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TriR serves as a repressor for a resistance-nodulation-cell division (RND) efflux pump TriABC involved in triclosan (TCS) resistance in Agrobacterium tumefaciens. The triR gene is transcribed divergently from the triABC operon. TriR specifically bound to the triR-triA intergenic region, at an imperfect 10 bp inverted repeat, 5'-TTGACTAttC-GgtTAGTCAA-3' (TriR box), that was revealed by DNase I footprinting and electrophoretic mobility shift assay. TCS treatment appeared to up-regulate triR and triABC expression, via preventing TriR binding to the triR-triA intergenic region. Promoter-lacZ fusions and β-galactosidase activity assay further demonstrated TriR-mediated repression of triABC and triR autoregulation. Site-directed mutagenesis confirmed the identified TriR box is essential for TriR repression. A. tumefaciens mutant strains disrupting either triR or triA were constructed to determine their biological functions. The triA mutant showed hypersensitivity to TCS and sodium dodecyl sulfate (SDS), whereas the triR mutant was hyper-resistant, compared to wild-type. In addition to TCS and SDS, overproduction of TriABC from a multi-copy plasmid conferred enhanced resistance to a quaternary ammonium compound, benzalkonium chloride. Molecular modelling was able to predict the model of TriR and docking simulations were able to anticipate plausible binding interactions between TriR and TCS ligand., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The Agrobacterium tumefaciens atu4217 gene, which encodes a TetR family transcription regulator, is a repressor of the atu4218-atu4219-atu4220 operon. The Atu4218 and Atu4219 proteins belong to the HlyD family (membrane fusion protein) and the AcrB/AcrD/AcrF family (inner membrane transporter), respectively, and may form an efflux pump. The atu4220 gene encodes a short-chain dehydrogenase. Quantitative real-time PCR analysis showed induction of atu4217 and atu4218 by NaOCl but not by N-ethylmaleimide or reactive oxygen species (ROS) including H 2 O 2 , menadione and cumene hydroperoxide; therefore, the atu4218 and atu4219 were named NaOCl-inducible efflux genes nieA and nieB, respectively. The atu4217 gene, which was named nieR, serves as a repressor of nieA and nieB. DNase I footprinting assays identified 20-bp imperfect inverted repeat (IR, underlined) motifs 5'-TAGATTTAGGATGCAATCTA-3' (box A) and 5'-TAGATTTCACTTGACATCTA-3' (box R) in the intergenic region of the divergent nieA and nieR genes; these motifs were recognized by the NieR protein. Electrophoretic mobility shift assays demonstrated that NieR specifically binds to the 20-bp IR motifs and that NaOCl prevents this NieR-DNA interaction. Promoter-lacZ fusions and mutagenesis of the NieR boxes (A and R) showed a more dominant role for box A than for box R in the repression of the nieA and nieR promoters. However, full repression of either promoter required both operators. The nieR mutant strain exhibited a small colony phenotype and was more sensitive than the wild-type to NaOCl and antibiotics, including ciprofloxacin, nalidixic acid, novobiocin, and tetracycline. By contrast, the nieAB mutant strain showed no phenotype changes under the tested conditions., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

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., 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 B.V. All rights reserved.)

Zinc uptake regulator (Zur) is a transcriptional regulator that represses zinc acquisition genes under high zinc conditions. The aim of this study was to identify and investigate the role of Zur-binding motifs (Zur boxes) in the differential regulation of Zur target genes, including the zinT, znuA, znuCB-zur operon, the troCBA operon, and yciC, in Agrobacterium tumefaciens. DNase I footprinting and gel shift assays were performed, confirming that Zur directly binds to 18-bp inverted repeat motifs found in the promoter of these Zur-regulated genes. Furthermore, promoter-lacZ fusions and mutagenesis of the identified Zur boxes were performed to assess the role of each Zur box. A Zur box found in the zinT promoter was required for zinc-dependent repression by Zur. The intergenic region between the znuA gene and the znuCB-zur operon contains two Zur boxes, named A and C, which immediately precede the genes znuA and znuC, respectively. Zur box A, but not Zur box C, was essential for the repression of the znuA promoter. Both Zur boxes A and C were implicated in the repression of the znuC promoter, in which mutation of either box alone was sufficient for full derepression of the znuC promoter. Three Zur boxes named T, M, and Y were identified in the intergenic region between the troCBA operon and the yciC gene. Zur box Y, which immediately precedes yciC, was shown to be responsible for Zur repression of the yciC promoter. In contrast, two Zur boxes, T and M, were essential for the complete repression of the troCBA operon, and full derepression of the troC promoter was exhibited when both Zur boxes were mutated simultaneously. Sequence analysis of the identified Zur boxes revealed a correlation between deviation from the core recognition sequence of the Zur box and the requirement of two Zur boxes for Zur regulation of distinctive promoters.

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 expression of the Agrobacterium tumefaciens emrAB operon, which encodes a membrane fusion protein and an inner membrane protein, is inducible by various flavonoids, including apigenin, genistein, luteolin, naringenin, and quercetin. Among these flavonoids, quercetin is the best inducer, followed by genistein. The emrR gene is divergently transcribed from the emrAB operon. The EmrR protein, which belongs to the TetR transcriptional regulator family, negatively regulates the expression of emrAB and of itself. Electrophoretic mobility shift assays and DNase I footprinting showed that EmrR binds directly at two EmrR-binding sites in the emrR-emrAB intergenic region and that quercetin inhibits the DNA-binding activity of EmrR. Promoter-lacZ fusion analyses and 5' rapid amplification of cDNA ends were performed to map the emrR and emrAB promoters. Compared with the wild-type strain, the emrA mutant strain exhibited similar levels of resistance to the tested antibiotics. In contrast, disruption of emrR conferred protection against nalidixic acid and novobiocin, but it rendered A. tumefaciens sensitive to tetracycline and erythromycin. The emrR mutation also destabilized the outer membrane of A. tumefaciens, resulting in increased sensitivity to SDS and low pH. These findings demonstrate that proper regulation of emrR-emrAB is required for free-living A. tumefaciens to survive in deleterious environments in which toxic compounds are present. Nonetheless, A. tumefaciens strains that lack emrR or emrA still have the ability to cause tumors when infecting Nicotiana benthamiana plants.

Analysis of the Agrobacterium tumefaciens C58 genome revealed a potential Zur (zinc uptake regulator) binding site (5'-GATATGTTATTACATTAC-3', the underlined letters are the center of symmetry of the inverted palindrome) located in the upstream region of atu3184, whose gene product is a member of the COG0523 subfamily of G3E GTPases. The specific interaction of the Zur protein with the 18-bp inverted repeat operator motif in the presence of zinc was demonstrated in vitro by a DNA band shift assay and a DNase I footprinting assay. A LacZ reporter fusion assay further confirmed that Zur negatively regulates atu3184 promoter activity in vivo. The expression of atu3184 was upregulated in response to zinc limitation in the wild-type strain, but the zur mutant strain exhibited high-level constitutive expression of atu3184 under all conditions, irrespective of the zinc levels. It is likely that A. tumefaciens Zur senses zinc and directly regulates the atu3184 promoter by a molecular mechanism similar to that of Escherichia coli Zur, where the operator DNA is surrounded by four Zur monomers forming two dimers bound on the opposite sides of the DNA duplex. Disruption of atu3184 did not affect cell growth under metal-limited conditions and had no effect on the total cellular zinc content. Furthermore, an A. tumefaciens strain lacking atu3184 caused a tumor disease in a host plant., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Agrobacterium tumefaciens AcrR is the transcriptional repressor of the acrABR operon. The AcrAB efflux pump confers resistance to various toxic compounds, including antibiotics [ciprofloxacin (CIP), nalidixic acid (NAL), novobiocin (NOV) and tetracycline (TET)], a detergent [sodium dodecyl sulfate (SDS)] and a biocide [triclosan (TRI)]. The sequence to which AcrR specifically binds in the acrA promoter region was determined by EMSA and DNase I footprinting. The AcrR-DNA interaction was abolished by adding NAL, SDS and TRI. Quantitative real time-PCR analysis showed that induction of the acrA transcript occurred when wild-type cells were exposed to NAL, SDS and TRI. Indole is a signaling molecule that increases the antibiotic resistance of bacteria, at least in part, through activation of efflux pumps. Expression of the A. tumefaciens acrA transcript was also inducible by indole in a dose-dependent manner. Indole induced protection against CIP, NAL and SDS but enhanced susceptibility to NOV and TRI. Additionally, the TET resistance of A. tumefaciens was not apparently modulated by indole. A. tumefaciens AcrAB played a dominant role and was required for tolerance to high levels of the toxic compounds. Understanding the regulation of multidrug efflux pumps and bacterial adaptive responses to intracellular and extracellular signaling molecules for antibiotic resistance is essential. This information will be useful for the rational design of effective treatments for bacterial infection to overcome possible multidrug-resistant pathogens., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Analysis of the A. tumefaciens genome revealed estC, which encodes an esterase located next to its transcriptional regulator estR, a regulator of esterase in the MarR family. Inactivation of estC results in a small increase in the resistance to organic hydroperoxides, whereas a high level of expression of estC from an expression vector leads to a reduction in the resistance to organic hydroperoxides and menadione. The estC gene is transcribed divergently from its regulator, estR. Expression analysis showed that only high concentrations of cumene hydroperoxide (CHP, 1 mM) induced expression of both genes in an EstR-dependent manner. The EstR protein acts as a CHP sensor and a transcriptional repressor of both genes. EstR specifically binds to the operator sites OI and OII overlapping the promoter elements of estC and estR. This binding is responsible for transcription repression of both genes. Exposure to organic hydroperoxide results in oxidation of the sensing cysteine (Cys16) residue of EstR, leading to a release of the oxidized repressor from the operator sites, thereby allowing transcription and high levels of expression of both genes. The estC is the first organic hydroperoxide-inducible esterase-encoding gene in alphaproteobacteria., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: The Agrobacterium tumefaciens C58 genome harbors an operon containing the dmeR (Atu0890) and dmeF (Atu0891) genes, which encode a transcriptional regulatory protein belonging to the RcnR/CsoR family and a metal efflux protein belonging to the cation diffusion facilitator (CDF) family, respectively. The dmeRF operon is specifically induced by cobalt and nickel, with cobalt being the more potent inducer. Promoter-lacZ transcriptional fusion, an electrophoretic mobility shift assay, and DNase I footprinting assays revealed that DmeR represses dmeRF transcription through direct binding to the promoter region upstream of dmeR A strain lacking dmeF showed increased accumulation of intracellular cobalt and nickel and exhibited hypersensitivity to these metals; however, this strain displayed full virulence, comparable to that of the wild-type strain, when infecting a Nicotiana benthamiana plant model under the tested conditions. Cobalt, but not nickel, increased the expression of many iron-responsive genes and reduced the induction of the SoxR-regulated gene sodBII Furthermore, control of iron homeostasis via RirA is important for the ability of A. tumefaciens to cope with cobalt and nickel toxicity., Importance: The molecular mechanism of the regulation of dmeRF transcription by DmeR was demonstrated. This work provides evidence of a direct interaction of apo-DmeR with the corresponding DNA operator site in vitro The recognition site for apo-DmeR consists of 10-bp AT-rich inverted repeats separated by six C bases (5'-ATATAGTATACCCCCCTATAGTATAT-3'). Cobalt and nickel cause DmeR to dissociate from the dmeRF promoter, which leads to expression of the metal efflux gene dmeF This work also revealed a connection between iron homeostasis and cobalt/nickel resistance in A. tumefaciens., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Agrobacterium tumefaciens has a cluster of genes (Atu3178, Atu3179, and Atu3180) encoding an ABC-type transporter, here named troA, troB, and troC, respectively, which is shown here to be a zinc-specific uptake system. Reverse transcription (RT)-PCR analysis confirmed that troA, troB, and troC are cotranscribed, with troC as the first gene of the operon. The yciC (Atu3181) gene is transcribed in the opposite orientation to that of the troCBA operon and belongs to a metal-binding GTPase family. Expression of troCBA and yciC was inducible under zinc-limiting conditions and was controlled by the zinc uptake regulator, Zur. Compared to the wild type, the mutant strain lacking troC was hypersensitive to a metal chelator, EDTA, and the phenotype could be rescued by the addition of zinc, while the strain with a single yciC mutation showed no phenotype. However, yciC was important for survival under zinc limitation when either troC or zinT was inactivated. The periplasmic zinc-binding protein, ZinT, could not function when TroC was inactivated, suggesting that ZinT may interact with TroCBA in zinc uptake. Unlike many other bacteria, the ABC-type transporter ZnuABC was not the major zinc uptake system in A. tumefaciens However, the important role of A. tumefaciens ZnuABC was revealed when TroCBA was impaired. The strain containing double mutations in the znuA and troC genes exhibited a growth defect in minimal medium. A. tumefaciens requires cooperation of zinc uptake systems and zinc chaperones, including TroCBA, ZnuABC, ZinT, and YciC, for survival under a wide range of zinc-limiting conditions., Importance: Both host and pathogen battle over access to essential metals, including zinc. In low-zinc environments, physiological responses that make it possible to acquire enough zinc are important for bacterial survival and could determine the outcome of host-pathogen interactions. A. tumefaciens was found to operate a novel pathway for zinc uptake in which ZinT functions in concert with the high-affinity zinc importer TroCBA., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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.

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.

An Agrobacterium tumefaciens membrane-bound ferritin (mbfA) mutant was generated to assess the physiological functions of mbfA in response to iron and hydrogen peroxide (H(2) O(2) ) stresses. Wild-type and the mbfA mutant strains showed similar growth under high- and low-iron conditions. The mbfA mutant was more sensitive to H(2) O(2) than wild-type strain. Expression of a functional mbfA gene could complement the H(2) O(2) -hypersensitive phenotype of the mbfA mutant and a rhizobial iron regulator (rirA) mutant, suggesting that MbfA protects cells from H(2) O(2) toxicity by sequestering intracellular free iron, thus preventing the Fenton reaction. The expression of mbfA could be induced in response to iron and to H(2) O(2) treatment. The iron response regulator (irr) also acted as a repressor of mbfA expression. An irr mutant had high constitutive expression of mbfA, which partly contributed to the H(2) O(2) -hyperresistant phenotype of the irr mutant. The data reported here demonstrate an important role of A. tumefaciens MbfA in the cellular defence against iron and H(2) O(2) stresses., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the -35 and -10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

Thioredoxin-like protein (TlpA) is a membrane-anchored periplasmic protein that contains a thioredoxin domain at its C-terminus. An Agrobacterium tumefaciens mutant lacking functional tlpA shows increased sensitivity to a superoxide generator, increased resistance to H2O2, and reduced cytochrome c oxidase activity. Whereas the levels of antioxidant enzymes, including total superoxide dismutase (SOD) and catalases, are unaltered, high expression of periplasmic SOD partially restores the superoxide hypersensitivity of the mutant, suggesting an accumulation of superoxide anions in the periplasm. The change in the ability of the mutant to cope with H2O2 stress is independent of OxyR, an H2O2 sensor and transcription regulator. The presented data indicate that TlpA not only is involved in the proper assembly of cytochrome c but is also implicated in the bacterial oxidative stress response.

Sco (for the synthesis of cytochrome c oxidase) is a mitochondrial membrane protein essential for the correct assembly of cytochrome c oxidase. sco homolog genes exist in a wide variety of bacterial species. Inactivation of Agrobacterium tumefaciens sco leads to markedly decreased cytochrome c oxidase activity. This phenotype can be complemented by either supplementing the culture medium with copper or by a plasmid containing sco. The sco mutant also alters resistance to a superoxide generator menadione and H2O2. Mutational analysis of conserved cysteine residues and a histidine residue within the putative copper ions binding motif of Sco indicates that these residues are essential for the biological activity of Sco. To summarize, we find that A. tumefaciens sco is required for the delivery of copper into active cytochrome c oxidase and in maintaining optimal resistance levels to oxidative stress.

The analysis of genetics and physiological functions of Agrobacterium tumefaciens RirA (rhizobial iron regulator) has shown that it is a transcription regulator and a repressor of iron uptake systems. The rirA mutant strain (NTLrirA) overproduced siderophores and exhibited a highly constitutive expression of genes involved in iron uptake (fhuA, irp6A, and fbpA) compared to that of the wild-type strain (NTL4). The deregulation in the iron control of iron uptake in NTLrirA led to iron overload in the cell, which was supported by the observation that the NTLrirA mutant was more sensitive than wild-type NTL4 to an iron-activated antibiotic, streptonigrin. The NTLrirA mutant was more sensitive than the parental strain to oxidants, including hydrogen peroxide, organic hydroperoxide, and a superoxide generator, menadione. However, the addition of an iron chelator, 2,2'-dipyridyl, reversed the mutant hypersensitivity to H(2)O(2) and organic hydroperoxide, indicating the role of iron in peroxide toxicity. Meanwhile, the reduced level of superoxide dismutase (SodBIII) was partly responsible for the menadione-sensitive phenotype of the NTLrirA mutant. The NTLrirA mutant showed a defect in tumorigenesis on tobacco leaves, which likely resulted from the increased sensitivity of NTLrirA to oxidants and the decreased ability of NTLrirA to induce virulence genes (virB and virE). These data demonstrated that RirA is important for A. tumefaciens during plant-pathogen interactions.

Singlet oxygen is a highly reactive form of molecular oxygen that is harmful to biological systems. Here, the role of three iron-containing superoxide dismutase (sodB) genes is clearly shown in protecting Agrobacterium tumefaciens against singlet oxygen toxicity. A sodBI mutant was more sensitive to singlet oxygen than both wild-type bacteria and a double sodBII-sodBIII mutant strain. Moreover, a sodBI-sodBII double mutant had higher sensitivity to singlet oxygen than a single sodBI mutant, although the double mutant was comparable to a sodB null mutant. High-level expression of sodBI and sodBII fully complemented the singlet oxygen hypersensitivity phenotype of the sodB null mutant, while high-level expression of sodBIII encoding a periplasmic SOD only partially restored the phenotype. Taken together, our data suggest that SodBI and SodBII have novel protective roles against singlet oxygen toxicity through unknown mechanisms.

Agrobacterium tumefaciens possesses three iron-containing superoxide dismutases (FeSods) encoded by distinct genes with differential expression patterns. SodBI and SodBII are cytoplasmic isozymes, while SodBIII is a periplasmic isozyme. sodBI is expressed at a high levels throughout all growth phases. sodBII expression is highly induced upon exposure to superoxide anions in a SoxR-dependent manner. sodBIII is expressed only during stationary phase. Analysis of the physiological function of sods reveals that the inactivation of sodBI markedly reduced levels of resistance to a superoxide generator, menadione. A mutant lacking all three Sod enzymes is the most sensitive to menadione treatment, indicating that all sods contribute at various levels towards the overall menadione resistance level. Sods also have important roles in A. tumefaciens virulence toward a host plant. A sodBI but not a sodBII or sodBIII mutant showed marked reduction in its ability to induce tumors on tobacco leaf discs, while the triple sod null mutant is avirulent.

Exposure of Agrobacterium tumefaciens to menadione, cumene hydroperoxide, and diamide strongly induced trxA expression. The trxA mutant showed a reduction in the aerobic growth rate and plating efficiency and was cytochrome c oxidase negative. Atypically, the mutant has decreased resistance to menadione but an increased H2O2 resistance phenotype.

In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.

Inactivation of Agrobacterium tumefaciens soxR increases sensitivity to superoxide generators. soxR expression is highly induced by superoxide stress and is autoregulated. SoxR also directly regulates the superoxide-inducible expression of atu5152. Taken together, the physiological role of soxR and the mechanism by which it regulates expression of target genes make the A. tumefaciens SoxR system different from other bacterial systems.

The genes involved in organic hydroperoxide protection in Agrobacterium tumefaciens were functionally evaluated. Gene inactivation studies and functional analyses have identified ohr, encoding a thiol peroxidase, as the gene primarily responsible for organic hydroperoxide protection in A. tumefaciens. An ohr mutant was sensitive to organic hydroperoxide killing and had a reduced capacity to metabolize organic hydroperoxides. ohr is located next to, and is divergently transcribed from, ohrR, encoding a sensor and transcription regulator of organic hydroperoxide stress. Transcription of both ohr and ohrR was induced by exposure to organic hydroperoxides but not by exposure to other oxidants. This induction required functional ohrR. The results of gel mobility shift and DNase I footprinting assays with purified OhrR, combined with in vivo promoter deletion analyses, confirmed that OhrR regulated both ohrR and ohr by binding to a single OhrR binding box that overlapped the ohrR and ohr promoters. ohrR and ohr are both required for the establishment of a novel cumene hydroperoxide-induced adaptive response. Inactivation or overexpression of other Prx family genes (prx1, prx2, prx3, bcp1, and bcp2) did not affect either the resistance to, or the ability to degrade, organic hydroperoxide. Taken together, the results of biochemical, gene regulation and physiological studies support the role of ohrR and ohr as the primary system in sensing and protecting A. tumefaciens from organic hydroperoxide stress.

Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE. In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined. katA and catE were found to be independently regulated in a growth phase dependent manner. KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase. Only small increases in H2O2 resistance levels were detected as cells entering stationary phase. The katA mutant was more sensitive to H2O2 than the parental strain during both exponential and stationary phase. Inactivation of catE alone did not significantly change the level of H2O2 resistance. However, the katA catE double mutant was more sensitive to H2O2 during both exponential and stationary phase than either of the single catalase mutants. The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A. tumefaciens from H2O2 toxicity during all phases of growth. Catalase-peroxidase activity (KatA) was required for full H2O2 resistance. The expression patterns of the two catalases in A. tumefaciens reflect their physiological roles in the protection against H2O2 toxicity, which are different from other bacteria.

Agrobacterium tumefaciens is an aerobic plant pathogenic bacterium that is exposed to reactive oxygen species produced either as by-products of aerobic metabolism or by the defense systems of host plants. The physiological function of the bifunctional catalase-peroxidase (KatA) in the protection of A. tumefaciens from reactive oxygen species other than H(2)O(2) was evaluated in the katA mutant (PB102). Unexpectedly, PB102 was highly sensitive to the superoxide generator menadione. The expression of katA from a plasmid vector complemented the menadione-hypersensitive phenotype. A. tumefaciens possesses an additional catalase gene, a monofunctional catalase encoded by catE. Neither inactivation nor high-level expression of the catE gene altered the menadione resistance level. Moreover, heterologous expression of the catalase-peroxidase-encoding gene katG from Burkholderia pseudomallei, but not the monofunctional catalase gene katE from Xanthomonas campestris could restore normal levels of menadione resistance to PB102. A recent observation suggests that the menadione resistance phenotype involves increased activities of organic peroxide-metabolizing enzymes. Heterologous expression of X. campestris alkyl hydroperoxide reductase from a plasmid vector failed to complement the menadione-sensitive phenotype of PB102. The level of menadione resistance shows a direct correlation with the level of peroxidase activity of KatA. This is a novel role for KatA and suggests that resistance to menadione toxicity is mediated by a new, and as yet unknown, mechanism in A. tumefaciens.

Physiological adaptive and cross-protection responses to oxidants were investigated in Agrobacterium tumefaciens. Exposure of A. tumefaciens to sublethal concentrations of H2O2 induced adaptive protection to lethal concentrations of H2O2. Similar treatments with organic peroxide and menadione did not produce adaptive protection to subsequent exposure to lethal concentrations of these oxidants. Pretreatment of A. tumefaciens with an inducing concentration of menadione conferred cross-protection against H2O2, but not to tert-butyl hydroperoxide (tBOOH), killing. The menadione induced cross-protection to H2O2 was due to the compound's ability to highly induce the peroxide scavenging enzyme, catalase. The levels of catalase directly correlated with the bacterium's ability to survive H2O2 treatment. Some aspects of the oxidative stress response of A. tumefaciens differ from other bacteria, and these differences may be important in plant/microbe interactions.

Induced adaptive and cross-protective responses to peroxide stress are important strategies used by bacteria to survive stressful environments. We have shown that exposure to low levels of peroxide (adaptive) and superoxide anions (cross-protection) induced high levels of resistance to peroxide killing in Agrobacterium tumefaciens. The mechanisms and genes involved in these processes have not been identified. Here, the roles played by peroxide (oxyR) and superoxide (soxR) global regulators and a catalase gene (katA) during these responses were investigated. H2O2-induced adaptive protection was completely abolished in both the oxyR and katA mutants. Superoxide generator (menadione)-induced cross-protection to H2O2 killing was observed in a soxR mutant, but not in either an oxyR or a katA mutant. In vivo analysis of the katA promoter, using a katA::lacZ transcriptional fusion, revealed that it could be induced by menadione in an oxyR-dependent manner. These results lead us to conclude that H2O2 and superoxide anions directly or indirectly oxidize OxyR and it is the resulting activation of katA expression that is responsible for the induced protection against lethal concentrations of H2O2.

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.