy.inra.fr ABSTRACT SUMMARY: To study the functions of the uncharacterized open reading frames identified in the Bacillus subtih genome, several vectors were constructed t o perform insertional mutagenesis in the chromosome. All the pMUTlN plasmids carry a lac2 reporter gene and an inducible Pspac promoter, which is tightly regulated and tan be induced about 1000-fold. The integration of a pMUTlN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lac2 becomes transcriptionally fused t o the gene, allowing its expression pattern to be monitored; (3) the Pspac promoter controls the transcription of downstream genes in an IPTG-dependent fashion. This last feature is important because B. subti/is genes are often organized in operons. The potential polar effects generated by the integration of the vectors can be alleviated by addition of IPTG. Also, conditional mutants of essential genes can be obtained by integrating pMUTlN vectors upstream of the target gene. The vectors are currently being used for systematic inactivation of genes without known function within the B. subtilis European consortium. pMUTlN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented. Keywords: integrational vector, Bacillus subtilis, inducible promoter, essential genes