Genome-Scale Investigation of the Regulation of azoR Expression in Escherichia coli Using Computational Analysis and Transposon Mutagenesis.

Bacterial azoreductases are enzymes that catalyze the reduction of ingested or industrial azo dyes. Although azoreductase genes have been well identified and characterized, the regulation of their expression has not been systematically investigated. To determine how different factors affect the expression of azoR, we extracted and analyzed transcriptional data from the Gene Expression Omnibus (GEO) resource, then confirmed computational predictions by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results showed that azoR expression was lower with higher glucose concentration, agitation speed, and incubation temperature, but higher at higher culture densities. Co-expression and clustering analysis indicated ten genes with similar expression patterns to azoR: melA, tpx, yhbW, yciK, fdnG, fpr, nfsA, nfsB, rutF, and chrR (yieF). In parallel, constructing a random transposon library in E. coli K-12 and screening 4320 of its colonies for altered methyl red (MR)-decolorizing activity identified another set of seven genes potentially involved in azoR regulation. Among these genes, arsC, relA, plsY, and trmM were confirmed as potential azoR regulators based on the phenotypic decolorization activity of their transposon mutants, and the expression of arsC and relA was confirmed, by qRT-PCR, to significantly increase in E. coli K-12 in response to different MR concentrations. Finally, the significant decrease in azoR transcription upon transposon insertion in arsC and relA (as compared to its expression in wild-type E. coli) suggests their probable involvement in azoR regulation. In conclusion, combining in silico analysis and random transposon mutagenesis suggested a set of potential regulators of azoR in E. coli.

Characteristics of major Escherichia coli reductases involved in aerobic nitro and azo reduction.

AIMS: Escherichia coli is able to reduce azo compounds such as methyl red (MR) and nitro compounds such as 7-nitrocoumarin-3-carboxylic acid (7NCCA). The aim of this study was to clarify the specificity of the major E. coli reductases. METHODS AND RESULTS: Enzymatic assays with pure enzymes obtained after cloning, overproduction and purification under native or denaturing conditions were performed on three enzymes: AzoR, NfsA and NfsB. Their dependence on putative cofactors such as flavin mononucleotide (FMN), NADH and NADPH was studied as well as the reductase capacity of E. coli mutants depleted for one, two or three of the corresponding genes. CONCLUSIONS: AzoR was able to reduce both MR and 7NCCA, whereas NfsA and NfsB could only reduce the nitro compound. AzoR and NfsB were strictly FMN dependent in contrast to NfsA. At a low oxygen concentration, the three proteins were not mandatory for azo reduction and nitro reduction, but in optimal aerobic conditions, azoR was essential for MR reduction, and an nfsA/nfsB combination was important for 7NCCA reduction. Overexpression of azoR gene was able to compensate for the loss of nfsA and nfsB under aerobic conditions. SIGNIFICANCE AND IMPACT OF STUDY: These data provide new insights into the substrate specificity of major E. coli nitroreductases and demonstrate that oxygen is an important parameter to take into account in studies of nitroreductase activity.

Exploration on the Cr(VI) resistance mechanism of a novel thermophilic Cr(VI)-reducing bacteria Anoxybacillus flavithermus ABF1 isolated from Tengchong geothermal region, China.

Hexavalent chromium resistance and reduction mechanisms of microorganism provide a critical guidance for Cr(VI) bioremediation. However, related researches are limited in mesophiles and deficient for thermophiles. In this work, a novel alkaline Cr(VI)-reducing thermophile Anoxybacillus flavithermus ABF1 was isolated from geothermal region. The mechanisms of Cr(VI) resistance and reduction were investigated. The results demonstrated that A. flavithermus ABF1 could survive in a wide temperature range from 50°C to 70°C and in pH range of 7.0-9.0. Strain ABF1 showed excellent growth activity and Cr(VI) removal performance when initial Cr(VI) concentration was lower than 200 mg L-1 . 93.71% of Cr(VI) was removed at initial concentration of 20 mg L-1 after 72 h. The majority of Cr(VI) was found to be reduced extracellularly by enzymes secreted by cells. XPS and Raman analysis further manifested that Cr2 O3 was the product of Cr(VI) reduction. Moreover, the Cr(VI) transportation-related gene cysP and Cr(VI) reduction-related gene azoR of A. flavithermus ABF1 played key roles in inhibiting Cr(VI) entering cells and promoting extracellular Cr(VI) reduction respectively. This work provides novel insight into the mechanisms of Cr(VI) resistance and detoxication of thermophiles, which leads to a promising alternative strategy for heavy metal bioremediation in areas with elevated temperature.

Bayesian Modeling and Intrabacterial Drug Metabolism Applied to Drug-Resistant Staphylococcus aureus.

We present the application of Bayesian modeling to identify chemical tools and/or drug discovery entities pertinent to drug-resistant Staphylococcus aureus infections. The quinoline JSF-3151 was predicted by modeling and then empirically demonstrated to be active against in vitro cultured clinical methicillin- and vancomycin-resistant strains while also exhibiting efficacy in a mouse peritonitis model of methicillin-resistant S. aureus infection. We highlight the utility of an intrabacterial drug metabolism (IBDM) approach to probe the mechanism by which JSF-3151 is transformed within the bacteria. We also identify and then validate two mechanisms of resistance in S. aureus: one mechanism involves increased expression of a lipocalin protein, and the other arises from the loss of function of an azoreductase. The computational and experimental approaches, discovery of an antibacterial agent, and elucidated resistance mechanisms collectively hold promise to advance our understanding of therapeutic regimens for drug-resistant S. aureus.