A highly sensitive biosensor with a single-copy evolved sensing cassette for chlorpyrifos pesticide detection.

A formylglycine-generating enzyme (FGE)-sulfatase-based whole-cell biosensor was genetically improved into a single-copy system by integrating the Sinorhizobium meliloti transcriptional activator ChpR and the chpA promoter-FGE-sulfatase fusion into the Escherichia coli chromosome. The sensitivity was further enhanced through a random mutagenesis of the chpR. The new integrated biosensor offered both a lower detection limit [5 nM chlorpyrifos (CPF)] and fluorescence background. The ready-to-use kit was developed using silica gel for on-field detection. The biosensor kit was stable for 20 days when stored at 4 °C. Moreover, a 1-(1-naphthylmethyl)-piperazine (NMP) efflux pump inhibitor can improve the sensitivity by 57 %.

Potential use of two aryl sulfotransferase cell-surface display systems to detoxify the endocrine disruptor bisphenol A.

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.

Cefoperazone induces esterase B expression by EstR and esterase B enhances cefoperazone activity at the periplasm.

The occurrence of antibiotic resistance bacteria has become a major threat to public health. We have recently discovered a transcriptional activator that belongs to MarR family, EstR, and an esterase B (EstB) with a newly proposed de-arenethiolase activity from Sphingobium sp. SM42. De-arenethiolase activity involves the removal of the small aromatic side chain of cephalosporin antibiotics as an excellent leaving group by the enzymatic CS bond cleavage. Here, we report the regulation of estB through EstR as an activator in response to a third generation cephalosporin, cefoperazone, antibiotic. Cefoperazone induced the expression of estB in wild type Sphingobium sp., but not in the estR knockout strain, and the induction was restored in the complemented strain. Moreover, we revealed the importance of EstB localization in periplasm. Since EsB has the ability to inactivate selected ß-lactam antibiotics in vitro, it is possible that the enzyme works at the periplasmic space of Gram negative bacteria similar to ß-lactamases. EstB was genetically engineered by incorporating NlpA binding motif, or OmpA signal sequence, or SpyTag-SpyCatcher to the estB gene to mobilize it to different compartments of periplasm; inner membrane, outer membrane, and periplasmic space, respectively. Surprisingly, we found that Sphingobium sp. SM42 and E. coli expressing EstB at the periplasm were more sensitive to cefoperazone. The possible drug enhancement mechanism by enzyme was proposed. This work might lead to a novel strategy to tackle antibiotic resistance problem.

The esterase B from Sphingobium sp. SM42 has the new de-arenethiolase activity against cephalosporin antibiotics.

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.

Identification of a repressor and an activator of azoreductase gene expression in Pseudomonas putida and Xanthomonas oryzae.

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.

Specific detection of the pesticide chlorpyrifos by a sensitive genetic-based whole cell biosensor.

The Sinorhizobium meliloti chpA promoter is highly induced in the presence of the pesticide chlorpyrifos (CPF) through the action of the transcriptional activator, ChpR. A whole-cell biosensor for the detection of CPF was developed and is composed of an Escherichia coli strain carrying a chpR expression vector and a chpA promoter-atsBA transcriptional fusion plasmid encoding sulfatase (atsA) and formylglycine generating enzyme (atsB) from Klebsiella sp. The sulfatase is posttranslationally activated by formylglycine generating enzyme (FGE) and then converts 4-methylumbelliferyl sulfate (4-MUS) to the fluorescent product, 4-methyllumbelliferone (4-MU). This biosensor system exhibited a linear response range from 25 to 500 nM CPF.

Acquired resistance of Stenotrophomonas maltophilia to antimicrobials induced by herbicide paraquat dichloride.

Stenotrophomonas maltophilia, a ubiquitous environmental bacterium, is an important cause of nosocomial infections. Although banned in some countries, paraquat (PQ) is commonly used to control weeds. In this study, we investigated the effects of increasing concentrations of PQ on S. maltophilia and its antimicrobial resistance. The sequential exposure of S. maltophilia K279a to increasing concentrations of PQ induces the formation of strains with increased resistance to PQ. Among the 400 PQ-resistant isolates tested, 70 clones were resistant to 16 µg/ml ciprofloxacin (CIP), and around 18% of the PQ/CIP-resistant isolates showed increased resistance to all the tested antimicrobials including, the aminoglycosides, quinolones, cephalosporin, chloramphenicol, and co-trimoxazole. The results of the expression analysis of the antimicrobial resistance genes in the five selected PQ/CIP-resistant isolates demonstrated the high expression of genes encoding efflux pumps (smeYZ, smaAB, smaCDEF, smeDEF, smeVWX, and smtcrA) and the enzymes aph(3')-IIc, blaL1, and blaL2. However, expression of the genes known for PQ resistance (i.e., mfsA and sod) were not altered relative to the wild-type levels. Whole genome sequence analysis identified gene mutations that could account for the antimicrobial resistance, namely, smeT (TetR family regulatory protein), rplA (ribosomal protein L1), and acnA (aconitase A). Ectopic expression of wild-type AcnA partially complemented the fluoroquinolone-resistant phenotype of the mutant with mutated acnA, which suggests the role of aconitase A in antimicrobial susceptibility. Exposure of S. maltophilia to PQ thus induces the development of strains that increase resistance to multiple antimicrobials.

Quantitative distribution of antibiotic resistance genes and crAssphage in a tropical urbanized watershed.

As antimicrobial resistance continues to pose a significant threat to global health, this study provided a focused examination of the prevalence and behavior of key antibiotic resistance genes in aquatic environments. We investigated the quantitative distribution of intI1, sul1, blaTEM, blaNDM, blaVIM, mcr-1, tetQ, and crAssphage within wastewater influents (n = 12), effluents (n = 12) and river water samples (n = 12), from three municipal wastewater treatment plants and three river locations in an urbanized watershed in Central Thailand over dry and wet seasons. The qPCR method demonstrated that intI1, sul1, blaTEM, and tetQ were the most abundant in all samples (2.71-7.89 mean log10 copies/100 mL), with all genes exhibiting consistently uniform levels across diverse locations, suggesting the potential for any site to act as a monitoring sentinel. Although there is a significant reduction of ARG concentrations by WWTPs (0.62 - >4.05 LRV), the persistence of these genes in effluents points to the limited effectiveness of existing treatment methodologies. Temporal data indicated stable ARG concentrations over time, but tetQ levels rose during the wet season, in alignment with the monsoonal climate in Thailand. Additionally, we identified crAssphage, a marker of human sewage contamination, exhibited strong correlations with the more abundant ARGs (rho 0.65 - 0.81), implying that human waste contributes significantly to the environmental burden of ARGs. The results of this research highlight the widespread nature of ARGs in water systems and the need for improved treatment and sanitation strategies to mitigate the public health threat posed by antimicrobial resistance.

Analysis of mutations that alter HO sensing and transcription regulation properties of a global peroxide regulator OxyR in Xanthomonas campestris pv. phaseoli.

OxyR5, from a Xanthomonas campestris pv. phaseoli H(2)O(2)-resistant mutant, contains the two mutations G197D and L301R. The protein exists in its oxidized-like form in the absence of oxidants as judged by the protein's ability to activate the ahpC promoter. Analysis of DNase I footprint patterns indicates that under reducing conditions OxyR5 and OxyRG197D bind to the target site in the ahpC promoter in a manner similar to oxidized wild-type OxyR. Site-directed mutagenesis showed that OxyR5 behaves like oxidized OxyR, independent of the highly conserved C residues at positions 199 and 208 where, in normal OxyR, a disulfide bond between these residues converts the protein from its reduced to the oxidized form. The presence of aspartic acid or valine residue at position 197 caused OxyR to behave like the oxidized form in uninduced cells. Changing D197 to A or T in OxyR5 resulted in proteins with similar properties to native OxyR. In vivo, OxyR5 probably locked in an oxidized-like conformation, resulting in continuous high-level activation of target genes in the OxyR regulon.

The Burkholderia pseudomallei oxyR gene: expression analysis and mutant characterization.

Burkholderia pseudomallei (Bp) is the causative agent of the life-threatening melioidosis in humans. The global transcription factor oxyR gene was isolated and characterized. It is located between recG, encoding a putative DNA helicase, and katG, encoding a putative catalase-peroxidase. oxyR is expressed as a monocistronic 1 kb mRNA and is induced by oxidative stress compounds. Northern, primer extension, and transcription reporter fusion analyses showed that oxyR mRNA is induced by 0.2 mM menadione, 2 mM paraquat, and 10 mM H(2)O(2). Two knockout mutants of oxyR were constructed, by single- and double-crossover recombination, and found to be hypersensitive to H(2)O(2) and paraquat. Bp lacking OxyR exhibited autoaggregation when cultured in liquid broth and an increased ability to form biofilms in minimal medium, but not in Luria-Bertani broth. The oxyR mutants also have a decreased level of extracellular protease activity. The altered phenotypes of oxyR deficient mutants were complemented when a copy of oxyR was transposed into the mutant chromosomes on the mini-Tn5 transposon.

Regulation of the katG-dpsA operon and the importance of KatG in survival of Burkholderia pseudomallei exposed to oxidative stress.

Homologues of the catalase-peroxidase gene katG and the gene for the non-specific DNA binding protein dpsA were identified downstream of oxyR in Burkholderia pseudomallei. Northern experiments revealed that both katG and dpsA are co-transcribed during oxidative stress. Under conditions where the katG promoter is not highly induced, dpsA is transcribed from a second promoter located within the katG-dpsA intergenic region. A katG insertion mutant was found to be hypersensitive to various oxidants. Analysis of katG expression in the oxyR mutant indicates that OxyR is a dual function regulator that represses the expression of katG during normal growth and activates katG during exposure to oxidative stress. Both reduced and oxidized OxyR were shown to bind to the katG promoter.

Gene cloning and characterization of a novel highly organic solvent tolerant lipase from Proteus sp. SW1 and its application for biodiesel production.

Proteus sp. SW1 was found to produce an extracellular solvent tolerant lipase. The gene, lipA, encoding a bacterial lipase, was cloned from total Proteus sp. SW1 DNA. lipA was predicted to encode a 287 amino acid protein of 31.2 kDa belonging to the Group I proteobacterial lipases. Purified His-tagged LipA exhibited optimal activity at pH 10.0 and 55°C. It was highly stable in organic solvents retaining 112% of its activity in 100% isopropanol after 24 h, and exhibited more than 200% of its initial activity upon exposure to 60% acetone, ethanol, and hexane for 18 h. Biodiesel synthesis reactions, using a single step addition of 13% an acyl acceptor ethanol, showed that LipA was highly effective at converting palm oil into biodiesel.

The hdhA gene encodes a haloacid dehalogenase that is regulated by the LysR-type regulator, HdhR, in Sinorhizobium meliloti.

The plasmid pSymA, in the nitrogen-fixing soil bacterium, Sinorhizobium meliloti, carries a 750-bp ORF (SMa1978) designated, hdhA, which encodes a novel dehalogenase that can detoxify haloacid compounds, showing a preference for haloacetic acids. Purified His-tagged HdhA demonstrated the apparent ability to dehalogenate chloroacetic acid and trifluoroacetic acid. In addition, upstream of hdhA, a gene encoding a lysR-type transcription regulator denoted, hdhR (SMa1979), has been identified to be a transcriptional repressor of hdhA expression. In an hdhR knockout mutant, hdhA promoter activity was markedly increased. Purified 32-kDa His-tagged HdhR repressed expression of hdhA by specifically binding to the promoter region of hdhA, as demonstrated by gel mobility shift assay and DNase I foot printing experiments. Moreover, the pesticide, pentachlorophenol, was also found to induce hdhA expression via HdhR. Site-directed mutants, in which the Cys residues at positions 160 and 192 in HdhR were changed to Ser, were constructed. C160S and C192S single mutants showed diminished HdhR-mediated repression of hdhA expression, while a C160S:C192S double mutant could no longer repress expression of hdhA.

ChpR is a chlorpyrifos-responsive transcription regulator in Sinorhizobium meliloti.

The broad-spectrum organophosphate insecticide chlorpyrifos (CPF)-inducible locus, chpAB, was identified on the endogenous plasmid pSymB in Sinorhizobium meliloti. The S. meliloti chpA promoter was highly induced by CPF and was induced at much lower levels by diazinon and ethion. Transcription of chpA was dependent on chpR, a CadC family transcriptional regulator located upstream of, and divergently transcribed from, chpAB. ChpR was able to mediate the CPF-inducible expression of the S. melilotichpA promoter in Escherichia coli through direct interaction with the chpAB promoter. The chpR-chpA intergenic regions of several bacterial chpRAB operons were aligned and a putative ChpR-binding sequence was proposed. Both the ChpR transcription factor and chpA promoter constitute a good candidate system for genetic-based biosensor development.

HpdR is a transcriptional activator of Sinorhizobium meliloti hpdA, which encodes a herbicide-targeted 4-hydroxyphenylpyruvate dioxygenase.

Sinorhizobium meliloti hpdA, which encodes the herbicide target 4-hydroxyphenylpyruvate dioxygenase, is positively regulated by HpdR. Gel mobility shift and DNase I footprinting analyses revealed that HpdR binds to a region that spans two conserved direct-repeat sequences within the hpdR-hpdA intergenic space. HpdR-dependent hpdA transcription occurs in the presence of 4-hydroxyphenylpyruvate, tyrosine, and phenylalanine, as well as during starvation.

The unique glutathione reductase from Xanthomonas campestris: gene expression and enzyme characterization.

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.

Important role for methionine sulfoxide reductase in the oxidative stress response of Xanthomonas campestris pv. phaseoli.

A methionine sulfoxide reductase gene (msrA) from Xanthomonas campestris pv. phaseoli has unique expression patterns and physiological function. msrA expression is growth dependent and is highly induced by exposure to oxidants and N-ethylmaleimide in an OxyR- and OhrR-independent manner. An msrA mutant showed increased sensitivity to oxidants but only during stationary phase.

Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli.

katA encodes the major catalase that accounts for 90 % of the total catalase activity present in Xanthomonas campestris pv. phaseoli. katA is located upstream of an ORF designated ankA encoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis of katA and ankA identified two katA transcripts: a major monocistronic katA transcript and a minor bicistronic katA-ankA transcript. KatA expression was induced in the presence of various oxidants including H2O2, organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of the katA promoter region showed a putative OxyR binding site located upstream of an Escherichia coli-like sigma70 -35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to the katA promoter. A katA mutant was highly sensitive to H2O2 during both the exponential and stationary phases of growth. This phenotype could be complemented by functional katA, confirming the essential role of the gene in protecting X. campestris from H2O2 toxicity. Unexpectedly, inactivation of ankA also significantly reduced resistance to H2O2 and the phenotype could be complemented by plasmid-borne expression of ankA. Physiological analyses showed that katA plays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris.

A suppressor of the menadione-hypersensitive phenotype of a Xanthomonas campestris pv. phaseoli oxyR mutant reveals a novel mechanism of toxicity and the protective role of alkyl hydroperoxide reductase.

We isolated menadione-resistant mutants of Xanthomonas campestris pv. phaseoli oxyR (oxyR(Xp)). The oxyRR2(Xp) mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H(2)O(2) and tert-butyl hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the oxyRR2(Xp) mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of ahpC in the oxyRR2(Xp) mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned ahpC and ahpF in the oxyR(Xp) mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that alkyl hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in X. campestris.

Compensatory increase in ahpC gene expression and its role in protecting Burkholderia pseudomallei against reactive nitrogen intermediates.

In the human pathogen Burkholderia pseudomallei, katG encodes the antioxidant defense enzyme catalase-peroxidase. Interestingly, a B. pseudomallei mutant, disrupted in katG, is hyperresistant to organic hydroperoxide. This hyperresistance is due to the compensatory expression of the alkyl hydroperoxide reductase gene ( ahpC) and depends on a global regulator OxyR. The KatG-deficient mutant is also highly resistant to reactive nitrogen intermediates (RNI). When overproduced, the B. pseudomallei AhpC protein, protected cells against killing by RNI. The levels of resistance to both organic peroxide and RNI returned to those of the wild-type when the katG mutant was complemented with katG. These studies establish the partially overlapping defensive activities of KatG and AhpC.