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Bisphenol A (BPA) is one of the most common toxic endocrine disruptors in the environment. A fast, efficient and environmental-friendly method for BPA detoxification is urgently needed. In this study, we show that the enzymatic transformation of BPA into a non-estrogenic BPA sulfate can be performed by the aryl sulfotransferase (ASTB) from Desulfitobacterium hafniense. We developed and compared two Escherichia coli ASTB cell-surface displaying systems using the outer membrane porin F (OprF) and the lipoprotein outer membrane A (Lpp-OmpA) as carriers. The surface localization of both fusion proteins was confirmed by Western blot and flow cytometry analysis as well as the enzymatic activity assay of the outer membrane fractions. Unfortunately, Lpp-OmpA-ASTB cells had an adverse effect on cell growth. In contrast, the OprF-ASTB cell biocatalyst was stable, expressing 70% of enzyme activity for 7 days. It also efficiently sulfated 90% of 5 mM BPA (1 mg/mL) in wastewater within 6 h., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The esterase B (EstB) from Sphingobium sp. SM42, which was previously reported to be active towards dibutyl phthalate, can cleave some small aromatic ring side chains from cephalosporin derivatives. A new name, de-arenethiolase, has been proposed to represent this activity. We present the in vitro characterization of the activity of purified EstB toward cephalosporin substrates. Interestingly, EstB was highly active against cefoperazone and cefazolin resulting in 83 and 67% decreases in killing zone diameter, respectively. EstB also demonstrated a moderate activity towards ceftriaxone (18%) and cefotaxime (16%) while exhibiting no activity against cephalosporin C and cefixime. HPLC analysis indicated that EstB catalyzed the cleavage of the C-S bond found in cephalosporin derivatives to release the corresponding free aromatic ring side chains., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Genes responsible for the production of azoreductase enzymes in 2 gram-negative bacteria, the soil bacterium Pseudomonas putida (AzoP) and the plant pathogen Xanthomonas oryzae (AzoX), were identified. The deduced amino acid sequences of AzoP and AzoX, share 46% amino acid identity to each other. Two different bacterial transcription factors, a repressor (AzoPR) and an activator (AzoXR), in P. putida and X. oryzae, respectively, were found to be divergently oriented to their respective azoreductase genes. Both regulators are LysR-type transcriptional regulators (LTTR) that respond to the azo dye inducer, methyl red (MR). AzoPR represses transcription of azoP in P. putida, which is reversed when cells are exposed to MR. Interestingly, in X. oryzae, AzoXR positively regulates azoX transcription upon MR induction. Moreover, despite their similarity, with 51% amino acid sequence identity, azoPR and azoXR are expressed differently in response to MR. The transcription of azoPR is increased in a dye concentration-dependent manner, while azoXR transcription is constitutive and relatively higher than azoPR. Both regulators are autoregulatory. Gel mobility shift assays (EMSA) verified the binding between the regulators and their corresponding promoter regions. Additionally, binding only occurred under reduced conditions in the presence of 0.5 mM dithiothreitol (DTT), indicating that the proteins are active in their reduced form., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The glutathione reductase gene, gor, was cloned from the plant pathogen Xanthomonas campestris pv. phaseoli. Its gene expression and enzyme characteristics were found to be different from those of previously studied homologues. Northern blot hybridization, promoter-lacZ fusion, and enzyme assay experiments revealed that its expression, unlike in Escherichia coli, is OxyR-independent and constitutive upon oxidative stress conditions. The deduced amino acid sequence shows a unique NADPH binding motif where the most highly conserved arginine residue, which is critical for NADPH binding, is replaced by glutamine. Interestingly, a search of the available Gor amino acid sequences from various sources, including other Xanthomonas species, revealed that this replacement is specific to the genus Xanthomonas. Recombinant Gor enzyme was purified and characterized, and was found to have a novel ability to use both, NADPH and NADH, as electron donor. A gor knockout mutant was constructed and shown to have increased expression of the organic peroxide-inducible regulator gene, ohrR.

The gene for Agrobacterium tumefaciens OxyR, a peroxide sensor and transcriptional regulator, was characterized. Phylogenetic analysis of bacterial OxyR showed that the protein could be divided into four clades. The A. tumefaciens OxyR grouped in clade III that consists primarily of OxyRs of Alphaproteobacteria displayed the highest homology to OxyR from Rhizobium leguminosarum. oxyR is located next to, and is divergently transcribed from, a bifunctional catalase-peroxidase gene (katA). An A. tumefaciens oxyR mutant was constructed and shown to be hyper-sensitive to H2O2, but not to the superoxide generator, menadione, or an organic hydroperoxide. Exposure of A. tumefaciens to H2O2 resulted in induction of the catalase-peroxidase enzyme. This induction was abolished in the oxyR mutant. In vivo analysis of a katA::lacZ promoter fusion confirmed the results of enzyme assays and indicated that induction of the katA promoter by H2O2 was dependent on functional OxyR. We also examined the regulation of oxyR in A. tumefaciens. Exposure to H2O2 did not induce expression of the gene but simply changed OxyR from a reduced to an oxidized form. The in vivo oxyR promoter analysis showed that the promoter was auto-regulated and that transcription was not induced by H2O2.

Alkyl hydroperoxide reductase (ahpC) and organic hydroperoxide resistance (ohr) are distinct genes, structurally and regulatory, but have similar physiological functions. In Xanthomonas campestris pv. phaseoli inactivation of either gene results in increased sensitivity to killing with organic peroxides. An ahpC1-ohr double mutant was highly sensitive to both growth inhibition and killing treatment with organic peroxides. High level expression of ahpC or ohr only partially complemented the phenotype of the double mutant, suggesting that these genes function synergistically, but through different pathways, to protect Xanthomonas from organic peroxide toxicity. Functional analyses of Ohr and AhpC abilities to degrade organic hydroperoxides revealed that both Ohr and AhpC could degrade tert-butyl hydroperoxide (tBOOH) while the former was more efficient at degrading cumene hydroperoxide (CuOOH). Expression analysis of these genes in the mutants showed no compensatory alterations in the levels of AhpC or Ohr. However, CuOOH induced expression of these genes in the mutants was affected. CuOOH induced ahpC expression was higher in the ohr mutant than in the parental strain; in contrast, the ahpC mutation has no effect on the level of induced ohr expression. These analyses reveal complex physiological roles and expression patterns of seemingly functionally similar genes.

A talA gene encoded transaldolase, a rate-limiting enzyme in the non-oxidative branch of the pentose-phosphate pathway, was cloned from Xanthomonas campestris pv. phaseoli. talA located in a region of the bacterial genome rich in genes involved in oxidative stress protection and regulation. TalA from X. campestris pv. phaseoli showed a high degree of homology to many previously reported transaldolases from both prokaryotic and eukaryotic sources. The expression of X. campestris pv. phaseoli talA was high at log-phase of growth, then declined at stationary phase, and could not be induced by oxidants. A talA mutant constructed by insertional inactivation did not possess any detectable transaldolase activity. Lack of a functional talA gene did not affect bacterial growth in a rich medium containing glucose or sucrose as a carbon source. However, the talA knockout mutant showed increased sensitivity to the superoxide generator menadione, but not to other oxidants. This increased menadione sensitivity phenotype could be complemented by expression of talA in a plasmid vector. The data demonstrated a novel and essential role of transaldolase in protection against menadione toxicity in X. campestris.