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Journal of Bacteriology, July 2000, p. 3794-3801, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Defining a rob Regulon in Escherichia coli by Using Transposon Mutagenesis

Marjon H. J. Bennik,1,2,* Pablo J. Pomposiello,1 Derek F. Thorne,1 and Bruce Demple1

Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115,1 and Agrotechnological Research Institute (ATO), Wageningen University Research Centre, Wageningen, The Netherlands2

Received 10 January 2000/Accepted 12 April 2000

The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a rob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB, ybaO, mdlA, yfhD, and ybiS). One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of micF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor.

* Corresponding author. Mailing address: Agrotechnological Research Institute (ATO), Wageningen University Research Centre, P.O. Box 17, 6700 AA, Wageningen, The Netherlands. Phone: 31-317-475108. Fax: 31-317-475347. E-mail: m.h.j.bennik{at}ato.wag-ur.nl.

Journal of Bacteriology, July 2000, p. 3794-3801, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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