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.

FIG. 1

Expression profiles of E. coli strains with MarA deleted and constitutively expressing MarA. Identical arrays were probed with labeled ³³P-cDNA populations prepared from total RNA from mar-deleted, AG100Kan[pMAK705] (A), and mar-expressing, AG100Kan[pAS10] (B), strains. Columns (1 to 24) and rows (A to P) forming the primary grid in field 1 of the autoradiogram are shown. Fields 2 and 3 are similar in format to field 1 and are not shown. The four spots in the four corners of each field are genomic DNA. Boxes underneath are expanded views of representative areas shown in panels A and B, where changes in expression levels are visible for several genes (seven of the differentially expressed genes are labeled as examples).

FIG. 2

Chromosomal distribution and location of the different genes affected by MarA expression. The internal circle represents the chromosome of E. coli K-12 MG1655 divided in intervals of 1 min, while the external circle is divided in intervals of 100,000 nucleotide residues (adapted from Blattner et al. [8]). Genes induced by constitutive expression of MarA are plotted to face the exterior of the chromosome, and genes repressed by MarA are plotted to face the interior of the chromosome. Boldface genes read in the clockwise direction, while lightface genes are on the opposite strand (8). Genes that are in the immediate vicinity of each other are together over the same designation line.

FIG. 3

Northern blot analysis of genes affected by constitutive expression of MarA. Eight genes up-regulated by MarA, acnA, gshB, hemB, mdaA, tpx, mglB, nfnB, and yadG, and two genes down-regulated by MarA, aceE and ndh, were selected from those listed in Table 1. Samples were prepared and run in duplicate from mar-expressing (mar⁺) and mar-deleted (Δmar) cells. RNA samples were transferred to nylon membranes and hybridized to ³²P-labeled PCR-amplified probes of the genes in the study.