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. 2001 Sep 15;29(18):3804-13.
doi: 10.1093/nar/29.18.3804.

DNA Microarray Analysis of Bacillus Subtilis DegU, ComA and PhoP Regulons: An Approach to Comprehensive Analysis of B.subtilis Two-Component Regulatory Systems

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DNA Microarray Analysis of Bacillus Subtilis DegU, ComA and PhoP Regulons: An Approach to Comprehensive Analysis of B.subtilis Two-Component Regulatory Systems

M Ogura et al. Nucleic Acids Res. .
Free PMC article

Abstract

We have analyzed the regulons of the Bacillus subtilis two-component regulators DegU, ComA and PhoP by using whole genome DNA microarrays. For these experiments we took the strategy that the response regulator genes were cloned downstream of an isopropyl-beta-D-thiogalactopyranoside-inducible promoter on a multicopy plasmid and expressed in disruptants of the cognate sensor kinase genes, degS, comP and phoR, respectively. The feasibility of this experimental design to detect target genes was demonstrated by the following two results. First, expression of lacZ fusions of aprE, srfA and ydhF, the target genes of DegU, ComA and PhoP, respectively, was stimulated in their cognate sensor kinase-deficient mutants upon overproduction of the regulators. Secondly, by microarray analysis most of the known target genes for the regulators were detected and, where unknown genes were found, the regulator dependency of several of them was demonstrated. As the mutants used were deficient in the kinase genes, these results show that target candidates can be detected without signal transduction. Using this experimental design, we identified many genes whose dependency on the regulators for expression had not been known. These results suggest the applicability of the strategy to the comprehensive transcription analysis of the B.subtilis two-component systems.

Figures

Figure 1
Figure 1
Effect of overexpression of response regulator genes on target gene expression. Cells were grown as described in Materials and Methods, except that the total culture volume was 50 ml. After the addition of IPTG (1 mM) at T-1, T-1 and T-2.5 for the aprE-lacZ, srfA-lacZ and ydhF-lacZ experiments, respectively, samples were withdrawn at the indicated times and processed as previously described (24). The numbers on abscissa indicate the growth time in hours relative to the end of vegetative growth (T0). Open and closed symbols indicate the β-galactosidase activities in the cells grown without and with the addition of IPTG, respectively. (A) TT7291 carrying pDG148-degU. (B) OSM102 carrying pDG148-comA. (C) OAM137 carrying pDG148-phoP.
Figure 2
Figure 2
Effect of disruption of degU and comA on expression of bpr-lacZ, yukL-lacZ, ycdA-lacZ and rapF-lacZ, respectively. Cells were grown in Shaeffer’s medium, and the samples were taken at the indicated times for the determination of β-galactosidase activities. Open and closed symbols indicate the β-galactosidase activities in disruption mutants of degU (AC) and comA (D), and those in the wild-type strains, respectively.
Figure 3
Figure 3
Northern analysis of dhbA (A) and murD (B) expression. RNAs were isolated from 20 ml cultures at the indicated times as described in Materials and Methods, and 10 µg of RNA was subjected to gel electrophoresis. Specific RNA was detected with DIG-labeled probes prepared by PCR using primer sets dhbF1 and dhbR1, and murD1 and murD2 for dhbA and murD, respectively.

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