title: Genome engineering reveals large dispensable regions in Bacillus subtilis.
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 nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 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 (SPbeta, 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.
authors.affiliation: Department of Pharmaceutical Biology, University of Groningen, Groningen, the Netherlands.
authors.forename: Helga
authors.initials: H
authors.lastname: Westers
authors.forename: Ronald
authors.initials: R
authors.lastname: Dorenbos
authors.forename: Jan Maarten
authors.initials: JM
authors.lastname: van Dijl
authors.forename: Jorrit
authors.initials: J
authors.lastname: Kabel
authors.forename: Tony
authors.initials: T
authors.lastname: Flanagan
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Devine
authors.forename: Florence
authors.initials: F
authors.lastname: Jude
authors.forename: Simone J
authors.initials: SJ
authors.lastname: Seror
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Beekman
authors.forename: Elise
authors.initials: E
authors.lastname: Darmon
authors.forename: Caroline
authors.initials: C
authors.lastname: Eschevins
authors.forename: Anne
authors.initials: A
authors.lastname: de Jong
authors.forename: Sierd
authors.initials: S
authors.lastname: Bron
authors.forename: Oscar P
authors.initials: OP
authors.lastname: Kuipers
authors.forename: Alessandra M
authors.initials: AM
authors.lastname: Albertini
authors.forename: Haike
authors.initials: H
authors.lastname: Antelmann
authors.forename: Michael
authors.initials: M
authors.lastname: Hecker
authors.forename: Nicola
authors.initials: N
authors.lastname: Zamboni
authors.forename: Uwe
authors.initials: U
authors.lastname: Sauer
authors.forename: Claude
authors.initials: C
authors.lastname: Bruand
authors.forename: Dusko S
authors.initials: DS
authors.lastname: Ehrlich
authors.forename: Juan C
authors.initials: JC
authors.lastname: Alonso
authors.forename: Margarita
authors.initials: M
authors.lastname: Salas
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
mesh_terms: D017103:Bacillus Phages / Q000235:genetics / Q000254:growth & development; D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development; D002876:Chromosomes, Bacterial; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D020161:Physical Chromosome Mapping; D010957:Plasmids; D013171:Spores, Bacterial / Q000235:genetics
chemical_list: D003470:Culture Media