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Journal of Bacteriology, April 2005, p. 2662-2672, Vol. 187, No. 8
0021-9193/05/$08.00+0     doi:10.1128/JB.187.8.2662-2672.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Bacillus subtilis SinR and RapA Developmental Regulators Are Responsible for Inhibition of Spore Development by Alcohol{dagger}

Natalia Gottig, María Eugenia Pedrido, Marcelo Méndez, Esteban Lombardía, Adrián Rovetto, Valeria Philippe, Lelia Orsaria, and Roberto Grau*

Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario and Instituto de Biología Molecular y Celular de Rosario, IBR-CONICET, Rosario, Argentina

Received 8 October 2004/ Accepted 12 January 2005

Even though there is a large body of information concerning the harmful effects of alcohol on different organisms, the mechanism(s) that affects developmental programs, at a single-cell level, has not been clearly identified. In this respect, the spore-forming bacterium Bacillus subtilis constitutes an excellent model to study universal questions of cell fate, cell differentiation, and morphogenesis. Here, we demonstrate that treatment with subinhibitory concentrations of alcohol that did not affect vegetative growth inhibited the initiation of spore development through a selective blockage of key developmental genes under the control of the master transcription factor Spo0A~P. Isopropyl-ß-D-thiogalactopyranoside-directed expression of a phosphorylation-independent form of Spo0A (Sad67) and the use of an in vivo mini-Tn10 insertional library permitted the identification of the developmental SinR repressor and RapA phosphatase as the effectors that mediated the inhibitory effect of alcohol on spore morphogenesis. A double rapA sinR mutant strain was completely resistant to the inhibitory effects of different-C-length alcohols on sporulation, indicating that the two cell fate determinants were the main or unique regulators responsible for the spo0 phenotype of wild-type cells in the presence of alcohol. Furthermore, treatment with alcohol produced a significant induction of rapA and sinR, while the stationary-phase induction of sinI, which codes for a SinR inhibitor, was completely turned off by alcohol. As a result, a dramatic repression of spo0A and the genes under its control occurred soon after alcohol addition, inhibiting the onset of sporulation and permitting the evaluation of alternative pathways required for cellular survival.

* Corresponding author. Mailing address: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario and Instituto de Biología Molecular y Celular de Rosario, IBR-CONICET, Suipacha 531, Rosario (2000), Argentina. Phone: (+54) 341-4353377. Fax: (+54) 341-4804601. E-mail: rrgrau{at}infovia.com.ar.

{dagger} Dedicated to the memory of Pierre Schaeffer, who was a clever mind and a visionary at the beginning of the era of sporulation study.

Journal of Bacteriology, April 2005, p. 2662-2672, Vol. 187, No. 8
0021-9193/05/$08.00+0     doi:10.1128/JB.187.8.2662-2672.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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