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Bacillus subtilis Gene Cluster Involved in Calcium Carbonate Biomineralization

Chiara Barabesi,¹ Alessandro Galizzi,² Giorgio Mastromei,^1* Mila Rossi,¹ Elena Tamburini,^1, and Brunella Perito¹

Dipartimento di Biologia Animale e Genetica Leo Pardi, Università degli Studi di Firenze, via Romana 17, 50125 Firenze, Italy,¹ Dipartimento di Genetica e Microbiologia A. Buzzati-Traverso, and Centro di Eccellenza in Biologia Applicata, Università degli Studi di Pavia, via Ferrata 1, 27100 Pavia, Italy²

Received 13 September 2006/ Accepted 27 October 2006

Calcium carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological implications. Nevertheless, little is known of the molecular mechanisms by which bacteria foster calcium carbonate mineralization. In our laboratory, we are studying calcite formation by Bacillus subtilis, in order to identify genes involved in the biomineralization process. A previous screening of UV mutants and of more than one thousand mutants obtained from the European B. subtilis Functional Analysis project allowed us to isolate strains altered in the precipitation phenotype. Starting from these results, we focused our attention on a cluster of five genes (lcfA, ysiA, ysiB, etfB, and etfA) called the lcfA operon. By insertional mutagenesis, mutant strains carrying each of the five genes were produced. All of them, with the exception of the strain carrying the mutated lcfA operon, were unable to form calcite crystals. By placing transcription under IPTG (isopropyl-ß-D-thiogalactopyranoside) control, the last gene, etfA, was identified as essential for the precipitation process. To verify cotranscription in the lcfA operon, reverse transcription-PCR experiments were performed and overlapping retrocotranscripts were found comprising three adjacent genes. The genes have putative functions linked to fatty acid metabolism. A link between calcium precipitation and fatty acid metabolism is suggested.

Published ahead of print on 3 November 2006.

Present address: Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e Biotecnologie Microbiche, Università degli Studi di Cagliari, Cagliari, Italy.