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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 %.

The Agrobacterium tumefaciens ActSR two-component regulatory system is a member of a homologous group of global redox-responsive regulatory systems that adjust the expression of energy-consuming and energy-supplying metabolic pathways in order to maintain cellular redox balance. In this study, the transcriptional organization of the hrpB-actSR locus was determined and the effect of actSR system inactivation on stress resistance was investigated. It was found that hrpB is transcribed as a monocistronic mRNA and actS is transcribed along with actR as a bicistronic mRNA, while actR is also transcribed as a monocistronic message. Each message is initiated from a separate promoter. Inactivation of actR resulted in decreased resistance to membrane stress (sodium dodecyl sulfate), acid stress (pH 5.5), iron starvation (bipyridyl) and iron excess (FeCl 3 ), and antibiotic stress (tetracycline and ciprofloxacin). Resistance to oxidative stress in the form of organic peroxide (cumene hydroperoxide) increased, while resistance to inorganic peroxide (H 2 O 2 ) decreased. An actR insertion mutant displayed reduced catalase activity, even though transcription of katA and catE remained unchanged. Complementation of the actR inactivation mutant with plasmid-encoded actR or overexpression of hemH , encoding ferrochelatase, restored wild-type catalase activity and H 2 O 2 resistance levels. Gel mobility shift and hemH promoter -lacZ fusion results indicated that ActR is a positive regulator of hemH that binds directly to the hemH promoter region. Thus, inactivation of the A. tumefaciens ActSR system affects resistance to multiple stresses, including reduced resistance to H 2 O 2 resulting from a reduction in catalase activity due to reduced expression of hemH .

The putative zinc exporters ZntA (a P1B-type ATPase) and ZntB (2-TM-GxN family) in Agrobacterium tumefaciens were characterized. The expression of the zntA gene is inducible by CdCl2, ZnCl2 and CoCl2, of which CdCl2 is the most potent inducer, whereas zntB is constitutively expressed. The metal-induced expression of zntA is controlled by the MerR-like regulator ZntR. The zntA and zntR mutants were highly sensitive to CdCl2 and ZnCl2, and CoCl2 sensitivity was demonstrated to a lesser extent. By contrast, the zntB mutant showed similar levels of metal resistance to the WT strain. Even in the zntA mutant background, zntB did not play an apparent role in metal resistance under the conditions tested. The inactivation of zntA increased the accumulation of intracellular cadmium and zinc, and conferred hyper-resistance to H2O2. Thus, the metal transporter ZntA and its regulator ZntR are important for controlling zinc homeostasis and cadmium and cobalt detoxification. The loss of either the zntA or zntR gene did not affect the virulence of A. tumefaciens in Nicotiana benthamiana.

The Agrobacterium tumefaciens zinc uptake regulator (Zur) was shown to negatively regulate the zinc uptake genes znuABC, encoding a zinc transport system belonging to the ATP-binding cassette (ABC) transporter family, and zinT, which encodes a periplasmic zinc-binding protein. The expression of znuABC and zinT was inducible when cells were grown in medium containing a metal chelator (EDTA), and this induction was shown to be specific for zinc depletion. The expression of znuABC was reduced in response to increased zinc in a dose-dependent manner, and zinT had a less pronounced but similar pattern of zinc-regulated expression. The inactivation of zur led to constitutively high expression of znuABC and zinT. In addition, a zur mutant had an increased total zinc content compared to the WT NTL4 strain, whereas the inactivation of zinT caused a reduction in the total zinc content. The zinT gene is shown to play a dominant role and to be more important than znuA and znuB for A. tumefaciens survival under zinc deprivation. ZinT can function even when ZnuABC is inactivated. However, mutations in zur, znuA, znuB or zinT did not affect the virulence of A. tumefaciens., (© 2014 The Authors.)

Agrobacterium tumefaciens membrane-bound ferritin (MbfA) is a member of the erythrin (Er)-vacuolar iron transport family. The MbfA protein has an Er or ferritin-like domain at its N terminus and has been predicted to have five transmembrane segments in its C-terminal region. Analysis of protein localization using PhoA and LacZ reporter proteins supported the view that the N-terminal di-iron site is located in the cytoplasm whilst the C-terminal end faces the periplasm. An A. tumefaciens mbfA mutant strain had 1.5-fold higher total iron content than the WT strain. Furthermore, multi-copy expression of mbfA reduced total iron content two- and threefold in WT and mbfA mutant backgrounds, respectively. These results suggest that MbfA may function as an iron exporter rather than an iron storage protein. The mbfA mutant showed 10-fold increased sensitivity to the iron-activated antibiotic streptonigrin, implying that the mutant had increased accumulation of intracellular free iron. Growth of the mbfA mutant was reduced in the presence of high iron under acidic conditions. The expression of mbfA was induced highly in cells grown in iron-replete medium at pH 5.5, further supporting the view that mbfA is involved in the response to iron under acidic conditions. A. tumefaciens MbfA may play a protective role against increased free iron in the cytoplasm through iron binding and export, thus preventing iron-induced toxicity via the Fenton reaction.

Copper (Cu)-based biocides are currently used as control measures for both fungal and bacterial diseases in agricultural fields. In this communication, we show that exposure of the bacterial plant pathogen Xanthomonas campestris to nonlethal concentrations of Cu(2+) ions (75 µM) enhanced expression of genes in OxyR, OhrR and IscR regulons. High levels of catalase, Ohr peroxidase and superoxide dismutase diminished Cu(2+)-induced gene expression, suggesting that the production of hydrogen peroxide (H2O2) and organic hydroperoxides is responsible for Cu(2+)-induced gene expression. Despite high expression of antioxidant genes, the CuCl2-treated cells were more susceptible to H2O2 killing treatment than the uninduced cells. This phenotype arose from lowered catalase activity in the CuCl2-pretreated cells. Thus, exposure to a nonlethal dose of Cu(2+) renders X. campestris vulnerable to H2O2, even when various genes for peroxide-metabolizing enzymes are highly expressed. Moreover, CuCl2-pretreated cells are sensitive to treatment with the redox cycling drug, menadione. No physiological cross-protection response was observed in CuCl2-treated cells in a subsequent challenge with killing concentrations of an organic hydroperoxide. As H2O2 production is an important initial plant immune response, defects in H2O2 protection are likely to reduce bacterial survival in plant hosts and enhance the usefulness of copper biocides in controlling bacterial pathogens.

The Agrobacterium tumefaciens genome contains a cluster of genes that are predicted to encode Fe-S cluster assembly proteins, and this cluster is known as the sufS2BCDS1XA operon. sufS2 is the first gene in the operon, and it was inactivated to determine its physiological function. The sufS2 mutant exhibited a small colony phenotype, grew slower than the wild-type strain and was more sensitive to various oxidants including peroxide, organic hydroperoxide and superoxide. The sufS2 gene was negatively regulated by iron response regulator (Irr) and rhizobial iron regulator (RirA) under low and high iron conditions, respectively, and was inducible in response to oxidative stress. The oxidant-induced expression of sufS2 was controlled by Irr, RirA and an additional but not yet identified mechanism. sufS2 was required for RirA activity in the repression of a sufS2 promoter-lacZ fusion. RirA may use Fe-S as its cofactor. sufS2 disruption may cause a defect in the Fe-S supply and could thereby affect the RirA activity. The three conserved cysteine residues (C91, C99 and C105) in RirA were predicted to coordinate with the Fe-S cluster and were shown to be essential for RirA repression of the sufS2-lacZ fusion. These results suggested that sufS2 is important for the survival of A. tumefaciens.

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.

Xanthomonas campestris Ohr (a protein involved in organic peroxide protection) and Escherichia coli OsmC (an osmotically inducible protein of unknown function) are related proteins. Database searches and phylogenetic analyses reveal that Ohr and OsmC homologues cluster into two related subfamilies of proteins widely distributed in both Gram-negative and Gram-positive bacteria. To determine if these two subfamilies are functionally distinct, ohr and osmC in Pseudomonas aeruginosa (a bacterium with one representative from each subfamily) were analysed. Only ohr mutants are hypersensitive to organic peroxide, and this phenotype can be restored by complementation with ohr but not osmC. In addition, expression of ohr was highly induced only by organic peroxides, and not by other oxidants or stresses. In contrast, osmC was induced by ethanol and osmotic stress. A similar pattern of regulation was observed for Ohr and OsmC homologues in the Gram-positive bacterium Deinococcus radiodurans, though uninduced expression was much higher and induction lower in this species. These data clearly support the conclusion that Ohr and OsmC define two functionally distinct subfamilies with distinct patterns of regulation.

The ability of XANTHOMONAS: campestris pv. phaseoli to protect itself against lethal concentrations of man-made (N:-ethylmaleimide, NEM) and endogenously produced (methylglyoxal, MG) electrophiles was investigated. Pretreatment of X. c. pv. phaseoli with a low concentration of NEM induced protection against lethal concentrations of NEM and MG. MG pretreatment weakly induced protection against NEM but not against MG itself. NEM-induced protection against electrophile killing required new protein synthesis and was abolished by the addition of a protein synthesis inhibitor. By contrast, MG-induced protection against NEM killing was independent of de novo protein synthesis. X. c. pv. phaseoli harbouring an expression vector carrying a catalase gene was over 100-fold more resistant to MG and NEM killing. High expression levels of genes for other peroxide-protective enzymes, such as those for alkyl hydroperoxide reductase (ahpC and ahpF) and ohr, failed to protect against electrophile killing. Thus, catalase appears to have a novel protective role(s) against electrophile toxicity. This finding suggests that in X. c. pv. phaseoli NEM and MG toxicity might involve accumulation and/or increased production of H(2)O(2). This idea was supported by the observation that addition of 10 mM sodium pyruvate, a compound that can react chemically with peroxide or hydroxyl radical scavengers (DMSO and glycerol), was found to protect XANTHOMONAS: from electrophile killing. The protective role of catalase and the role of H(2)O(2) in electrophile toxicity are novel observations and could be generally important in other bacteria. In addition, unlike other bacteria, XANTHOMONAS: in stationary phase was more susceptible to electrophile killing compared to cells in exponential phase.