A vector for systematic gene inactivation in Bacillus subtilis

Abstract

To study the functions of the uncharacterized open reading frames identified in the Bacillus subtilis genome, several vectors were constructed to perform insertional mutagenesis in the chromosome. All the pMUTIN plasmids carry a lacZ reporter gene and an inducible Pspac promoter, which is tightly regulated and can be induced about 1000-fold. The integration of a pMUTIN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lacZ becomes transcriptionally fused to 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. subtilis 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 pMUTIN 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. pMUTIN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented.