A vector for systematic gene inactivation in Bacillus subtilis

Abstract

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 t o 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.

Regulation of Bacillus subtilis bacillithiol biosynthesis operons by Spx

“…To explore the possibility of another promoter in the ylbQbshC intergenic region, the ylbQ and bshC genes were independently disrupted by Campbell integration using plasmids derived from pMUTIN4 (Vagner et al, 1998). Consistent with previous results (Gaballa et al, 2010), the bshC disruptant was highly sensitive to fosfomycin, indicative of a loss of BSH production.…”

“…To test the hypothesis that the ypjD promoter site is required for the transcription of all seven genes, we took advantage of the fact that the last two genes in the putative operon, cca and birA, are essential for growth. We integrated a pMUTIN4-derived plasmid by Campbell integration into the ypjD gene, which thereby places expression of downstream genes under IPTG control (Vagner et al, 1998). Growth of the resulting ypjD mutant strain was dependent on IPTG (Fig.…”

“…Site-directed mutagenesis was done by PCR and overlap extension as described by Ho et al (1989). Construction of mutations in ypjD, ylbQ and bshC was carried out by cloning an internal fragment from each ORF (lacking the promoter) into pMUTIN4 (Vagner et al, 1998) and inserting the plasmid into the locus using Campbell integration, which places downstream genes under the control of the IPTG-inducible P spac promoter. For disruption of ypjD, IPTG was added during transformation and growth to ensure the expression of the essential downstream genes.…”

Systematic study of gene expression and transcription organization in the gntZ–ywaA region of the Bacillus subtilis genome

“…Table 1 comprises a list of the B. subtilis strains through integrational mutagenesis of one of the pMUTIN plasmids into the chromosome of B. subtilis Marburg 168 trpC2 (Anagnostopoulos & Spizizen, 1961), the standard strain for the international project. The standard strain and pMUTIN plasmids [pMUTIN1, pMUTIN2 and pMUTIN4 (Vagner et al, 1998)] were obtained from Drs V. Vagner and S. D. Ehrlich (Institut National de la Recherche Agronomique, Jouy-en-Josas, France).…”

“…This inactivation is being carried out through integrational mutagenesis of a pMUTIN series of plasmids carrying lacZ as a reporter gene, as illustrated in Fig. 1 (Vagner et al, 1998). Segments of the target genes are cloned into one of the pMUTIN plasmids, and the genomic copy is inactivated through a single-or double-crossover event.…”