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 %.

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.

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.

Mutations of ferric uptake regulator (fur) impair iron homeostasis, growth, oxidative stress survival, and virulence of Xanthomonas campestris pv. campestris.

Iron is essential in numerous cellular functions. Intracellular iron homeostasis must be maintained for cell survival and protection against iron's toxic effects. Here, we characterize the roles of Xanthomonas campestris pv. campestris (Xcc) fur, which encodes an iron sensor and a transcriptional regulator that acts in iron homeostasis, oxidative stress, and virulence. Herein, we isolated spontaneous Xcc fur mutants that had high intracellular iron concentrations due to constitutively high siderophore levels and increased expression of iron transport genes. These mutants also had reduced aerobic plating efficiency and resistance to peroxide killing. Moreover, one fur mutant was attenuated on a host plant, thus indicating that fur has important roles in the virulence of X. campestris pv. campestris.

Detection of 2,4-dichlorophenoxyacetic acid herbicide using a FGE-sulfatase based whole-cell Agrobacterium biosensor.

2,4-Dichlorophenoxyacetic acid (2,4-D) has been widely used as a herbicide for agricultural purposes. Currently, the available methods for detecting 2,4-D require multi-step sample preparations and expensive instruments. The use of a whole cell biosensor is an interesting approach that is straightforward and simple to use. In this study, we constructed a genetic-based Agrobacterium tumefaciens biosensor based on a cadA promoter and cadR regulator from Bradyrhizobium sp. strain HW13 (2,4-D degrader) with a formylglycine generating enzyme (FGE)-sulfatase as the reporter gene. The performance of the biosensor was further improved through direct evolution of the cadR activator. The detection limit of cadR mutants for phenoxyacetic acid herbicides including 2,4-D and 4-Chlorophenoxyacetic acid (4-CPAA) were 1.56 µM (an eight-fold improvement compared to wild-type CadR). The biosensor could detect 2,4-D contamination in environmental samples without encountering interference from other complex compounds. The Agrobacterium biosensor was also stable after storing in a simple Luria-Bertani (LB) medium at 4 °C for 30 days where the activity remained at 82% when exposed to 100 µM of 2,4-D. This novel biosensor, with its high stability under simple storage conditions, exhibits promising potential to be used as an inexpensive and easy-to-use tool to screen for 2,4-D contamination in environmental sources.

Cloning of Toluene 4-Monooxygenase Genes and Application of Two-Phase System to the Production of the Anticancer Agent, Indirubin.

Indirubin is a strong inhibitor of several eukaryotic cell signaling pathways and shows promise as a treatment for myelocytic leukemia and Alzheimer's disease. The tmoABCDEF operon, encoding the components of a novel toluene 4-monooxygenase from the paint factory soil isolate, Pseudomonas sp. M4, was cloned and expressed in Escherichia coli. E. coli::pKSR12 expressing the tmo genes was used to develop a two-phase [dioctyl phthalate (DOP)/aqueous medium] culture system that was optimized to obtain maximal yields of indirubin from the starting substrate, indole. DOP was used as the organic phase to solubilize and sequester the toxic indole substrate, making possible the use of high indole concentrations that would otherwise interfere with growth in aqueous media. A 50 % (v/v) DOP two-phase system using tryptophan medium containing 3 mM cysteine, 5 mM indole, and 1 mM isatin yielded 102.4 mg/L of indirubin with no conversion of indole to the less valuable alternate product, indigo.

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.

Bacillus subtilis SSE4 produces subtulene A, a new lipopeptide antibiotic possessing an unusual C15 unsaturated beta-amino acid.

Subtulene A, a new cyclic lipopeptide, was isolated from the culture broth of Bacillus subtilis SSE4. This antibiotic compound contained the seven common alpha-amino acids, L-Asn-1, D-Tyr-2, D-Asn-3, L-Gln-4, L-Pro-5, D-Asn-6, L-Ser-7 and the unique beta-amino acid-8 present in the iturin family. 1D and 2D NMR, as well as MS analyses, identified the beta-amino acid as 3-amino-13-methyltetradec-8-enoic acid, an Iso C15 long chain beta-amino acid. B. subtilis SSE4 was also found to produce iturin A. B. subtilis SSE4 culture filtrate exhibited both antifungal and antibacterial activities.

The omlA gene is involved in multidrug resistance and its expression is inhibited by coumarins in Xanthomonas campestris pv. phaseoli.

A gene encoding the outer membrane lipoprotein, OmlA, from the bacterial phytopathogen Xanthomonas campestris pv. phaseoli was isolated and characterized. An omlA insertion mutant showed an increased susceptibility to novobiocin and coumermycin, antibiotics with gyrase inhibitor activity. The omlA mutant accumulated novobiocin. Additionally, the omlA mutant was more sensitive than the wild type to chloramphenicol, a protein synthesis inhibitor; SDS, a detergent; and menadione, a superoxide generator. The susceptibility of the mutant to unrelated chemicals indicated a general role for OmlA in maintaining membrane integrity. Transcription of omlA was downregulated in the presence of both gyrase inhibitors, suggesting that DNA supercoiling might regulate the synthesis of OmlA. The omlA gene was divergently transcribed from the gene encoding the ferric uptake regulator Fur. Although the promoters of omlA and fur overlapped, Fur did not play any regulatory role in the expression of omlA due to the fact that inactivation of Fur did not affect the expression of omlA either in the presence or absence of iron.

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.

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.

DpsA protects the human pathogen Burkholderia pseudomallei against organic hydroperoxide.

The human pathogen, Burkholderia pseudomalle, is able to survive and multiply in hostile environments such as within macrophages. In an attempt to understand its strategy to cope with oxidative stress, the physiological role and gene regulation of a nonspecific DNA-binding protein (DpsA) was investigated. Expression of dpsA increases in response to oxidative stress through increased transcription from the upstream katG (catalase-peroxidase) promoter, which is OxyR dependent. dpsA is also transcribed from its own promoter, which is activated by osmotic stress in an OxyR-independent manner. DpsA-deficient mutants are hypersensitive to tert-butyl hydroperoxide, while overexpression of DpsA leads to increased resistance to organic oxidants. B. pseudomallei DpsA can also protect Escherichia coli against organic hydroperoxide toxicity. The mechanism of DpsA-mediated resistance to organic hydroperoxides was shown to differ from that of alkyl hydroperoxide reductase.

OhrR, a transcription repressor that senses and responds to changes in organic peroxide levels in Xanthomonas campestris pv. phaseoli.

We report the physiological role of OhrR as an organic peroxide sensor and transcription repressor in Xanthomonas campestris pv. phaseoli. In vivo exposure of X. campestris pv. phaseoli to either tert-butyl or cumene hydroperoxides efficiently neutralized OhrR repression of expression from the OhrR-regulated P1 promoter. H2O2 was a weak and non-physiological inducer of the system while other oxidants and metabolites of organic peroxide metabolism did not induce the expression from the P1. Northern blotting results indicated a correlation between concentrations of tert-butyl hydroperoxide used in the treatment and the induction of ohr (an OhrR-regulated gene) expression. In addition, the levels of ohr mRNA in cultures induced by various concentrations of tert-butyl hydroperoxide were reduced in cells with high levels of an organic peroxide metabolising enzyme (AhpC-AhpF) but not in cells with high catalase levels suggesting that organic peroxide interacts with OhrR. DNA band shift experiments using purified OhrR and the P1 promoter fragment showed that organic peroxide treatment prevented binding of the protein to the P1 promoter by oxidation of OhrR, as the inhibition of binding to the P1 promoter was reversed by addition of a reducing agent, DTT. The highly conserved cysteine residue C22 of OhrR is required for organic peroxide inducible gene expression. A mutant protein, OhrRC22S can repress the P1 promoter activity but is insensitive to organic peroxide treatment. Thus, OhrR is the first transcription repressor characterized that appeared to evolve to physiologically sense organic peroxides.