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MBE Advance Access published online on August 29, 2003
Molecular Biology and Evolution, doi:10.1093/molbev/msg219
Molecular Biology and Evolution © Society for Molecular Biology and Evolution 2003; all rights reserved
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Accepted July 14, 2003
© 2003 Society for Molecular Biology and Evolution

Original Articles

Genome Engineering Reveals Large Dispensable Regions in Bacillus subtilis

Helga Westers 1, Ronald Dorenbos 1, Jan Maarten van Dijl 1*, Jorrit Kabel 1, Tony Flanagan 2, Kevin M. Devine 2, Florence Jude 3, Simone J. Séror 3, Aäron C. Beekman 4, Elise Darmon 4, Caroline Eschevins 4, Anne de Jong 4, Sierd Bron 4, Oscar P. Kuipers 4, Alessandra M. Albertini 5, Haike Antelmann 6, Michael Hecker 6, Nicola Zamboni 7, Uwe Sauer 7, Claude Bruand 8, Dusko S. Ehrlich 8, Juan C. Alonso 9, Margarita Salas 10, and Wim J. Quax 1

1 Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands
2 Department of Genetics, Smurfit Institute, Trinity College, Dublin 2, Ireland
3 Institut de Génétique et Microbiologie, Université Paris-Sud, 91405 Orsay Cedex, France
4 Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN Haren, the Netherlands
5 Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, Via Abbiategrasso, 207, 27100 Pavia, Italy
6 Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universität Greifswald, D-17487 Greifswald, Germany
7 Institute of Biotechnology, ETH Zürich, CH-8093 Zürich, Switzerland
8 Génétique Microbienne, INRA - Domaine de Vilvert, 78352 Jouy en Josas cedex, France
9 Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
10 Centro de Biología Molecular Severo Ochoa, CSIC, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

* To whom correspondence should be addressed. E-mail: J.M.van.Dijl{at}farm.rug.nl.


  Abstract

Bacterial genomes contain 250 to 500 essential genes as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining non-essential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, ~47% of the ~4100 genes of B. subtilis belong to paralogous gene families of 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 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. Step-wise deletion of 2 prophages (SP{beta}, PBSX), 3 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 towards an optimized Bacillus cell factory.

Key Words: Bacillus subtilis, PBSX, prophage, secretome, skin, SP{beta}


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