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One Perturbation of the Mother Cell Gene Regulatory Network Suppresses the Effects of Another during Sporulation of Bacillus subtilis ^,

Department of Microbiology & Molecular Genetics,¹ Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, Michigan 48824,² Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, Illinois 60153³

Received 8 August 2007/ Accepted 11 September 2007

In the mother cell of sporulating Bacillus subtilis, a regulatory network functions to control gene expression. Four transcription factors act sequentially in the order ^E, SpoIIID, ^K, GerE. ^E and ^K direct RNA polymerase to transcribe different regulons. SpoIIID and GerE are DNA-binding proteins that activate or repress transcription of many genes. Several negative regulatory loops add complexity to the network. First, transcriptionally active ^K RNA polymerase inhibits early sporulation gene expression, resulting in reduced accumulation of ^E and SpoIIID late during sporulation. Second, GerE represses sigK transcription, reducing ^K accumulation about twofold. Third, SpoIIID represses cotC, which encodes a spore coat protein, delaying its transcription by ^K RNA polymerase. Partially circumventing the first feedback loop, by engineering cells to maintain the SpoIIID level late during sporulation, causes spore defects. Here, the effects of circumventing the second feedback loop, by mutating the GerE binding sites in the sigK promoter region, are reported. Accumulation of pro-^K and ^K was increased, but no spore defects were detected. Expression of ^K-dependent reporter fusions was altered, increasing the expression of gerE-lacZ and cotC-lacZ and decreasing the expression of cotD-lacZ. Because these effects on gene expression were opposite those observed when the SpoIIID level was maintained late during sporulation, cells were engineered to both maintain the SpoIIID level and have elevated sigK expression late during sporulation. This restored the expression of ^K-dependent reporters to wild-type levels, and no spore defects were observed. Hence, circumventing the second feedback loop suppressed the effects of perturbing the first feedback loop. By feeding information back into the network, these two loops appear to optimize target gene expression and increase network robustness. Circumventing the third regulatory loop, by engineering cells to express cotC about 2 h earlier than normal, did not cause a detectable spore defect.

Published ahead of print on 21 September 2007.

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