Identification of essential genes in bacteria.
Essential genes are identified in duplicated regions of the bacterial chromosome. Transposition of a vector that forms operon fusions into a strain carrying a chromosomal duplication allows insertion of the transposon into essential genes because a second copy of the essential gene is present. When the duplication is allowed to segregate, only the segregant that carries the copy of the intact essential gene survives. The transposon insertion in the essential gene is maintained only in the duplication derivatives. A technique is described that uses a Tn10 derivative, Tn10dTc-araC(+), which contains a cloned copy of the Escherichia coli araC(+) gene, as a portable region of homology to generate large duplications of the Salmonella chromosome. The duplication is maintained in the population by growth in the presence of tetracycline. When the lac operon fusion vector, MudJ, is transposed into the duplicated region, removal of tetracycline from the growth media allows segregation of the duplication yielding (Ara(-)) haploid segregants which appear as red colonies or as red/white (Ara(-/+)) sectoring colonies on TTC arabinose indicator plates. However, if the insertion is in an essential gene, only segregants that lose the MudJ insertion in the essential gene survive. In this case, selection for the insertion in the essential gene yields solid white (Ara(+)) colonies in the absence of tetracycline. While the specific design presented uses Mud transposon insertions to generate lac operon (transcriptional) and lacZ gene (translational) fusions to essential genes, this technique can be used to generate transposon insertions of any kind into essential genes.
Hillyard
David R
DR
Department of Pathology, University of Utah, Salt Lake City, UT, USA.
Redd
Michael J
MJ
D002876:Chromosomes, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D020043:Genes, Essential