Genome engineering reveals large dispensable regions in Bacillus subtilis.
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.
Westers
Helga
H
Department of Pharmaceutical Biology, University of Groningen, Groningen, the Netherlands.
Dorenbos
Ronald
R
van Dijl
Jan Maarten
JM
Kabel
Jorrit
J
Flanagan
Tony
T
Devine
Kevin M
KM
Jude
Florence
F
Seror
Simone J
SJ
Beekman
Aaron C
AC
Darmon
Elise
E
Eschevins
Caroline
C
de Jong
Anne
A
Bron
Sierd
S
Kuipers
Oscar P
OP
Albertini
Alessandra M
AM
Antelmann
Haike
H
Hecker
Michael
M
Zamboni
Nicola
N
Sauer
Uwe
U
Bruand
Claude
C
Ehrlich
Dusko S
DS
Alonso
Juan C
JC
Salas
Margarita
M
Quax
Wim J
WJ
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
D003470:Culture Media