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In most bacteria, zinc depletion is sensed by Zur, whereas the surplus is sensed by different regulators to achieve zinc homeostasis. Here we present evidence that zinc-bound Zur not only represses genes for zinc acquisition but also induces the zitB gene encoding a zinc exporter in Streptomyces coelicolor, a model actinobacteria. Zinc-dependent gene regulation by Zur occurs in two phases. At sub-femtomolar zinc concentrations (phase I), dimeric Zur binds to the Zur-box motif immediately upstream of the zitB promoter, resulting in low zitB expression. At the same time, Zur represses genes for zinc uptake. At micromolar zinc concentrations (phase II), oligomeric Zur binding with footprint expansion upward from the Zur box results in high zitB induction. Our findings reveal a mode of zinc-dependent gene activation that uses a single metalloregulator to control genes for both uptake and export over a wide range of zinc concentrations., Zinc homeostasis in most bacteria is achieved by a set of regulators, each responding to a certain level of intracellular zinc. Here the authors show that, in Streptomyces coelicolor, the Zur regulator modulates the expression of genes for zinc import and export over a large range of zinc concentrations.

Summary SoxR is a [2Fe-2S]-containing sensor-regulator, which is activated through oxidation by redox-active compounds (RACs). SoxRs show differential sensitivity to RACs, partly due to different redox potentials, such that Escherichia coli (Ec) SoxR with lower potential respond to broader range of RACs than Streptomyces coelicolor (Sc) SoxR. In S. coelicolor, the RACs that do not activate ScSoxR did not inhibit growth, suggesting that ScSoxR is tuned to respond to growth-inhibitory RACs. Based on sequence comparison and mutation studies, two critical amino acids around the [2Fe-2S] binding site were proposed as key determinants of sensitivity. ScSoxR-like mutation (R127L/P131V) in EcSoxR changed its sensitivity profile as ScSoxR, whereas EcSoxR-like mutation (L126R/V130P) in ScSoxR caused relaxed response. In accordance, the redox potentials of EcSoxRR127L/P131V and ScSoxRL126R/V130P were estimated to be −192 ± 8 mV and −273 ± 10 mV, respectively, approaching that of ScSoxR (−185 mV) and EcSoxR (−290 mV). Molecular dynamics simulations revealed that the R127L and P131V substitutions in EcSoxR caused more electropositive environment around [2Fe-2S], making it harder to get oxidized. This reveals a mechanism to modulate redox-potential in [Fe-S]-containing sensors by point mutations and to evolve a sensor with differential sensitivity to achieve optimal cellular physiology.

Bacteria in natural habitats are exposed to myriad redox-active compounds (RACs), which include producers of reactive oxygen species (ROS) and reactive electrophile species (RES) that alkylate or oxidize thiols. RACs can induce oxidative stress in cells and activate response pathways by modulating the activity of sensitive regulators. However, the effect of a certain compound on the cell has been investigated primarily with respect to a specific regulatory pathway. Since a single compound can exert multiple chemical effects in the cell, its effect can be better understood by time-course monitoring of multiple sensitive regulatory pathways that the compound induces. We investigated the effect of representative RACs by monitoring the activity of three sensor-regulators in the model actinobacterium Streptomyces coelicolor; SoxR that senses reactive compounds directly through oxidation of its [2Fe–2S] cluster, CatR/PerR that senses peroxides through bound iron, and an anti-sigma factor RsrA that senses RES via disulfide formation. The time course and magnitude of induction of their target transcripts were monitored to predict the chemical activities of each compound in S. coelicolor. Phenazine methosulfate (PMS) was found to be an effective RAC that directly activated SoxR and an effective ROS-producer that induced CatR/PerR with little thiol-perturbing activity. p-Benzoquinone was an effective RAC that directly activated SoxR, with slower ROS-producing activity, and an effective RES that induced the RsrA-SigR system. Plumbagin was an effective RAC that activated SoxR, an effective ROS-producer, and a less agile but effective RES. Diamide was an RES that effectively formed disulfides and a weak RAC that activated SoxR. Monobromobimane was a moderately effective RES and a slow producer of ROS. Interestingly, benzoquinone induced the SigR system by forming adducts on cysteine thiols in RsrA, revealing a new pathway to modulate RsrA activity. Overall, this study showed that multiple chemical activities of a reactive compound can be conveniently monitored in vivo by examining the temporal response of multiple sensitive regulators in the cell to reveal novel activities of the chemicals.

Summary SoxR from Escherichia coli and related enterobacte- ria is activated by a broad range of redox-active compounds through oxidation or nitrosylation of its (2Fe-2S) cluster. Activated SoxR then induces SoxS, which subsequently activates more than 100 genes in response. In contrast, non-enteric SoxRs directly activate their target genes in response to redox-active compounds that include endogenously produced metabolites. We compared the respon- siveness of SoxRs from Streptomyces coelicolor (ScSoxR), Pseudomonas aeruginosa (PaSoxR) and E. coli (EcSoxR), all expressed in S. coelicolor, towards natural or synthetic redox-active com- pounds. EcSoxR responded to all compounds exam- ined, whereas ScSoxR was insensitive to oxidants such as paraquat (Eh −440 mV) and menadione sodium bisulphite (Eh −45 mV) and to NO generators. PaSoxR was insensitive only to some NO generators. Whole-cell EPR analysis of SoxRs expressed in E. coli revealed that the (2Fe-2S) 1+ of ScSoxR was not oxidizable by paraquat, differing from EcSoxR and PaSoxR. The mid-point redox potential of purified ScSoxR was determined to be −185 ± 10 mV, higher by ∼ 100 mV than those of EcSoxR and PaSoxR, supporting its limited response to paraquat. The overall sensitivity profile indicates that both redox potential and kinetic reactivity determine the differential responses of SoxRs towards various oxidants.

The reactive α-oxoaldehydes such as glyoxal (GO) and methylglyoxal (MG) are generated in vivo from sugars through oxidative stress. GO and MG are believed to be removed from cells by glutathione-dependent glyoxalases and other aldehyde reductases. We isolated a number of GO-resistant (GO r ) mutants from Escherichia coli strain MG1655 on LB plates containing 10 mM GO. By tagging the mutations with the transposon Tn phoA -132 and determining their cotransductional linkages, we were able to identify a locus to which most of the GO r mutations were mapped. DNA sequencing of the locus revealed that it contains the yqhC gene, which is predicted to encode an AraC-type transcriptional regulator of unknown function. The GO r mutations we identified result in missense changes in yqhC and were concentrated in the predicted regulatory domain of the protein, thereby constitutively activating the product of the adjacent gene yqhD . The transcriptional activation of yqhD by wild-type YqhC and its mutant forms was established by an assay with a β-galactosidase reporter fusion, as well as with real-time quantitative reverse transcription-PCR. We demonstrated that YqhC binds to the promoter region of yqhD and that this binding is abolished by a mutation in the potential target site, which is similar to the consensus sequence of its homolog SoxS. YqhD facilitates the removal of GO through its NADPH-dependent enzymatic reduction activity by converting it to ethadiol via glycolaldehyde, as detected by nuclear magnetic resonance, as well as by spectroscopic measurements. Therefore, we propose that YqhC is a transcriptional activator of YqhD, which acts as an aldehyde reductase with specificity for certain aldehydes, including GO.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria that serves as a barrier against harmful molecules, including antibiotics. The waaYZ locus that encodes the LPS core biosynthetic function in Escherichia coli was found to be induced strongly by superoxide generators but not by H 2 O 2 , ethanol, or heat shock. This induction was dependent on SoxRS, a superoxide and nitric oxide sensing system, through a soxbox in the waaY promoter that binds SoxS. A Δ waaYZ mutant became more sensitive to some superoxide generators, and the activation of SoxR by these drugs became more sensitized in the mutant. Through phenotypic microarray analysis, we found that the mutant became sensitive to a wide variety of chemicals not restricted to oxidizing agents. We found that the mutant is under envelope stress and is altered in LPS composition, as monitored by the level of σ E activation and changes in the electrophoretic mobility of LPS, respectively. waaY expression was also regulated by MarA (multiple-antibiotic resistance regulator), which shares a binding site (soxbox) with SoxS, and was induced by salicylate, a nonoxidative compound. These results demonstrate a novel way of protecting gram-negative bacteria against various compounds by modifying LPS, possibly through phosphorylation. Since either oxidant or nonoxidant compounds elicit resistance toward themselves and other toxic drugs, this mechanism could serve as an efficient way for pathogenic bacteria to enhance survival during antibiotic treatment within an oxidant-rich host immune environment.

Background/Aims: The serum cytokeratin-18 (CK-18) has been suggested to be a surrogate marker of non-alcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the correlation between CK-18 and metabolic parameter in NAFLD patients. Correlation between CK-18 and macronutrient composition was also assessed. Methods: A total of 212 subjects were recruited. Blood chemistry including fasting glucose, cholesterol level, AST, ALT, and CK-18 were compared. Data on calorie intake and carbohydrate consumption were acquired by five-day-diet diary using 24 hour recall method. Results: Plasma CK-18 were markedly increased in patient with NAFLD compared with control group (420.4±282.3 vs. 313.6±179, p