soxR, a locus governing a superoxide response regulon in Escherichia coli K-12

Abstract

The nfo (endonuclease IV) gene of Escherichia coli is induced by superoxide generators such as paraquat (methyl viologen). An nfo'-lacZ operon fusion was used to isolate extragenic mutations affecting its expression. The mutations also affected the expression of glucose 6-phosphate dehydrogenase, Mn2+-superoxide dismutase (sodA), and three lacZ fusions to soi (superoxide-inducible) genes of unknown function. The mutations were located 2 kilobases clockwise of ssb at 92 min on the current linkage map. One set of mutations, in a new gene designated soxR, caused constitutive overexpression of nfo and the other genes. It included insertions or deletions affecting the carboxyl end of a 17-kilodalton polypeptide. In a soxR mutant, the expression of sodA, unlike that of nfo, was also regulated independently by oxygen tension. Two other mutants were isolated in which the target genes were noninducible; they had an increased sensitivity to killing by superoxide-generating compounds. One had a TnlO insertion in or near soxR; the other had a multigene deletion encompassing soxR. Therefore, the region functions as a positive regulator because it encodes one or more products needed for the induction of nfo. Regulation is likely to be at the level of transcription because the mutations were able to affect the expression of an nfo'-lac operon fusion that contained the ribosome-binding site for lacZ. Some mutant plasmids that failed to suppress (or complement) constitutivity in trans had insertion mutations several hundred nucleotides upstream of soxR in the general region of a gene for a 13-kilodalton protein encoded by the opposite strand, raising the possibility of a second regulatory gene in this region. The results define a new regulon, controlled by soxR, mediating at least part of the global response to superoxide in E. coli.When Escherichia coli is exposed to redox reagents that generate superoxide radical anions through oxidative recycling, it responds with an increased synthesis of a specific set of proteins. Typical inducers are paraquat (methyl viologen), phenazine methosulfate, plumbagin, and menadione. The global response to superoxide involves genes for at least 33 proteins, most of which are of unknown function and which were detected by two-dimensional gel electrophoresis (16,51). The known enzymes that are induced by superoxide include Mn2+-superoxide dismutase (21), glucose 6-

Regulation of the soxRS Oxidative Stress Regulon

“…Plasmids containing truncated soxR alleles were constructed as follows. An HpaI-SmaI segment of the soxRS region (13,14) was ligated first into the SmaI site in the BamHI-SmaI-BamHI cloning site region of plasmid pHE6 (15), and the resulting new BamHI fragment was subcloned in pET11K to yield pET11K-soxR21. Another plasmid was constructed by subcloning a truncated soxR gene from one of a set of M13 recombinant plasmids containing terminal soxR deletions adjacent to the vector's EcoRI site (14).…”

“…Other Redox Cycling Agents-Plumbagin and paraquat are also inducers of the soxRS regulon (13,21). As expected, treatment of the IPTG-induced cells with 15 M plumbagin led to a disappearance of the EPR signal in vivo or a concentration of SoxR red of less than 1 M (results not shown).…”

Several pathways of hydrogen peroxide action that damage the E. coli genome

“…Although this system responds to oxidative stress when cells are exposed to superoxide radical-generating agents, it is not induced by H 2 O 2 (Chan and Tsaneva and Weiss, 1990;Hidalgo et al, 1997). However, it was demonstrated that in some conditions H 2 O 2 as well as singlet molecular oxygen could activate the SoxRS regulon in vivo (Manchado et al, 2000;Agnez-Lima et al, 2001).…”

“…Two key protective responses have been described in E. coli -one controlled by soxRS genes and the other by oxyR (Tsaneva and Weiss, 1990;Storz and Imlay, 1999;Gonzalez-Flecha and Demple, 2000).…”

Genetic definition of the Escherichia coli zwf "soxbox," the DNA binding site for SoxS-mediated induction of glucose 6-phosphate dehydrogenase in response to superoxide

“…Thus, unlike gnd, zwf expression is transcriptionally activated by SoxS during episodes of oxidative stress induced by exposure of E. coli to superoxide-generating agents such as paraquat (17,18,39). Indeed, E. coli strains deleted for zwf are hypersensitive to oxidative stress, thus making zwf a pivotal member of the soxRS regulon (18, 21).…”