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Mol. Biol. Evol. 20(12):2076-2090. 2003
DOI: 10.1093/molbev/msg219
© 2003 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

Genome Engineering Reveals Large Dispensable Regions in Bacillus subtilis

Helga Westers*, Ronald Dorenbos*, Jan Maarten van Dijl*,, Jorrit Kabel*, Tony Flanagan{dagger}, Kevin M. Devine{dagger}, Florence Jude{ddagger}, Simone J. Séror{ddagger}, Aäron C. Beekman§, Elise Darmon§, Caroline Eschevins§, Anne de Jong§, Sierd Bron§, Oscar P. Kuipers§, Alessandra M. Albertini||, Haike Antelmann, Michael Hecker, Nicola Zamboni#, Uwe Sauer#, Claude Bruand**, Dusko S. Ehrlich**, Juan C. Alonso{dagger}{dagger}, Margarita Salas{ddagger}{ddagger} and Wim J. Quax*

* Department of Pharmaceutical Biology, University of Groningen, Groningen, the Netherlands
{dagger} Department of Genetics, Smurfit Institute, Trinity College, Dublin, Ireland
{ddagger} Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France
§ Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Haren, the Netherlands
|| Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, Pavia, Italy
Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universität Greifswald, Greifswald, Germany
# Institute of Biotechnology, ETH Zürich, Zürich, Switzerland
** Génétique Microbienne, INRA—Domaine de Vilvert, Jouy en Josas, France
{dagger}{dagger} Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
{ddagger}{ddagger} Centro de Biología Molecular Severo Ochoa, CSIC, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

E-mail: j.m.van.dijl{at}farm.rug.nl.

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, ~47% of the ~4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPß, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.

Key Words: Bacillus subtilis • PBSX • prophage • secretome • skin • SPß


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