Differential Expression of over 60 Chromosomal Genes in Escherichia coli by Constitutive Expression of MarA

Abstract

In Escherichia coli, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the mar regulon, duplicate macroarrays containing 4,290 open reading frames of the E. coli genome were hybridized to radiolabeled cDNA populations derived from mar-deleted and mar-expressing E. coli. Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the soxRS regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described.The chromosomal multiple-antibiotic-resistance (mar) locus, first described for Escherichia coli (22), is also present among other enteric bacteria (14). Molecular characterization of this locus has been performed in E. coli (11), Salmonella enterica serovar Typhimurium (51), and more recently Shigella flexneri (T. M. Barbosa and S. B. Levy, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. A-42, p. 9, 1999). In all three genera, the locus consists of two divergently transcribed units, marC and marRAB, which are regulated from independent promoters (Pmar I and Pmar II , respectively) located in the marC-marR intergenic promoter/operator region. MarC has characteristics of a putative integral inner membrane protein whose function is unknown. marRAB specifies two regulatory proteins, MarR, the repressor of the operon, and MarA, a transcriptional activator. The function of MarB has not yet been defined. Increased expression of the marRAB operon results from mutations in marO or marR or from inactivation of MarR following exposure to different inducing agents, such as salicylate (1, 12). The resultant Mar phenotype includes resistance to structurally unrelated antibiotics (21, 43), organic solvents (6, 54), oxidative stress agents (4), and disinfectant products (40,42).The Mar phenotype is achieved through the differential expression of many chromosomal genes within the mar regulon. Regulation by MarA is achieved by its binding to a specific DNA sequence, "marbox," in the vicinity of the promoters of controlled genes (37) or by other mechanisms yet to be identified.Considering the broad Mar phenotype, we hypothesized that MarA affected the expression of a much wider collection of genes than is currently known. Using E. coli Panorama gene macroarrays we identified a large number of genes differentially expressed by constitutive expression of MarA, whose products may be involved in the cell's response to different environmental stresses. MATERI...

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