soxR Mutant Research Articles

The role of oxidative stress genes and effect of pH on methylene blue sensitized photooxidation of Escherichia coli.

In this study, the survival time of wild type E. coli W3110 and 11 mutants was analysed with a plate count method in methylene blue added or control groups under daylight fluoroscence illumination (4950 lux) at different pH values (5.0, 6.0, 7.0, and 8.0) in phosphate buffer. As a result, while the number of bacteria did not decrease under photooxidative stress at pH 5.0 and 6.0 during a 6-hour incubation, the wild type and all mutants decreased more than 2 log. at pH 8.0, and approximately one log. at pH 7.0. It was determined that a 2 log decrease in wild type E. coli takes 3.7 h according to t99 value at pH 8, these values were 2.39 h in the katE mutant, 2.64 h in the soxR mutant, 2.67 h in the oxyR mutant, 2.71 h in the sodB mutant, 3 h in the btuE mutant, 3.38 h in the zwf mutant and 3.40 h in the soxS mutant, respectively (p < 0.05). The roles of these genes were proved with complement tests. Finally, it is found that the effectiveness of photooxidative stress is in direct relation with pH, and the katE, soxR, oxyR, sodB, btuE, zwf, and soxS genes are important for the protection against this stress.

Regulation by SoxR of mfsA, Which Encodes a Major Facilitator Protein Involved in Paraquat Resistance in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia MfsA (Smlt1083) is an efflux pump in the major facilitator superfamily (MFS). Deletion of mfsA renders the strain more susceptible to paraquat, but no alteration in the susceptibility levels of other oxidants is observed. The expression of mfsA is inducible upon challenge with redox cycling/superoxide-generating drug (paraquat, menadione and plumbagin) treatments and is directly regulated by SoxR, which is a transcription regulator and sensor of superoxide-generating agents. Analysis of mfsA expression patterns in wild-type and a soxR mutant suggests that oxidized SoxR functions as a transcription activator of the gene. soxR (smlt1084) is located in a head-to-head fashion with mfsA, and these genes share the -10 motif of their promoter sequences. Purified SoxR specifically binds to the putative mfsA promoter motifs that contain a region that is highly homologous to the consensus SoxR binding site, and mutation of the SoxR binding site abolishes binding of purified SoxR protein. The SoxR box is located between the putative -35 and -10 promoter motifs of mfsA; and this position is typical for a promoter in which SoxR acts as a transcriptional activator. At the soxR promoter, the SoxR binding site covers the transcription start site of the soxR transcript; thus, binding of SoxR auto-represses its own transcription. Taken together, our results reveal for the first time that mfsA is a novel member of the SoxR regulon and that SoxR binds and directly regulates its expression.

Investigation into the effect of soxR and soxS genes deletion on the central metabolism of Escherichia coli based on gene expressions and enzyme activities

Effect of soxR and soxS genes deletion on the central metabolism of Escherichia coli was investigated by combining the information of fermentation characteristics, gene expressions and enzyme activities. Twenty-one different enzyme activities in the central metabolic pathways were assayed in both mutants and its parent E. coli. Semi-quantitative RT-PCR was performed to detect the transcriptional levels of 79 different genes. The deletion of soxR and soxS genes reduced the specific glucose uptake rate and specific growth rate. On the other hand, acetate production rate was increased significantly in the soxS mutant as compared to that in the parent strain, which is partly due to down-regulation of tricarboxylic acid (TCA) cycle. It was found that the pntA (membrane bound transhydrogenase) transcripts, which is involved in NADPH generation were up-regulated in both soxR and soxS mutants by 2.0-fold. The reason was mainly due to the down-regulation of NADPH generating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in pentose phosphate (PP) pathway, and isocitrate dehydrogenase (ICDH) in TCA cycle, since NADPH plays a significant role to reduce oxidative stress. On the other hand, udhA (soluble transhydrogenase) transcripts which is involved in the reoxidation of NADPH were found to be down-regulated by 0.8-fold in the soxR mutant but up-regulated by 1.7-fold in the soxS mutant compared to that in the parent strain. This result was consistent with the measurement of transhydrogenase activity, indicating that soxR mutant suffered from insufficient reoxidation of reducing power. This caused the slow growth for the soxR mutant. It was also found that the expression levels of cyoA and ndh genes were down-regulated in both mutants, demonstrating that soxR and soxS genes deletion adversely affected respiratory system and electron transport chain.

Effects of Acetyl Salicylate and Ibuprofen on Fluoroquinolone MICs on Salmonella enterica Serovar typhimurium In Vitro

In this study, the effects of acetylsalicylate and ibuprofen at 2, 4 and 8 mM concentration were investigated on ofloxacin, ciprofloxacin, levofloxacin and pefloxacin minimum inhibitory concentrations (MICs) for 14 Salmonella enterica serovar typhimurium clinical isolates, one standard strain (SZH KUEN 557), SH7616 (acr mutant), SH5014 (parent strain of acr mutant) and PP120 (soxRS mutant) strains.All isolates were susceptible to the 4 fluoroquinolones. In the presence of 2, 4 and 8 mM acetylsalicylate and ibuprofen, 2- to 8-fold increases were observed in fluor oquinolone MICs. This rise was higher, especially in the presence of acetylsalicylate. In spite of this rise, none of the MICs were in the range of resistance limits in vitro. Except for a 2-fold increase in levofloxacin MICs, we did not observe any difference in MICs of ofloxacin, ciprofloxacin, and pefloxacin in the presence of 2, 4 and 8 mM acetylsalicylate and ibuprofen for SH7616 and PP120 strains.According to the in vitro results of this study, it can be suggested that use of acetylsalicylate or ibuprofen together with clinical treatment of bacteria, especially bacteria which show intermediate resistance, will cause resistance. However, since clinical data are insufficient, further studies are needed.

Oxidant-inducible resistance to hydrogen peroxide killing in Agrobacterium tumefaciens requires the global peroxide sensor-regulator OxyR and KatA

Induced adaptive and cross-protective responses to peroxide stress are important strategies used by bacteria to survive stressful environments. We have shown that exposure to low levels of peroxide (adaptive) and superoxide anions (cross-protection) induced high levels of resistance to peroxide killing in Agrobacterium tumefaciens. The mechanisms and genes involved in these processes have not been identified. Here, the roles played by peroxide ( oxyR) and superoxide ( soxR) global regulators and a catalase gene ( katA) during these responses were investigated. H 2O 2-induced adaptive protection was completely abolished in both the oxyR and katA mutants. Superoxide generator (menadione)-induced cross-protection to H 2O 2 killing was observed in a soxR mutant, but not in either an oxyR or a katA mutant. In vivo analysis of the katA promoter, using a katA::lacZ transcriptional fusion, revealed that it could be induced by menadione in an oxyR-dependent manner. These results lead us to conclude that H 2O 2 and superoxide anions directly or indirectly oxidize OxyR and it is the resulting activation of katA expression that is responsible for the induced protection against lethal concentrations of H 2O 2.

The effect of nitric oxide combined with fluoroquinolones against Salmonella enterica serovar Typhimurium in vitro.

Two regulons, soxRS and marRAB, are associated with resistance to quinolones or multiple antibiotic in Salmonella enterica serovar Typhimurium. These regulons are activated by nitric oxide and redox-cycling drugs, such as paraquat and cause on activation of the acrAB-encoded efflux pump. In this study, we investigated the effect of nitric oxide (NO) alone and in combination with ofloxacin, ciprofloxacin, and pefloxacin against S. typhimurium clinical isolates and mutant strains in vitro. We did not observe synergistic effect against clinical isolates and SH5014 (parent strain of acr mutant), while we found synergistic effect against PP120 (soxRS mutant) and SH7616 (an acr mutant) S. typhimurium for all quinolones. Our results suggest that the efficiencies of some antibiotics, including ofloxacin, ciprofloxacin, and pefloxacin are decreased via activation of soxRS and marRAB regulons by NO in S. enterica serovar Typhimurium. Further studies are warranted to establish the interaction of NO with the genes of Salmonella and, with multiple antibiotic resistance.

Polyamines reduce paraquat-induced soxS and its regulon expression in Escherichia coli.

Polyamines, ubiquitous polycationic compounds, are involved in many cellular responses and relieve paraquat-induced cytotoxicity in Escherichia coli. We constructed a new E. coli mutant strain, JIL528, which is deficient in the biosynthesis of both putrescine and spermidine, to examine the physiological role of polyamines under oxidative stress caused by paraquat. Putrescine and spermidine downregulate the expression of soxS induced by paraquat in a concentration-dependent manner. The product of SoxS is a key regulator governing cellular responses against oxidative stress in E. coli. The downregulation of soxS expression by polyamines was not shown in the soxR mutant background. Glucose-6-phosphate dehydrogenase (G6PDH; encoded by zwf) and manganese-containing superoxide dismutase (Mn-SOD; encoded by sodA) activities induced by paraquat were decreased by exogenous polyamines. The induction of the zwf expression by paraquat was also decreased by exogenous polyamines. The polyamine-deficient mutant strain JIL528 showed a higher soxS expression than its parent polyamine-proficient wild type BW1157, on exogenous supplementation of paraquat concentrations below 1 micromol/L. While the growth rate of the mutant was decreased, soxS expression was increased in a concentration-dependent manner above 0.01 micromol/L of paraquat. In contrast, growth inhibition of the mutant by paraquat was relieved, and soxS was no longer induced by exogenous putrescine (1 mmol/L). In conclusion, polyamines protect against paraquat-induced toxicity but downregulate soxS expression, suggesting that the protective role of polyamines against oxidative damage induced by paraquat results in soxS downregulation.

Mechanism of Regulation of 8-Hydroxyguanine Endonuclease by Oxidative Stress: Roles of FNR, ArcA, and Fur

We found previously that 8-hydroxyguanine (oh 8Gua) endonuclease in E. coli is induced in response to oxidative stress in a fashion similar to the oxidative response of the Mn-superoxide dismutase (MnSOD). In this study, attempts were made to identify the genes involved in the co-regulation of E. coli endonuclease and MnSOD ( sodA). oh 8Gua nuclease is induced by molecular oxygen and a superoxide radical generator (paraquat) but not by H 2O 2, suggesting that the regulation of this endonuclease is dependent on SoxRS but independent of OxyR. This enzyme was induced by paraquat in all of the soxRS mutant strains used ( soxR −, soxS − and soxR c), whereas glucose-6-phosphate dehydrogenase (a member of the soxRS regulon) showed the expected responses; therefore, this possibility was excluded. The presence of metal chelators in the growth medium caused the induction of this enzyme, and this induction was suppressed by the addition of Fe ++. Consistent with this finding, this enzyme was expressed under anaerobiosis in all of the mutant strains of fnr in particular, as well as fur, arcA, and combinations thereof. These findings suggest that the oxidative regulation of oh 8Gua endonuclease is under control of fnr, fur, and arcA, where fnr plays a predominant role. The multiple involvement of regulatory genes as well as co-regulation with antioxidant enzyme will enhance the efficiency of cellular growth and survival in the aerobic environment.

Hypochlorous acid activates the heat shock and soxRS systems of Escherichia coli.

A series of plasmids, containing fusions of different stress promoters to lux reporter genes, was used in an attempt to monitor the defense circuits activated upon exposure of Escherichia coli to sublethal doses of free chlorine. A significant level of activation was exhibited by promoters of three heat shock genes (grpE, dnaK, and lon), in an rpoH-dependent manner. The promoter of micF, a gene under the control of the soxRS regulon, was also strongly induced, but not in a soxR mutant. This induction was not affected by sodA and sodB mutations, implying that it did not involve oxygen radical activity. Free-chlorine activation of both heat shock and soxRS regulons required an exposure of less then I s in duration. The oxyR or the SOS regulons were apparently not induced by free chlorine (as judged by lack of activation of katG and recA, respectively), and neither was the universal stress (uspA) protein.

Induction of the SOS response and mutations by reactive oxygen-generating compounds in various Escherichia coli mutants defective in the mutM, mutY or soxRS loci.

Derivatives of E. coli WP2s (uvrA trpE) defective in 7,8-dihydro-8-oxoguanine (8-OG) DNA glycosylase activity (mutM), MutY glycosylase activity on an A:8-OG mispair (mutY), and/or an adaptive response to oxidative stress by superoxide (soxRS) were constructed to compare the mutability to various reactive oxygen-generating compounds. Induction of Trp+ reversion was assayed both in the presence and absence of plasmid pKM101. Phenazine methosulfate and phenazine ethosulfate showed mutagenic activity at a relatively low dose in soxRS mutants. In comparison to the parent strain WP2s, however, the introduction of mutM, mutY, or soxRS mutations, in any combination, did not make the strain hypersensitive in terms of mutability (i.e. mutation induction at relatively low doses) to hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or phenylhydrazine. Mutagenicity of formaldehyde was detected only in the pKM101-carrying strains. On the other hand, bleomycin, menadione, plumbagin, paraquat, and diquat were not mutagenic to any strain, with or without pKM101. The SOS-response inducing activity was measured by monitoring the expression of a umu'-'lacZ fusion gene, carried on the plasmid pSK1002. The induction of the SOS response by hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, formaldehyde, phenylhydrazine, and bleomycin was of almost the same magnitude between the parent strain and a mutM or soxRS mutant. Phenazine methosulfate and phenazine ethosulfate induced the SOS response only in the soxRS derivatives. No induction was detected by treatment with redox-cycling compounds, such as menadione, plumbagin, paraquat, or diquat.

Two-stage induction of the soxRS (superoxide response) regulon of Escherichia coli.

soxR and soxS are adjacent genes that govern a superoxide response regulon. Previous studies revealed that induction of the regulon is accompanied by increased transcription of soxS, which can activate the target genes. Therefore, induction may occur in two stages: the soxR-dependent activation of soxS, followed by the soxS-dependent induction of other genes. However, the requirement for soxR was unproven because the only existing soxR mutations either were of the regulon-constitutive type or also involved soxS. Therefore, we produced an insertion mutation that was shown by complementation to inactivate only soxR. In confirmation of the two-stage model, soxR was required for the induction by paraquat of the target genes studied (nfo, zwf, and sodA), for paraquat resistance, and for the 47- to 76-fold induction of soxS-lacZ gene fusions. Paraquat did not affect the expression of soxR-lacZ gene fusions. In a soxRS deletion mutant, the regulon was constitutively activated by a runaway soxS+ plasmid. However, a lower-copy-number plasmid failed to activate nfo, zwf, or sodA but did increase the paraquat resistance of a soxRS mutant. Therefore, there is a differential response of the regulon genes to soxS overproduction. A soxR regulon-constitutive mutation was suppressed by a soxR+ plasmid, suggesting a competition between native and activated forms of SoxR. It is proposed that to enhance the sensitivity of the response, the cell minimizes such potential competition by manufacturing only a small amount of this sensor protein, thereby necessitating signal amplification via induction of soxS.

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

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 Tn10 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 result define a new regulon, controlled by soxR, mediating at least part of the global response to superoxide in E. coli.

On the regulation of gene expression during Escherichia coli adaptation to hydrogen peroxide

We found previously that 8-hydroxyguanine (oh8Gua) endonuclease in E. coli is induced in response to oxidative stress in a fashion similar to the oxidative response of the Mn-superoxide dismutase (MnSOD). In this study, attempts were made to identify the genes involved in the co-regulation of E. coli endonuclease and MnSOD (sodA). oh8Gua nuclease is induced by molecular oxygen and a superoxide radical generator (paraquat) but not by H2O2, suggesting that the regulation of this endonuclease is dependent on SoxRS but independent of OxyR. This enzyme was induced by paraquat in all of the soxRS mutant strains used (soxR−, soxS− and soxRc), whereas glucose-6-phosphate dehydrogenase (a member of the soxRS regulon) showed the expected responses; therefore, this possibility was excluded. The presence of metal chelators in the growth medium caused the induction of this enzyme, and this induction was suppressed by the addition of Fe++. Consistent with this finding, this enzyme was expressed under anaerobiosis in all of the mutant strains of fnr in particular, as well as fur, arcA, and combinations thereof. These findings suggest that the oxidative regulation of oh8Gua endonuclease is under control of fnr, fur, and arcA, where fnr plays a predominant role. The multiple involvement of regulatory genes as well as co-regulation with antioxidant enzyme will enhance the efficiency of cellular growth and survival in the aerobic environment.