This article was downloaded by: [Middle Tennessee State University] On: 17 November 2014, At: 02:34 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20

Transcriptional response machineries of Bacillus subtilis conducive to plant growth promotion Kazutake Hirooka

a

a

Department of Biotechnology, Faculty of Life Science and Biotechnology, Fukuyama University, Fukuyama, Hiroshima, Japan Published online: 11 Sep 2014.

To cite this article: Kazutake Hirooka (2014) Transcriptional response machineries of Bacillus subtilis conducive to plant growth promotion, Bioscience, Biotechnology, and Biochemistry, 78:9, 1471-1484, DOI: 10.1080/09168451.2014.943689 To link to this article: http://dx.doi.org/10.1080/09168451.2014.943689

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 9, 1471–1484

Award Review

Transcriptional response machineries of Bacillus subtilis conducive to plant growth promotion Kazutake Hirooka* Department of Biotechnology, Faculty of Life Science and Biotechnology, Fukuyama University, Fukuyama, Hiroshima, Japan Received May 15, 2014; accepted June 29, 2014

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

http://dx.doi.org/10.1080/09168451.2014.943689

Bacillus subtilis collectively inhabits the rhizosphere, where it contributes to the promotion of plant growth, although it does not have a direct symbiotic relationship to plants as observed in the case of rhizobia between leguminous plants. As rhizobia sense the flavonoids released from their host roots through the NodD transcriptional factor, which triggers transcription of the nod genes involved in the symbiotic processes, we supposed that B. subtilis utilizes certain flavonoids as signaling molecules to perceive and adapt to the rhizospheric environment that it is in. Our approaches to identify the flavonoid-responsive transcriptional regulatory system from B. subtilis resulted in the findings that three transcriptional factors (LmrA/QdoR, YetL, and Fur) are responsive to flavonoids, with the modes of action being different from each other. We also revealed a unique regulatory system by two transcriptional factors, YcnK and CsoR, for copper homeostasis in B. subtilis. In this review, we summarize the molecular mechanisms of these regulatory systems with the relevant information and discuss their physiological significances in the mutually beneficial interaction between B. subtilis and plants, considering the possibility of their application for plant cultivation. Key words:

Bacillus subtilis; transcriptional regulation; flavonoid; copper; plant growthpromoting rhizobacteria

Rhizosphere is defined as the surface region of the soil that is directly influenced by root exudates and associated soil microorganisms. Root exudates can be roughly classified into two groups, low- and high-molecular mass compounds. Mucilages, consisting mainly of polysaccharides, are the major high-molecular mass exudates and they serve not only to maintain hydration around roots under dry conditions but also to provide a suitable inhabited environment for the microorganisms. The lowmolecular mass exudates include directly nutritious

organic compounds, such as sugars, amino acids, and lipids. Thus, diverse microorganisms grow thickly to feed on them in the favorable habitat of the rhizosphere and they survive coordinately or hostilely with each other. Microbial populations react to root exudates, and their numbers in rhizosphere can vary by as much as 10–100fold from those found in normal soil conditions.1–4) Some rhizospheric microorganisms are deleterious (pathogenic) for plants, and others exert a beneficial effect on them in return for receiving sufficient nutrients (symbiosis). It is well known that beneficial mycorrhizal fungi provide the host plant with a root surface with the enhanced capacity for absorbing water and minerals, particularly phosphate.5) Additionally, rhizobia in nodules of leguminous plants convert atmospheric nitrogen to ammonium that can be used by the host plant for amino acid biosynthesis.1) In addition to these symbiotic microorganisms, some non-phytopathogenic microorganisms associated with roots enhance the adaptive potential of the plants and increase their growth; these are called plant growth-promoting rhizobacteria (PGPR) or plant growthpromoting fungi (PGPF). PGPR and PGPF often serve as bio-control agents to suppress diseases caused by phytopathogens through producing a wide range of antimicrobial compounds, competition in colonization of the niche and for nutrients with the pathogens, and activation of the host defense system by induced systemic resistance (ISR). Their stimulatory effects on plant growth may also be achieved by increasing the availability of nutrients such as nitrogen, phosphorus, and amino acids.4,6) Bacillus subtilis has been extensively and closely studied as a Gram-positive model bacterium. Although B. subtilis is well known as a soil-dwelling bacterium, it is widely distributed and thus can be isolated from numerous terrestrial and aquatic environments. In Japan, B. subtilis subsp. natto has been used for the production of “natto,” a traditional Japanese food of fermented soybeans. In the classical procedure of natto production, fermentation of boiled soybeans is initiated by wrapping them with pasteurized rice straws, to which the B. subtilis species are dominantly attached

*Email: [email protected] This review was written in response to the author’s receipt of the JSBBA Award for Young Scientists in 2013. Abbreviations: PGPR, plant growth-promoting rhizobacteria; PGPF, plant growth-promoting fungi; ISR, induced systemic resistance; SAR, systemic acquired resistance; HTH, helix-turn-helix; PCA, protocatechuate; FAD, flavin adenine dinucleotide. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

1472

K. Hirooka

because they intrinsically prefer to inhabit the plant surface and they can withstand high temperatures in the form of thermotolerant endospores.7) B. subtilis species have also been isolated from the rhizosphere of various plants in greater numbers than most other spore-forming bacteria.8) These facts indicate that B. subtilis preferentially grows in close association with plants. Several proofs have been reported that B. subtilis in the rhizosphere acts as PGPR.9,10) It was found that B. subtilis has broad suppressive properties for more than 23 types of phytopathogens in vitro because it is capable of producing a great abundance of antibiotics with a variety of structures and activities, such as surfactin, iturin, and fengycin.9) B. subtilis also colonizes in contact with roots with a biofilm formation, which deprives other phytopathogens of habitat and nutrients, resulting in suppression of their propagation. In addition, the root-associated B. subtilis can enhance the plant resistance against phytopathogens by eliciting ISR.10) It is interesting to note that B. subtilis is also a saprophytic bacterium that secretes various degrading enzymes, including those able to hydrolyze polysaccharides constituting the plant cell wall, although it does not prey on the living plant cells.11) Intensive studies have been done on signaling mechanisms underlying the beneficial interaction between B. subtilis and plants in the rhizosphere. Bais’s group reported a signaling mechanism between B. subtilis and Arabidopsis thaliana in response to the phytopathogen infection.12,13) When the leaves of A. thaliana were infected with a phytopathogen, Pseudomonas syringae pv. tomato DC3000, the expression of ALMT1 encoding a malate transporter was induced in the roots independent of the salicylate signaling pathway that controls systemic acquired resistance (SAR), leading to malate secretion from the roots. The secreted malate attracted B. subtilis strain FB17 to form a biofilm on the root surface. The biofilm formation, in turn, invoked the abscisic acid and salicylate signaling pathways to close stomata, which restricted the phytopathogen entry through the stomatal aperture.12) Although the SAR caused by the phytopathogen infection on leaves also activated the defense system in roots, B. subtilis strain FB17 suppressed this root-defense response in order to facilitate its root colonization, in which TasA, a protein component of the extracellular matrix of the B. subtilis biofilm, was suggested to be involved.13) Thus, on the plant side, the signaling mechanism committed to this defense response is being unveiled; yet how B. subtilis modulates its gene expressions in response to signals released from the roots remains unclear. It is unlikely that malate is the sole signaling molecule that B. subtilis receives to enter into the beneficial interaction with the plant roots. In this review, we focused on the transcriptional mechanisms of B. subtilis in response to the substances found in the rhizosphere, such as flavonoids and metal ions, which are probably related to the mutually beneficial interaction between B. subtilis and plants. Understanding these mechanisms, together with the information about the other signaling mechanisms in the rhizosphere, is expected to offer clues for the

practical use of not only B. subtilis but also the other soil bacteria for the promotion of plant growth.

I. Dual regulatory system by LmrA and QdoR in B. subtilis, first identified as flavonoid-responsive transcriptional repressors in genus Bacillus In addition to the nutrient materials, plant root cells release various secondary metabolites as low-molecular mass exudates, and flavonoids constitute a large portion of them. A metabolomic analysis of the root exudates of A. thaliana demonstrated that flavonoids accounted for 37% of all secondary metabolites.14) Although flavonoids are pooled mainly as glycosylated forms in the plant cell vacuoles, most flavonoids present in the rhizosphere are not glycosylated, but aglycones.15) Quantitative measurements performed by different groups showed that the roots of the soybean plant (Glycine max) released flavonoids, which were mainly isoflavones (genistein, daidzein, and coumestrol), at tens to hundreds of pmol d−1 per root.15) Through experiments with membrane vesicles isolated from the soybean roots, it was suggested that genistein and daidzein are transported by an ABC transporter having a preference for aglycones over the glycosylated forms.16) On the other hand, it was reported that a β-glucosidase specifically hydrolyzing isoflavone glycosides is localized in the apoplast of the soybean roots.17) Thus, in this case, isoflavone aglycones are likely to be actively released into the rhizosphere through two routes: isoflavone aglycones, resulting from hydrolysis of their glycosides inside the cell, are exported via the ABC transporter; or isoflavone glycosides are transferred from vacuole to apoplast via exocytosis, followed by their hydrolysis by extracellular enzymes from plants and microorganisms. In many plant species, biosynthesis of flavonoids and their release into the rhizosphere are enhanced by various stresses, such as deficiency of nutrients (e.g. phosphorus, nitrogen, and iron), wounding, pathogen infection, and oxidative stress.15) In some cases, these two ways of controlled release might play a role in an adaptive response, although it is also possible that flavonoids simply leak due to impaired membrane integrity induced by the stresses.15) Flavonoids can serve various plant-defensive functions which include feeding deterrence, allelopathic growth suppression to competitors, and antibiotic activity to pathogens.18) In contrast to these defensive actions of plants against other organisms, beneficial interactions between plants and microorganisms through flavonoids as a signaling molecule are also found in the rhizosphere. In the symbiosis of rhizobia and leguminous plants under nitrogen starvation, certain flavonoids released from the roots specifically trigger the expressions of rhizobial genes, such as the nod genes, required for nodulation.18) The regulation of the nod genes is primarily mediated by NodD, a transcriptional factor belonging to the LysR family. The NodD protein binds as a homotetramer to the conserved DNA motifs (nod boxes) located adjacent to the promoter regions of the nod genes, even in the absence of the

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

B. subtilis responses conducive to plant growth

inducing flavonoid. Upon interaction with the inducer, NodD activates the transcription from the target promoter while maintaining its DNA binding; the conformational change of the DNA-bound NodD, caused by the inducer interaction, sharpens the DNA bend at the promoter region, which probably facilitates the access of RNA polymerase onto the promoter.19) A number of nod gene products are the enzymes involved in the synthesis of rhizobial species-specific Nod factors, lipochitin oligosaccharides required for nodule formation in the host plant. In the host-specific manner, rhizobia secreting Nod factors stimulate the reorientation of cell wall growth of the root hair cells, resulting in curled root hairs. Within these curled root hairs, Nod factors act to promote the formation of infected threads, through which the rhizobia enter the root tissues, eventually bacteroids being formed in the nodule cells for nitrogen fixation.18,20) On the other hand, the symbiosis between mycorrhizal fungi and land plants is promoted by phosphorus deficiency in the soil. After spore germination, mycorrhizal fungi turn to the branched hyphae and then penetrate the host root tissue to form ecto- or endo-mycorrhizal invasion structures. Some of the host exudates that stimulate spore germination, hyphal branching in the soil, and root colonization, often in a symbiont-specific manner, have been identified as flavonoids,1,15,18) whereas strigolactones have been identified as non-flavonoid branching factors for arbuscular mycorrhizal fungi.5) As mentioned above, it

1473

was observed that the release of flavonoids into the rhizosphere is elevated under starvation of nitrogen or phosphorus, by which the host plants should effectively attract their symbiotic counterparts (Fig. 1). Since flavonoids also have chemical properties to function as metal chelators and reducing agents, it is possible that they improve mobilization of poorly soluble phosphates by chelating or reducing the counter metal cations.15) We speculated that, as with rhizobia and mycorrhizal fungi, B. subtilis utilizes certain flavonoids as signaling molecules to perceive and adapt to the rhizospheric environment that it is in, and we tried to identify the flavonoid-responsive transcriptional regulatory system from B. subtilis that might be involved not only in its adaptation to the rhizospheric environment but also in its beneficial interaction with plants. The LmrA/QdoR regulatory system is the first characterized system in the genus Bacillus that utilizes flavonoids as an effector molecule to induce the transcription of their target genes.21) The lmrAB operon, one of the LmrA/QdoR regulon members, encodes a transcriptional factor (LmrA) belonging to the TetR family and a multidrug efflux transporter (LmrB) belonging to the major facilitator superfamily. The isolation and analysis of the spontaneous B. subtilis mutants that confer resistance to lincomycin, a lincosamide antibiotic derived from Streptomyces lincolnensis, revealed that the obtained mutants, exhibiting resistance to several drugs besides lincomycin, elevate the lmrAB expression and that they

Fig. 1. Interaction between plants and microorganisms through flavonoids as signaling molecules in the rhizosphere. Note: Responses directly related to flavonoids are indicated by boldface type. Beneficial interactions are indicated by a pair of pale green arrows, while a competitive interaction is indicated by a pair of red arrows.

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

1474

K. Hirooka

have the mutations in the lmrA promoter or coding regions. Moreover, introduction of the lmrB disruption into each mutant abolished the multidrug resistance.22) The subsequent DNA-binding experiments showed that LmrA interacts with a cis-sequence in the lmrA promoter region. These findings suggested that LmrA acts as an autorepressor by binding to the lmrA promoter region. However, the DNA binding of LmrA was not inhibited by lincomycin, and the inducer compound interacting with LmrA was not determined at that point.23) The DNA microarray analysis of the wild type and the lmrA disruptant, combined with genome-wide computational sequence analysis, found the qdoI (formerly yxaG)-yxaH operon to be another LmrA target. Through the DNA-binding experiments, it was confirmed that LmrA interacts with two-tandem cissequences in the qdoI promoter, each of which is similar to that in the lmrA promoter region.23) Additionally, it was reported that qdoI encodes a quercetin 2,3-dioxygenase (EC 1.13.11.24) that converts quercetin to 2-protocatechuoyl-phloroglucinol carboxylic acid and carbon monoxide.24) Further detail characterization revealed that this enzyme is also able to convert other flavonols, such as fisetin, tamarixetin, and galangin, to the corresponding depsides by a similar C-ring cleavage reaction, although its catalytic activity toward fisetin is relatively low.25) In contrast to qdoI, there is little information about yxaH, except that it is predicted to encode a membrane protein by the sequence information. The qdoR (formerly yxaF) gene encoding the TetR-type transcriptional factor is located immediately upstream of the qdoI-yxaH operon and oriented in the same direction as qdoI-yxaH. The LmrA and QdoR proteins exhibit 56% identity in their amino acid sequences, and especially their N-terminal regions including the helix-turn-helix (HTH) motif for DNA binding are highly conserved. Based on these findings, we assumed that LmrA and QdoR recognize the same cis-sequences located in the promoter regions of the lmrAB and qdoI-yxaH operons and that the DNA binding of LmrA and QdoR is effectively inhibited by certain flavonoids, such as quercetin, and thus the transcription is induced. The DNase I footprinting demonstrated that LmrA and QdoR interact with almost the same regions, including the single and tandem cissequences, respectively located in the lmrA and qdoI promoters, and that these regulator proteins also interact with the qdoR promoter region, in which an additional cis-sequence was identified. These four cis-sequences, termed LmrA/QdoR boxes, comprise 18 bp of a modestly conserved sequence of AWTATAtagaNYGgTCTA (W, Y, and N stand for A or T, C or T, and any base, respectively, and lower-case letters stand for a 3-out-of-4 match). A gel retardation assay with the two regulator proteins and the DNA probes corresponding to the lmrA and qdoI promoter regions covering the respective LmrA/QdoR boxes indicated that, out of the eight flavonoids tested, quercetin, fisetin, and (+)-catechin are most inhibitory to the DNA binding of LmrA, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory to that of QdoR. A reporter assay of the B. subtilis strains carrying the promoter-lacZ fusions and the lmrA and/or

qdoR disruptions essentially supported the in vitro results, except that galangin did not activate the lmrA and qdoI promoters, probably due to its poor incorporation into cells. Taken together, we considered that LmrA and QdoR recognize and bind to the LmrA/ QdoR boxes in the lmrA, qdoI, and qdoR promoter regions to repress the transcription from these promoters and that the binding of the two regulators is inhibited efficiently and distinctly by the flavonoids with specific structural features, leading to transcriptional derepression (the relationship between structures and effects of flavonoids on these regulators is discussed in our previous report21)). Our attempt to find the other candidate genes repressed by LmrA and QdoR, using the DNA microarray analysis of the wild type and the lmrA and qdoR double disruptant, was unsuccessful. Thus, the LmrA/QdoR regulon presumably comprises the lmrAB operon, the qdoR gene, and the qdoI-yxaH operon (Fig. 2).21) The gel retardation assay showed that QdoR binds most tightly to the tandem LmrA/QdoR boxes in the qdoI promoter region. In addition, along with the reporter assay, the measurements of the quercetin degradation and the lincomycin resistance of the mutant strains grown in the absence of the inducer flavonoids indicated that the lmrA disruption significantly enhances both lmrA and qdoI promoter activities, whereas the qdoR disruption predominantly affects the qdoI promoter activity.21) These results suggest that LmrA represses the lmrAB and qdoI-yxaH operons, but that QdoR represses qdoI-yxaH more preferentially. It is intriguing that B. subtilis utilizes flavonoids as signaling molecules to induce resistance toward structurally unrelated antibiotics such as lincomycin through the LmrA/QdoR regulatory system. We assume that this might be one of the strategies that B. subtilis uses in its struggle against other microorganisms in the mixed microbiological flora in the rhizosphere, the environmental conditions of which B. subtilis perceives through the abundant flavonoids (Fig. 1). A similar situation was observed for the habitat of Staphylococcus aureus, in which gene expression of the QacA major facilitator superfamily pump controlled by QacR, a member of the TetR family, is induced in response to the plant alkaloid berberine.26) In the course of studying the regulatory mechanisms of LmrA and QdoR, it was found that a B. subtilis strain carrying the lmrA and qdoR double disruption is much more susceptible to quercetin than the wild type, and the viability of the mutant strain was affected. This was likely due to constitutively expressed qdoI, but not yxaH or lmrB, because introduction of the qdoI disruption into the mutant strain canceled this quercetin-sensitive phenotype, while the disruption of yxaH or lmrB did not.21) We also examined the susceptibility to the other seven flavonoids of B. subtilis strains with different levels of qdoI expression, and found that only the strains with high QdoI activity were highly sensitive to quercetin, but that the growth of these strains was not seriously affected by the other seven flavonoids. Further analyses, including a co-cultivation experiment with the wild-type and mutant strains, led to the conclusion that intracellular accumulation of protocatechuate (PCA), which is derived from the rapid

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

B. subtilis responses conducive to plant growth

1475

Fig. 2. Organization of the LmrA/QdoR Regulon. Note: The five gene members of the regulon are indicated by large open arrows; three promoters and three hairpin structures, probably functioning as ρ-independent transcription terminators, are indicated by bent arrows and stem loops, respectively. The LmrA and QdoR proteins, forming a dimer (two ovals), bind to the four LmrA/QdoR boxes (cross-hatched boxes) located in the promoter regions repressing the regulon members. The binding of LmrA and QdoR to the LmrA/QdoR boxes is inhibited by certain flavonoids, which leads to the derepression of the regulon members. The qdoI gene, one of the LmrA/QdoR regulon members, encodes quercetin 2,3-dioxygenase, which forms a dimer (two rotundate rectangles)24) and catalyzes the C-ring cleavage of flavonols, as illustrated.29)

decomposition of quercetin by the increased QdoI and an endogenous esterase, exerts an adverse effect on cell viability. In addition, we demonstrated that the sensitivity of the mutant B. subtilis strain toward quercetin was quenched by the repression of qdoI by the ectopically introduced lmrA or qdoR gene.25) Although primary role of QdoI is detoxification of flavonols including quercetin, the excess QdoI activity appears rather harmful in the presence of quercetin, causing rapid production of PCA, apparently more toxic than quercetin, in the cells. Hence, we have identified the physiological significance of the dual-regulation system by LmrA and QdoR, which is designed to regulate qdoI expression rigidly for adjusting the QdoI activity to a suitable level as necessary (Fig. 2). The binding of both regulators to the LmrA/QdoR boxes is inhibited by several flavonoids, but they show considerably different responsive properties, which depend not only on the regulator type but also on the target DNA sequence. Quercetin and fisetin effectively inhibit the binding of both regulators to the lmrA and qdoI promoter regions including the single- and the tandem-LmrA/QdoR boxes, respectively, whereas (+)-catechin has an inhibitory effect only on LmrA binding to the promoters and it does not affect QdoR binding. Moreover, in the in vitro DNA-binding experiment, QdoR was readily released from the lmrA promoter region by tamarixetin and galangin, but these flavonoids did not release QdoR from the qdoI promoter region, suggesting that their inhibitory effect also depends on the higher order architecture of the regulator-DNA complex.21)

LmrA and QdoR belong to the TetR family of bacterial transcriptional factors, known typically to possess two functional domains, a highly conserved N-terminal DNA-binding domain and a less conserved C-terminal domain functioning in both dimerization and effector binding.27) The crystal structure of the QdoR protein showed that it forms a dimer structure in which the Cterminal domains are associated with each other while the respective N-terminal regions appear to constitute the core of the DNA-binding domain including the HTH motif.28) Based on structural information on QdoR and other transcriptional factors belonging to the TetR family, eight residues were selected as possibly critical to the ligand response in QdoR (Fig. 3). To confirm the significance of these residues, eight QdoR mutants in which each of the residues was replaced by alanine were produced, and the DNA-binding affinity of these single mutants and their sensitivity toward various flavonoids were evaluated by a gel retardation assay and compared with those of wild-type QdoR. The three mutants, carrying the alanine substitution at Phe87, Trp131, or Phe135, showed features distinctly different from those of the wild type and from each other. We further examined the in vivo function of the mutant with alanine substitution at Trp131 by a reporter assay, which largely supported the corresponding in vitro results.29) These in vitro and in vivo results suggest that Phe87, Trp131, and Phe135, forming a hydrophobic cluster in QdoR, play crucial roles in the DNA binding, flavonoid accommodation, and/or conformational change triggered by ligand binding (Fig. 3). A QdoR mutant capable of responding to

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

1476

K. Hirooka

Fig. 3. Three-dimensional structure of QdoR. Note: Crystallographic analysis revealed that QdoR forms a homodimer (PDB ID, 1SGM).28) One subunit is depicted by ribbons, where the Nterminal domain for DNA binding and the C-terminal domain for dimerization are pink and pale blue, respectively, and the other subunit is depicted by a wireframe. The eight residues into which the alanine substitution was introduced in our previous study are depicted by sticks.29) The three aromatic residues found to be important to the QdoR function are presented in orange, and the other five residues, whose replacement with alanine did not significantly affect the QdoR function, are presented in dark green.29)

various flavonoids can be created by substituting the residues in the flavonoid-interacting domain probably including these three aromatic residues, and this mutant would be available as a novel biosensor to detect various flavonoids and related compounds in such a way as to introduce its gene into the B. subtilis strain carrying a reporter-fusion construct similar to that used in this study.29) Siedler et al. reported that a biosensor composed of QdoR and the qdoI promoter-gfp fusion functions in the Escherichia coli cells to detect flavonoids such as quercetin and kaempferol by the greenfluorescent signal, which is expected to be applied for isolation of the genes involved in the flavonoid biosynthetic pathways in plants.30)

II. B. subtilis YetL and Fur-regulatory systems, flavonoid responses of which were newly identified by DNA microarray analysis The two paralogous TetR-type transcriptional factors, LmrA and QdoR, were first characterized to show the flavonoid responses in the genus Bacillus. On the other hand, NodD regulators, which belong to the LysR family and control transcription of the nod genes involved in nodulation of rhizobia in response to the flavonoid signals released by their leguminous hosts, have been characterized in detail as mentioned above. Additionally, in Pseudomonas putida DOT-T1E, both the resistance-nodulation-cell division family transporter TtgABC and its cognate TetR family repressor of TtgR constitute a multidrug recognition system, and several flavonoids are substrates of TtgABC and trigger the pump expression through their binding to the

TtgR-operator complex to dissociate it.31) Since it does not seem to be rare that flavonoids function as signaling molecules for communication among rhizobacteria and plants, it was expected that, in addition to the LmrA/QdoR regulon, B. subtilis possesses genes involved in flavonoid degradation or another physiological function for intercellular communication via flavonoids, which are under the control of flavonoidresponsive transcriptional factors other than LmrA/ QdoR. In order to elucidate the comprehensive regulatory network for the expression of the genes responsive to flavonoids in B. subtilis, we tried to identify additional genes whose expression is significantly induced by flavonoid addition by means of DNA microarray analysis. Among the new candidate flavonoid-inducible genes found, we focused on the yetM gene encoding a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase and on its transcriptional regulatory mechanism.32) DNA microarray analysis involving the wild-type strain and a yetL disruptant, performed in the framework of the Japan Functional Analysis Network for B. subtilis (http://bacillus.genome.jp/), suggested that the product of the yetL gene, which encodes a transcriptional factor of the MarR family and is located immediately upstream of the yetM gene in the opposite direction, negatively regulates yetM transcription, which is induced by certain flavonoids. DNA-binding experiments including DNase I footprinting and gel retardation indicated that YetL binds specifically to the respective single sites in the divergent yetL and yetM promoter regions, with particularly higher affinity for the latter region; the former overlaps the Shine–Dalgarno sequence of yetL, and the latter contains a perfect 18-bp palindromic sequence

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

B. subtilis responses conducive to plant growth

(TAGTTAGGCGCCTAACTA). An in vitro gel retardation assay with the YetL protein and the DNA probes corresponding to the yetL and yetM promoter regions and an in vivo reporter assay of the B. subtilis strains carrying the yetL or yetM promoter-lacZ fusion indicated that the DNA binding of YetL is inhibited effectively by flavonoids such as kaempferol, apigenin, and luteolin, and its weaker interruption by flavonoids such as quercetin and fisetin appears to be different from the case of LmrA/QdoR (the responsive property of YetL to flavonoids with different structural features is discussed in our previous report32)). After our publication of the YetL regulatory system, some bacterial transcriptional factors that recognize and respond to flavonoids have been additionally reported, including TetR members of Bradyrhizobium japonicum FrrA33) and Sinorhizobium meliloti EmrR,34) and LysR members of Lactobacillus brevis KaeR35) and Herbaspirillum seropedicae strain SmR1 FdeR.36) However, to our knowledge, YetL is the only MarR member that specifically responds to flavonoids among those reported thus far. Most MarR members act as a transcriptional repressor, and many of them are categorized into two groups: one regulates gene expression through ligand-mediated release of DNA binding, while the other regulates adaptive response to oxidative stress through oxidative modification of its cysteine residue(s) that affects DNA binding.37) E. coli MarR, the prototype of the MarR family, can be dissociated from the operator DNA of the marRAB operon, which is involved in multidrug resistance, through interaction with a broad range of drugs containing salicylate.26) A high concentration of salicylate was required to obtain the crystal structure of MarR, in which two salicylate molecules per monomer are located within the DNA-binding motif and are relatively surface exposed.38) Salicylate was also found to facilitate crystal packing of the other MarR members, which led to co-crystallization of several MarR members with salicylate molecules such as Salmonella enterica SlyA and Methanobacterium thermoautotrophicum MTH313, though it is unlikely that salicylate is the natural ligand for these MarR members, as salicylate associates with these members and impairs their DNA binding only at high concentrations.39,40) The crystal structures of these MarR members revealed that they form a dimer structure with a common triangular shape, at the two corners of which winged HTH motifs for DNA binding are located. These DNA-binding motifs consist of the internal region of each subunit, and their N and C termini are intertwined with each other to form a core domain. As YetL dimerization in solution was confirmed by gel filtration,32) it is assumed that YetL shares this structural feature. Recently, Davis et al. reported crystal structures of the apo- and PCA-bound forms of Streptomyces coelicolor PcaV, a MarR-type transcriptional repressor that regulates the pcaIJFHGBL operon encoding enzymes involved in the PCA cleavage of the β-ketoadipate pathway, as well as the pcaV gene itself, the promoter of which is oppositely oriented and adjacent to the pcaI promoter.41) Besides the fact that PCA is a substrate of the enzymes encoded in the PcaV regulon, the in vitro experiments showed that PCA has a high affinity to PcaV with a stoichiometry of one molecule per mono-

1477

mer. Thus, PCA is regarded as a physiologically relevant ligand of PcaV. The in vitro and in vivo mutational analyses revealed that an Arg residue, located in the N-terminal region and forming a part of the ligand-binding pocket of PcaV, is not only critical to the ligand coordination but also required for the DNA binding. Comparison of the PcaV–PCA complex with the SlyA-salicylate and MTH313-salicylate complexes indicated that one of the salicylates of both complexes is accommodated in a pocket in a similar manner to PCA in the pocket of PcaV, suggesting that a common ligand-binding pocket exists in the crevice between the dimerization domain and the DNA-binding domain. In addition to hydrophobic interactions and hydrogen bonds between the ligand and the protein, the carboxylate moiety of salicylate is coordinated by an Arg residue in the N-terminal region of these MarR members.39–41) Moreover, it was reported that an alanine substitution of the N-terminal Arg residue in E. coli MarR resulted in a significantly reduced binding affinity to salicylate.42) In the case of B. subtilis YetL, it does not appear that an Arg residue directly interacts with the ligand flavonoids in the same manner, since they do not possess the carboxylate moiety, and indeed, such an Arg residue was not found in the N-terminal region of YetL. However, it is persuasive that the ligand flavonoid molecule is accommodated in a pocket located in a niche of the dimerization and DNA-binding domains accompanied by hydrophobic interactions and hydrogen bonds with the residues of YetL, as observed in the ligand–protein complex of the abovementioned MarR members. As described in the previous section, B. subtilis possesses the qdoI gene, one of the LmrA/QdoR regulon members, which encodes quercetin 2,3-dioxygenase capable of catalyzing the C-ring cleavage of several flavonols, such as quercetin, to yield the corresponding depsides and carbon monoxide.24,25) The resultant depsides can be hydrolyzed to a pair of the respective aromatic carboxylates by an endogenous esterase with broad substrate specificity. It was reported that B. subtilis also possesses the yfiE gene, whose expression is induced by catechol and which encodes a catechol-2,3dioxygenase capable of converting catechol into 2-hydroxymuconic semialdehyde,43) and the ywhB gene which encodes a 4-oxalocrotonate tautomerase capable of catalyzing the interconversion of 2-hydroxymuconate and 4-oxalocrotonate.44) This implies that catechol and its derivatives are degraded through the meta-cleavage pathway via the dehydrogenation route.45) Alternatively, highly electrophilic aromatic compounds, such as catechol and 2-methylhydroquinone, can form Sadducts with cellular thiols. These S-adducts are assumed to be subjected to thiol-dependent ring cleavage for detoxification by multiple dioxygenase/glyoxalase family enzymes encoded by mhqA, mhqO, and mhqE, which respond to thiol stress and are regulated by MhqR, a MarR-type repressor with an unknown derepression mechanism.46) The yetM gene, identified as the YetL target, is predicted to encode a FAD-dependent monooxygenase containing FAD- and NADHbinding motifs. The superfamily that contains YetM also includes salicylate monooxygenase, which converts salicylate to catechol.47) In addition, it was

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

1478

K. Hirooka

reported that an enzyme from an Asteraceae plant species, Chrysanthemum segetum, catalyzes hydroxylation at position 8 of flavonols and flavones using FAD and NADPH as co-factors.48) Thus, we speculate that YetM catalyzes the conversion of the salicylate derivatives generated from the flavonols through degradation by QdoI, or the direct hydroxylation of flavonols followed by degradation by QdoI, yielding the catechol derivatives, which would flow into the catechol metabolic pathways described above. Further close scrutiny of the data of the DNA microarray analysis that led to the identification of the YetL regulatory system allowed us to find the additional candidate genes, the expression of which can be induced by flavonoids. We found that the fisetin addition significantly (over threefold) elevated the transcription of many genes such as the dhbACEBF, fhuBCG, yclNOPQ, and yfiZyfhA operons and the yusV and ycgT genes (http://www.genome.jp/kegg/expression),32) which have been originally known to be under the control of Fur, an iron-sensing global transcriptional repressor.49) Fur orthologs are found in many Gram-positive and Gram-negative bacteria and constitute a unique family with other metalloregulatory proteins such as Zur and PerR.50) In B. subtilis, Fur represses at least 20 operons including approximately 40 genes, most of which are involved in iron uptake and homeostasis machineries, through its binding to the Fur boxes located in the regulatory regions of the target genes under iron-sufficient conditions. The Fur box is classically represented as a 19-bp inverted repeat, but it was later reconsidered that this sequence comprises two overlapping operator sites with a 7-1-7 minimal recognition element (TGATAATNATTATCA) and that two Fur dimers bind to opposite faces of the DNA duplex at the 19-bp Fur box.51) Upon iron starvation, a ferrous ion is dissociated from the metal-sensing site of Fur, which impairs the binding affinity of Fur to the Fur box, leading to derepression of the target genes. To test whether the DNA binding of Fur is affected by certain flavonoids, such as fisetin, a gel retardation assay was performed by using the Fur protein and the DNA probe corresponding to the promoter region containing the Fur box of the dhbACEBF operon encoding biosynthetic enzymes for bacillibactin (trimeric lactone of 2,3-dihydroxybenzoyl-Gly-Thr), which is categorized into a catecholate-type of siderophore, one of the highaffinity iron-chelating agents.52,53) The gel retardation assay showed that the Fur binding to the DNA probe was modestly but distinctly inhibited not only by fisetin but also by other flavonoids such as quercetin, kaempferol, luteolin, and apigenin at relatively high concentrations (mM levels). Moreover, the reporter assay of a B. subtilis strain carrying the dhbA promoter region corresponding to the DNA probe fused to lacZ demonstrated that the dhbA promoter was moderately induced by the respective addition of these flavonoids to the minimal medium. These reporter activities were much lower than those obtained by the same strain grown in the minimal medium minus FeCl3 and by the strain carrying the fur disruption along with the same promoterlacZ fusion grown in the normal minimal medium, and thus it was suggested that these flavonoids are not so effective that the repression by Fur is completely

released. We also observed that the in vitro DNA binding of Fur was inhibited by a metal-chelating agent EDTA and that this inhibitory effect was counteracted by the excess addition of ferrous ion in the mixture. By contrast, the inhibitory effect of fisetin on the DNA binding of Fur was not interrupted by the additional ferrous ion. In the reporter assay, the inducing effect of fisetin was not affected by the excess ferric ion in the medium; when the strain carrying fur+ and the dhbA promoter-lacZ was grown in the minimal medium containing 250 μM FeCl3, which is a 10-fold higher concentration than normal, the reporter activity was induced by fisetin to a level comparable with that obtained by the same strain grown in the normal minimal medium (Hirooka, K., unpublished results). These results suggest that specific flavonoids affect the DNA binding of Fur, which is independent of the concentration of ferrous ion sensed by Fur. Although the crystal structure of B. subtilis Fur has not been determined, its homology model was constructed and its metal-binding sites were assigned based on the structures of several members of the Fur family previously reported and on the metal-binding experiments of the various Fur mutants.54) It is assumed that, like a typical Fur member, the B. subtilis Fur protein folds into two domains consisting of the N-terminal DNA-binding domain linked by a hinge region to the C-terminal dimerization domain, and that it possesses three distinct metal-binding sites per monomer: site 1, a Cys4-Zn2+ structural site that is located near the C terminus and consists of two Cys-X-X-Cys motifs; site 2, at the junction of the DNA-binding domain and the dimerization domain; and site 3, adjacent to site 2 and also close to the dimer interface. While the zinc ion at site 1 is structural and non-exchangeable, the DNA-binding affinity can be modulated by the metal binding of site 2 and site 3, i.e. the highest DNA-binding affinity was obtained by metal binding at both sites, and site 2 likely plays the primary role in sensing the ferrous ion under the physiological condition.54) Identification of the flavonoidbinding site in Fur is the first challenge to be addressed for elucidation of the molecular mechanism by which flavonoids cause the release of the DNA binding of Fur independently of the ferrous ion (Fig. 4). Although iron is an essential micronutrient for nearly all living organisms, including plants and rhizospheric microorganisms, the iron availability in soil is quite limited because, in aerobic environments, the predominant ferric form is extremely insoluble, and even the slightly emerged free ferric ion is competitively incorporated by both roots and microorganisms. In order to efficiently capture ferric ions, microorganisms and Poaceae plants synthesize siderophores with various structures, and many microorganisms are equipped with machineries to import other xenosiderophores for iron uptake in addition to the transporters for the endogenously produced ones and the ferric ion itself.49,55) All higher plants except Poaceae have developed a strategy for iron acquisition, which involves acidification of the rhizosphere by excretion of protons and organic acids from roots, resulting in an increased solubility of the ferric ion, its reduction to the more soluble ferrous ion by a ferric-chelate reductase, and incorporation of the ferrous ion into the cytosol.56) The bacterial

B. subtilis responses conducive to plant growth

1479

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

Fig. 4. Proposed derepression mechanism of Fur regulon by specific flavonoids. Note: Two Fur dimers (a pair of two ovals) bind to opposite faces of the DNA duplex at the 19-bp Fur box located in the regulatory regions of the target genes under iron-sufficient conditions. Upon iron starvation, a ferrous ion is dissociated from the metal-sensing site (site 2) of Fur, which causes the conformational change and the decline in the DNA-binding affinity of Fur, leading to derepression of the target genes. Specific flavonoids likely affect the DNA binding of Fur by altering the conformation of the DNA-bound form of Fur partially, which is independent of the concentration of ferrous ion sensed by Fur.

siderophore-complexed ferric ion is likely to be utilized for the plant iron acquisition through this strategy.57) Collateral experimental proofs have been provided to support the physiological response that flavonoid exudation from roots is enhanced by iron deficiency.15,58) While flavonoids might act as chelating and reducing agents to improve the iron mobilization directly under iron-starved conditions,15), we hypothesize that plants encourage rhizobacteria such as B. subtilis to synthesize siderophores, having a much higher iron-chelating activity than flavonoids, through the active excretion of flavonoids, which cause derepression of the Fur regulon. The siderophores secreted into the rhizosphere can be utilized for iron acquisition not only by the producing bacteria but also by plants. Since iron is an essential co-factor of many proteins involved in major metabolic processes in plants, such as the energy-yielding electron transfer reactions of respiration and photosynthesis, sufficient iron supply to these proteins is expected to promote plant growth and the release of nutrients from roots, which are available for microbes in the rhizosphere. The flavonoid response of Fur should play a key role in this favorable relationship between plants and the relevant rhizobacteria (Fig. 1). It was also reported that B. subtilis serves to acidify the rhizosphere, which facilitates the solubilization of ferric ions, by increasing proton release from roots and by direct emission of volatile organic compounds, and that it up-regulates the plant’s own iron acquisition machinery by inducing the expression of a plant transcriptional factor involved in iron homeostasis.59) Thus, B. subtilis is likely to contribute to the plant iron uptake by these mechanisms as well as siderophore secretion.

III. Unique regulatory system for copper homeostasis through two transcriptional repressors, YcnK and CsoR, in B. subtilis In addition to the iron ion, the copper ion is an essential co-factor for many proteins involved in various physiological processes, such as respiration, oxidative stress response, and redox reactions in metabolic pathways. Two useful features of the copper ion as an active center of these proteins are that it is able to tightly bind to polar functional groups of the proteins and that it is

interconvertible between +1 and +2 oxidation states. However, these features also make the copper ion highly toxic when the free copper ion increases in the cell, as it undergoes undesirable redox reactions to generate reactive oxygen species and inappropriately binds to the other metal binding sites of the proteins, thereby altering their properties. Recent studies suggest that the cytotoxicity of the copper ion is primarily derived from its destabilizing effect on iron–sulfur clusters.60,61) To avoid the potential toxicity of the copper ion, organisms have evolved their systems to strictly control the cytoplasmic concentration of the copper ion as well as its intracellular trafficking to target proteins.62) Copper compounds are often used as a nutritional supplement for farm animals63) and a fungicide for various crop plants.64) Thus, the farmland soil might be at risk of copper contamination by the excrement of animals fed the excess copper supplement or by the excess spray of the copper fungicide, in addition to the contamination resulting from improper disposal of waste soil of cooper mining These copper contaminations are serious concerns about the impact on the ecosystem as well as the risk to human health raised by taking the crops grown in the highly polluted soil. Moreover, copper-resistant bacteria, which are equipped with the enhanced function of copper efflux, occasionally emerge from such coppercontaminated soils. Some of them have been subjected to study bacterial copper efflux systems.65) In B. subtilis, when the copper ion is in excess in its cytoplasm, a specific efflux system encoded by the copZA operon is induced.66) The CopA protein, belonging to the integral membrane protein family of P-type ATPases, functions to specifically translocate Cu+ across the cytoplasmic membrane, while CopZ is an Atx1-like soluble protein playing a role in the Cu+ transfer to the N-terminal domain of the CopA protein; this domain shows structural similarity to CopZ and transiently interacts with it when the transfer occurs.67–69) It seems that CopZ also serves as a copper chaperone with high binding affinity, so that the reactive free copper ion is kept at a very low level in the B. subtilis cell, as demonstrated for the yeast cell.70) Eukaryotic Sco proteins play a copper chaperone role in addition to their major role in the assembly of the two copper ions forming a dinuclear center (CuA) in subunit II of cytochrome c oxidase.71) However, the B. subtilis Sco protein is

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

1480

K. Hirooka

unlikely to act as a copper chaperone, although it fulfills a redox role in the CuA assembly.71) copZA transcription is controlled by a copper-sensing repressor, CsoR, dependent on copper availability within the cytoplasm.72,73) The gene encoding CsoR is located immediately upstream of the copZA operon and aligned in the same direction as copZA. The CsoR protein forms a tetramer, and two molecules of the CsoR tetramers cooperatively bind with high affinity to an operator site that includes a palindrome sequence (TACCcTACggggGTAtGGTA, mismatched bases are indicated in lower case letters) overlapping the copZA promoter. The DNA binding of CsoR is specifically inhibited by the addition of copper salts, and it was found that CsoR binds to one mol-equivalent Cu+ per monomer. These results indicate that the copZA is derepressed by the release of CsoR from the operator when the cytosolic copper ion is in excess (Fig. 5). Studies on copper resistance in P. syringae strains infective to tomato plants revealed that the copABCD operon, located on a plasmid, confers copper resistance and that it is regulated by the CopRS two-component system. The genes encoding CopRS are located immediately downstream from and in the same direction as copABCD, but the copRS genes are transcribed from a distinct constitutive promoter.74) Similar genes for copper resistance (pcoABCD) and for its regulatory system (pcoRS) were found in a gene cluster (pcoABCDRSE) on a plasmid of an E. coli strain that was isolated from

a copper-rich habitat.63) Despite the high similarity between these two systems, the mechanisms that afford copper resistance are quite different, i.e. the copper resistance of the E. coli pco system is achieved by an energy-dependent efflux of copper, whereas the mechanism taken by the P. syringae cop system is copper sequestration outside the cytoplasm.75) The B. subtilis YcnJ protein was found as a homolog of P. syringae CopCD; CopC and CopD are considered to be a periplasmic copper-binding protein and an inner membrane protein, respectively, and the N- and C-terminal parts of YcnJ show high homology to CopC and CopD.75–77) It was demonstrated that copCD expression in a P. syringae host, which does not have copper resistance, causes copper hypersensitivity and hyperaccumulation, suggesting that CopCD is also capable of copper uptake.76) In B. subtilis, disruption of ycnJ resulted in a growth-defective phenotype under copper limitation and a reduced intracellular copper content. Moreover, the ycnJ transcript increased greatly under copper limitation.77) These findings indicated that the primary role of B. subtilis YcnJ is associated with copper uptake. Northern blot and primer extension analyses revealed that ycnJ is a component of the ycnKJI operon and that the ycnL gene, located immediately upstream of the ycnKJI operon and oriented divergently, is monocistronic. The ycnK gene encodes a DeoR-type transcriptional factor, and, on the basis of sequence similarities,

Fig. 5. Regulatory system for homeostasis of the intracellular copper ion in B. subtilis. Note: The coding regions of the ycnKJI operon, the ycnL gene, the csoR gene, and the copZA operon are indicated by large open arrows; four promoters and four hairpin structures, probably functioning as ρ-independent transcription terminators, are indicated by bent arrows and stem loops, respectively. The YcnK protein, forming a dimer (two ovals), binds to the intergenic region between ycnK and ycnL to repress the ycnKJI operon effectively and the ycnL gene weakly. When the cytosolic copper ion (Cu+) is depleted, the binding affinity of YcnK is impaired, which leads to derepression of, at least, the ycnKJI operon. The YcnJ protein, predicted to possess nine transmembrane domains (http://bp.nuap.nagoya-u.ac.jp/so sui/),85) functions to import the copper ion from outside the cell. The CsoR protein forms a tetramer (four circles), and two molecules of the CsoR tetramer bind to the regulatory region of the copZA operon to repress its expression. When the cytosolic copper ion is in excess, CsoR is detached from the regulatory region, causing derepression of the copZA operon. The CopZ protein acts as a copper chaperone and transfers the copper ion (Cu+) to the N-terminal domain of the CopA protein. The CopA protein, predicted to possess seven transmembrane domains, functions to export the excess copper ion outside the cell, coupling with ATP hydrolysis (Copyright © American Society for Microbiology).78)

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

B. subtilis responses conducive to plant growth

the ycnI and ycnL genes are predicted to encode a membrane protein and a reductase or disulfide isomerase, respectively, although the actual functions of these gene products remain unclear. The DNA-binding experiments showed that the YcnK protein specifically binds to the ycnK-ycnL intergenic region including a 16-bp direct repeat (CACATTTTCACATTTT) that is essential for the high binding affinity of YcnK and that a copper-specific chelator, bathocuproine disulfonate, significantly inhibits the DNA binding of YcnK. Moreover, the lacZ-reporter assay showed that the ycnK promoter activity is elevated by ycnK disruption or copper limitation caused by the addition of bathocuproine disulfonate to the minimal medium. These results are consistent with YcnK’s functioning as a copper-responsive repressor that derepresses the ycnKJI expression under copper limitation. On the other hand, the ycnL promoter activity was hardly induced by copper limitation, but ycnK disruption resulted in a slight activation of the ycnL promoter, suggesting that YcnK also weakly represses the ycnL gene. While the CsoR protein did not bind to the ycnK-ycnL intergenic region, the lacZ-reporter assay demonstrated that csoR disruption increases the ycnK promoter activity only in the presence of intact ycnK and copZA genes. Since the copZA operon is involved in copper export and repressed by CsoR, it appears that csoR disruption indirectly enhances the ycnK promoter via CopZA and YcnK in such a manner that YcnK senses the intracellular depletion of the copper ion caused by overproduced CopZA (Fig. 5).78) According to its primary structure, YcnK is classified into the DeoR family of bacterial transcriptional factors.79) To date, many members of the DeoR family from various Gram-positive and Gram-negative bacteria have been characterized and were found to share several common features. They contain a highly conserved region near the N terminus that includes an HTH DNA-binding motif. YcnK also possesses this N-terminal region forming the DNA-binding domain, which shows high similarity to those of other members previously characterized.78,80) The C-terminal region of the DeoR family members is assumed to be responsible for oligomerization and effector binding. Most of the members thus far reported act as repressors involved in sugar and nucleoside metabolism, and their effectors are usually phosphorylated intermediates in the metabolic pathways that they control.80) These DeoR members possess a conserved region near their C terminus that is structurally related to E. coli D-ribose-5-phosphate isomerase, implying that this C-terminal region functions as the effector sensor.81) Distinct from these members, the Cterminal region of YcnK does not retain such a conserved sugar–phosphate recognition region; rather, it resembles the C-terminal regions of the NosL proteins, which are considered to act as a copper chaperone involved in the metallo-center assembly of nitrous oxide reductase.82) Thus, we speculate that the C-terminal region of the YcnK subunit functions to accommodate the copper ion probably as a monovalent cation. Our ortholog clustering search indicated that the ycnKJI operon is conserved in a narrow subgroup of the genus Bacillus, comprising B. subtilis, B. amyloliquefaciens, B. atrophaeus, and B. licheniformis (http://mbgd.genome.

1481

83)

Together with the characteristic C-terminal ad.jp/). region of YcnK, this limited conservation accentuates uniqueness of the control system of copper uptake by the ycnKJI operon in B. subtilis and its limited related species. While the bacterial copper-exporting systems have been extensively studied, a limited amount of information has been obtained about copper uptake systems in bacteria,84) which lends greater value to the findings on the ycnKJI operon.

IV. Conclusion and prospects As reported in our previous studies and described here, B. subtilis is the first Gram-positive bacterium that was demonstrated to possess multiple flavonoidresponsive transcriptional regulatory systems,21,32) while it was also reported that, in addition to the NodD system, rhizobia that are classified as Gram-negative bacteria possess the other transcriptional regulatory systems in response to flavonoids.33,34) The LmrA/QdoR system is first characterized in the genus Bacillus as the system that utilizes flavonoids as an effector molecule for the transcriptional induction of their regulon.21) Subsequently, the YetL system was selected as the candidate by the DNA microarray analysis, and it was found that YetL shows a flavonoid-responsive property that appears different from that of LmrA/QdoR.32) The DNA microarray analysis also suggested that Fur, originally known as an iron-sensing transcriptional repressor, responds to certain flavonoids. According to the results of the in vitro and in vivo characterizations, the flavonoid responsiveness of Fur is likely independent of the ferrous ion concentration (Hirooka, K., unpublished results), but further study by approaches such as mutational analysis and structural analysis is required to clarify the molecular mechanism of the response of Fur to flavonoids. While our attempt to find yet another flavonoid-responsive regulatory system from B. subtilis is still ongoing, it is also intriguing to examine whether regulatory systems analogous to the LmrA/QdoR and YetL systems exist in other Gram-positive rhizobacteria. The physiological roles of flavonoid-inducible LmrA/QdoR, YetL, and Fur regulons in the inhabitation of B. subtilis in the rhizosphere are roughly grasped, but not fully understood. Along with elucidation of the physiological significance of each regulatory system, contributions of these systems to the mutually beneficial interaction between B. subtilis and plants are to be evaluated by methods such as the co-cultivation of plants with B. subtilis mutants lacking either of these systems, aiming for their application to plant growth promotion in the future. The ycnKJI operon, which is involved in copper uptake in B. subtilis, is directly regulated by the copper-sensing repressor YcnK. Another copper-sensing repressor, CsoR, indirectly affects the expression of this operon via its direct target, copZA, in such a way that if CsoR unnecessarily releases the copZA repression causing copper depletion by the overproduced CopZA copper efflux system, YcnK senses the depletion and derepresses the ycnKJI expression for an increase in the YcnJ copper importer, leading to the maintenance of the intracellular copper availability at the proper

1482

K. Hirooka

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

78)

level. We expect that the copper homeostasis machinery of B. subtilis is applicable to a bioremediation for soil contaminated with copper using a B. subtilis strain carrying the ycnK and copA deletions. As the ycnK deletion constitutively releases the ycnJ repression, this strain can produce an excess amount of the YcnJ protein, through which the copper ion should be efficiently incorporated into the cells. At the same time, excretion of the copper ion should be interfered with by the deletion of copA encoding the copper exporter. Thus, the mutant strain is anticipated to highly accumulate copper ions in the cells. To alleviate cytotoxicity of a high concentration of the copper ion, the CopZ protein is expected to function as a copper chaperone to capture the free copper ion, the expression of which should be induced by the release of the repression by CsoR under high copper concentration. By spraying this strain onto the copper-contaminated soil, it is assumed that copper is prevented from being excessively incorporated into the plant roots cultivated there. If YcnJ also has the ability to import heavy metal ions other than copper, this bioabsorption system may be available for such metal ions. Of course, it is necessary to devise an effective strategy for blocking the excretion of these metal ions as well as for avoidance of their cytotoxicities.

[7]

[8]

[9] [10]

[11]

[12]

[13]

[14]

[15]

Acknowledgments I am profoundly grateful to Professor Yasutaro Fujita (Fukuyama University) for helpful advice and discussion and for continuous encouragement. I would like to acknowledge Professor Mamoru Yamada (Yamaguchi University) for recommending me for the JSBBA Award for Young Scientists. I also appreciate very much the past and present members of our laboratory (Fukuyama University) and all of our collaborators, in particular, Professor Ken-ichi Yoshida (Kobe University), Dr Takenori Satomura (Fukui University), Dr Hiroshi Matsuoka (Fukuyama University), Dr Satoshi Kunikane-Doi (Mikakuto Co., Ltd.), and Dr Shigeo Tojo (Hiroshima Institute of Technology), for their invaluable help.

[16]

[17]

[18]

[19]

[20] [21]

References [1] Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu. Rev. Plant Biol. 2006;57:233–266. [2] Carminati A, Vetterlein D. Plasticity of rhizosphere hydraulic properties as a key for efficient utilization of scarce resources. Ann. Bot. 2013;112:277–290. [3] Driouich A, Follet-Gueye ML, Vicré-Gibouin M, Hawes M. Root border cells and secretions as critical elements in plant host defense. Curr. Opin. Plant Biol. 2013;16:489–495. [4] Ortíz-Castro R, Contreras-Cornejo HA, Macías-Rodríguez L, López-Bucio J. The role of microbial signals in plant growth and development. Plant Signal Behav. 2009;4:701–712. [5] Fusconi A. Regulation of root morphogenesis in arbuscular mycorrhizae: what role do fungal exudates, phosphate, sugars and hormones play in lateral root formation? Ann. Bot. 2014;113:19–33. [6] Vacheron J, Desbrosses G, Bouffaud ML, Touraine B, MoënneLoccoz Y, Muller D, Legendre L, Wisniewski-Dyé F, Prigent-

[22]

[23]

[24]

[25]

Combaret C. Plant growth-promoting rhizobacteria and root system functioning. Front. Plant Sci. 2013;4:356. Kubo Y, Rooney AP, Tsukakoshi Y, Nakagawa R, Hasegawa H, Kimura K. Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production. Appl. Environ. Microbiol. 2011;77:6463–6469. Fall R, Kinsinger RF, Wheeler KA. A simple method to isolate biofilm-forming Bacillus subtilis and related species from plant roots. Syst. Appl. Microbiol. 2004;27:372–379. Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol. 2005;56:845–857. Nagórska K, Bikowski M, Obuchowski M. Multicellular behaviour and production of a wide variety of toxic substances support usage of Bacillus subtilis as a powerful biocontrol agent. Acta Biochim. Pol. 2007;54:495–508. Ochiai A, Itoh T, Kawamata A, Hashimoto W, Murata K. Plant cell wall degradation by saprophytic Bacillus subtilis strains: gene clusters responsible for rhamnogalacturonan depolymerization. Appl. Environ. Microbiol. 2007;73:3803–3813. Kumar AS, Lakshmanan V, Caplan JL, Powell D, Czymmek KJ, Levia DF, Bais HP. Rhizobacteria Bacillus subtilis restricts foliar pathogen entry through stomata. Plant J. 2012;72:694–706. Lakshmanan V, Kitto SL, Caplan JL, Hsueh YH, Kearns DB, Wu YS, Bais HP. Microbe-associated molecular patterns-triggered root responses mediate beneficial rhizobacterial recruitment in Arabidopsis. Plant Physiol. 2012;160:1642–1661. Narasimhan K, Basheer C, Bajic VB, Swarup S. Enhancement of plant-microbe interactions using a rhizosphere metabolomicsdriven approach and its application in the removal of polychlorinated biphenyls. Plant Physiol. 2003;132:146–153. Cesco S, Neumann G, Tomasi N, Pinton R, Weisskopf L. Release of plant-borne flavonoids into the rhizosphere and their role in plant nutrition. Plant Soil. 2010;329:1–25. Sugiyama A, Shitan N, Yazaki K. Involvement of a soybean ATP-binding cassette-type transporter in the secretion of genistein, a signal flavonoid in legume-Rhizobium symbiosis. Plant Physiol. 2007;144:2000–2008. Suzuki H, Takahashi S, Watanabe R, Fukushima Y, Fujita N, Noguchi A, Yokoyama R, Nishitani K, Nishino T, Nakayama T. An isoflavone conjugate-hydrolyzing β-glucosidase from the roots of soybean (Glycine max) seedlings: purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 2006;281:30251–30259. Hassan S, Mathesius U. The role of flavonoids in root-rhizosphere signalling: opportunities and challenges for improving plant-microbe interactions. J. Exp. Bot. 2012;63:3429–3444. Chen XC, Feng J, Hou BH, Li FQ, Li Q, Hong GF. Modulating DNA bending affects NodD-mediated transcriptional control in Rhizobium leguminosarum. Nucleic Acids Res. 2005;33:2540– 2548. Perret X, Staehelin C, Broughton WJ. Molecular basis of symbiotic promiscuity. Microbiol. Mol. Biol. Rev. 2000;64:180–201. Hirooka K, Kunikane S, Matsuoka H, Yoshida K, Kumamoto K, Tojo S, Fujita Y. Dual regulation of the Bacillus subtilis regulon comprising the lmrAB and yxaGH operons and yxaF gene by two transcriptional repressors, LmrA and YxaF, in response to flavonoids. J. Bacteriol. 2007;189:5170–5182. Murata M, Ohno S, Kumano M, Yamane K, Ohki R. Multidrug resistant phenotype of Bacillus subtilis spontaneous mutants isolated in the presence of puromycin and lincomycin. Can. J. Microbiol. 2003;49:71–77. Yoshida K, Ohki YH, Murata M, Kinehara M, Matsuoka H, Satomura T, Ohki R, Kumano M, Yamane K, Fujita Y. Bacillus subtilis LmrA is a repressor of the lmrAB and yxaGH operons: identification of its binding site and functional analysis of lmrB and yxaGH. J. Bacteriol. 2004;186:5640–5648. Gopal B, Madan LL, Betz SF, Kossiakoff AA. The crystal structure of a quercetin 2,3-dioxygenase from Bacillus subtilis suggests modulation of enzyme activity by a change in the metal ion at the active site(s). Biochemistry. 2005;44:193–201. Hirooka K, Fujita Y. Excess production of Bacillus subtilis quercetin 2,3-dioxygenase affects cell viability in the presence of quercetin. Biosci. Biotechnol. Biochem. 2010;74:1030–1038.

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

B. subtilis responses conducive to plant growth [26] Grkovic S, Brown MH, Skurray RA. Regulation of bacterial drug export systems. Microbiol. Mol. Biol. Rev. 2002;66:671– 701. [27] Ramos JL, Martinez-Bueno M, Molina-Henares AJ, Teran W, Watanabe K, Zhang X, Gallegos MT, Brennan R, Tobes R. The TetR family of transcriptional repressors. Microbiol. Mol. Biol. Rev. 2005;69:326–356. [28] Seetharaman J, Kumaran D, Bonanno JB, Burley SK, Swaminathan S. Crystal structure of a putative HTH-type transcriptional regulator yxaF from Bacillus subtilis. Proteins. 2006;63:1087–1091. [29] Hirooka K, Fujita Y. Identification of aromatic residues critical to the DNA binding and ligand response of the Bacillus subtilis QdoR (YxaF) repressor antagonized by flavonoids. Biosci. Biotechnol. Biochem. 2011;75:1325–1334. [30] Siedler S, Stahlhut SG, Malla S, Maury J, Neves AR. Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli. Metab. Eng. 2014;21:2–8. [31] Teran W, Krell T, Ramos JL, Gallegos MT. Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression. J. Biol. Chem. 2006;281:7102–7109. [32] Hirooka K, Danjo Y, Hanano Y, Kunikane S, Matsuoka H, Tojo S, Fujita Y. Regulation of the Bacillus subtilis divergent yetL and yetM genes by a transcriptional repressor, YetL, in response to flavonoids. J. Bacteriol. 2009;191:3685–3697. [33] Wenzel M, Lang K, Gunther T, Bhandari A, Weiss A, Lulchev P, Szentgyorgyi E, Kranzusch B, Gottfert M. Characterization of the flavonoid-responsive regulator FrrA and its binding sites. J. Bacteriol. 2012;194:2363–2370. [34] Rossbach S, Kunze K, Albert S, Zehner S, Göttfert M. The Sinorhizobium meliloti EmrAB efflux system is regulated by flavonoids through a TetR-like regulator (EmrR). Mol. Plant Microbe Interact. 2014;27:379–387. [35] Pande SG, Pagliai FA, Gardner CL, Wrench A, Narvel R, Gonzalez CF, Lorca GL. Lactobacillus brevis responds to flavonoids through KaeR, a LysR-type of transcriptional regulator. Mol. Microbiol. 2011;81:1623–1639. [36] Marin AM, Souza EM, Pedrosa FO, Souza LM, Sassaki GL, Baura VA, Yates MG, Wassem R, Monteiro RA. Naringenin degradation by the endophytic diazotroph Herbaspirillum seropedicae SmR1. Microbiology. 2013;159:167–175. [37] Perera IC, Grove A. Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators. J. Mol. Cell Biol. 2010;2:243–254. [38] Alekshun MN, Levy SB, Mealy TR, Seaton BA, Head JF. The crystal structure of MarR, a regulator of multiple antibiotic resistance, at 2.3 A resolution. Nat. Struct. Biol. 2001;8:710–714. [39] Dolan KT, Duguid EM, He C. Crystal structures of SlyA protein, a master virulence regulator of Salmonella, in free and DNA-bound states. J. Biol. Chem. 2011;286:22178–22185. [40] Saridakis V, Shahinas D, Xu X, Christendat D. Structural insight on the mechanism of regulation of the MarR family of proteins: high-resolution crystal structure of a transcriptional repressor from Methanobacterium thermoautotrophicum. J. Mol. Biol. 2008;377:655–667. [41] Davis JR, Brown BL, Page R, Sello JK. Study of PcaV from Streptomyces coelicolor yields new insights into ligand-responsive MarR family transcription factors. Nucleic Acids Res. 2013;41:3888–3900. [42] Duval V, McMurry LM, Foster K, Head JF, Levy SB. Mutational analysis of the multiple-antibiotic resistance regulator MarR reveals a ligand binding pocket at the interface between the dimerization and DNA binding domains. J. Bacteriol. 2013;195:3341–3351. [43] Tam LT, Eymann C, Albrecht D, Sietmann R, Schauer F, Hecker M, Antelmann H. Differential gene expression in response to phenol and catechol reveals different metabolic activities for the degradation of aromatic compounds in Bacillus subtilis. Environ. Microbiol. 2006;8:1408–1427. [44] Wang SC, Johnson WH Jr, Czerwinski RM, Stamps SL, Whitman CP. Kinetic and stereochemical analysis of YwhB, a 4-oxalocrotonate tautomerase homologue in Bacillus subtilis:

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56] [57]

[58]

[59]

[60]

[61]

[62]

[63]

[64] [65]

1483

mechanistic implications for the YwhB- and 4-oxalocrotonate tautomerase-catalyzed reactions. Biochemistry. 2007;46:11919– 11929. Shingler V, Powlowski J, Marklund U. Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. J. Bacteriol. 1992;174:711–724. Töwe S, Leelakriangsak M, Kobayashi K, Van Duy N, Hecker M, Zuber P, Antelmann H. The MarR-type repressor MhqR (YkvE) regulates multiple dioxygenases/glyoxalases and an azoreductase which confer resistance to 2-methylhydroquinone and catechol in Bacillus subtilis. Mol. Microbiol. 2007;66:40– 54. Lee J, Min KR, Kim YC, Kim CK, Lim JY, Yoon H, Min KH, Lee KS, Kim Y. Cloning of salicylate hydroxylase gene and catechol 2,3-dioxygenase gene and sequencing of an intergenic sequence between the two genes of Pseudomonas putida KF715. Biochem. Biophys. Res. Commun. 1995;211:382–388. Halbwirth H, Stich K. An NADPH and FAD dependent enzyme catalyzes hydroxylation of flavonoids in position 8. Phytochemistry. 2006;67:1080–1087. Ollinger J, Song KB, Antelmann H, Hecker M, Helmann JD. Role of the Fur regulon in iron transport in Bacillus subtilis. J. Bacteriol. 2006;188:3664–3673. Fillat MF. The FUR (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators. Arch. Biochem. Biophys. 2014;546:41–52. Baichoo N, Helmann JD. Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence. J. Bacteriol. 2002;184:5826–5832. Bsat N, Helmann JD. Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo. J. Bacteriol. 1999;181:4299–4307. May JJ, Wendrich TM, Marahiel MA. The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin. J. Biol. Chem. 2001;276:7209–7217. Ma Z, Faulkner MJ, Helmann JD. Origins of specificity and cross-talk in metal ion sensing by Bacillus subtilis Fur. Mol. Microbiol. 2012;86:1144–1155. Zawadzka AM, Kim Y, Maltseva N, Nichiporuk R, Fan Y, Joachimiak A, Raymond KN. Characterization of a Bacillus subtilis transporter for petrobactin, an anthrax stealth siderophore. Proc. Natl. Acad. Sci. USA. 2009;106:21854–21859. Kim SA, Guerinot ML. Mining iron: iron uptake and transport in plants. FEBS Lett. 2007;581:2273–2280. Nagata T, Oobo T, Aozasa O. Efficacy of a bacterial siderophore, pyoverdine, to supply iron to Solanum lycopersicum plants. J. Biosci. Bioeng. 2013;115:686–690. Zamboni A, Zanin L, Tomasi N, Pezzotti M, Pinton R, Varanini Z, Cesco S. Genome-wide microarray analysis of tomato roots showed defined responses to iron deficiency. BMC Genomics. 2012;13:101. Zhang H, Sun Y, Xie X, Kim MS, Dowd SE, Paré PW. A soil bacterium regulates plant acquisition of iron via deficiencyinducible mechanisms. Plant J. 2009;58:568–577. Macomber L, Imlay JA. The iron–sulfur clusters of dehydratases are primary intracellular targets of copper toxicity. Proc. Natl. Acad. Sci. USA. 2009;106:8344–8349. Chillappagari S, Seubert A, Trip H, Kuipers OP, Marahiel MA, Miethke M. Copper stress affects iron homeostasis by destabilizing iron–sulfur cluster formation in Bacillus subtilis. J. Bacteriol. 2010;192:2512–2524. Davis AV, O’Halloran TV. A place for thioether chemistry in cellular copper ion recognition and trafficking. Nat. Chem. Biol. 2008;4:148–151. Rouch DA, Brown NL. Copper-inducible transcriptional regulation at two promoters in the Escherichia coli copper resistance determinant pco. Microbiology. 1997;143:1191–1202. Mackie KA, Müller T, Kandeler E. Remediation of copper in vineyards—a mini review. Environ. Pollut. 2012;167:16–26. Altimira F, Yáñez C, Bravo G, González M, Rojas LA, Seeger M. Characterization of copper-resistant bacteria and bacterial

1484

[66]

[67]

[68]

[69]

Downloaded by [Middle Tennessee State University] at 02:34 17 November 2014

[70]

[71]

[72]

[73]

[74]

[75]

K. Hirooka communities from copper-polluted agricultural soils of central Chile. BMC Microbiol. 2012;12:193. Gaballa A, Helmann JD. Bacillus subtilis CPx-type ATPases: characterization of Cd, Zn, Co and Cu efflux systems. BioMetals. 2003;16:497–505. Banci L, Bertini I, Del Conte R, Markey J, Ruiz-Dueñas FJ. Copper trafficking: the solution structure of Bacillus subtilis CopZ. Biochemistry. 2001;40:15660–15668. Singleton C, Banci L, Ciofi-Baffoni S, Tenori L, Kihlken MA, Boetzel R, Le Brun NE. Structure and Cu(I)-binding properties of the N-terminal soluble domains of Bacillus subtilis CopA. Biochem J. 2008;411:571–579. Singleton C, Hearnshaw S, Zhou L, Le Brun NE, Hemmings AM. Mechanistic insights into Cu(I) cluster transfer between the chaperone CopZ and its cognate Cu(I)-transporting P-type ATPase. Biochem. J. 2009;424:347–356. Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV. Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science. 1999;284:805–808. Banci L, Bertini I, Cavallaro G, Ciofi-Baffoni S. Seeking the determinants of the elusive functions of Sco proteins. FEBS J. 2011;278:2244–2262. Smaldone GT, Helmann JD. CsoR regulates the copper efflux operon copZA in Bacillus subtilis. Microbiology. 2007;153:4123–4128. Ma Z, Cowart DM, Scott RA, Giedroc DP. Molecular insights into the metal selectivity of the copper(I)-sensing repressor CsoR from Bacillus subtilis. Biochemistry. 2009;48:3325–3334. Mills SD, Jasalavich CA, Cooksey DA. A two-component regulatory system required for copper-inducible expression of the copper resistance operon of Pseudomonas syringae. J. Bacteriol. 1993;175:1656–1664. Cha JS, Cooksey DA. Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins. Proc. Natl. Acad. Sci. USA. 1991;88:8915–8919.

[76] Cha JS, Cooksey DA. Copper hypersensitivity and uptake in Pseudomonas syringae containing cloned components of the copper resistance operon. Appl. Environ. Microbiol. 1993;59:1671–1674. [77] Chillappagari S, Miethke M, Trip H, Kuipers OP, Marahiel MA. Copper acquisition is mediated by YcnJ and regulated by YcnK and CsoR in Bacillus subtilis. J. Bacteriol. 2009;191:2362–2370. [78] Hirooka K, Edahiro T, Kimura K, Fujita Y. Direct and indirect regulation of the ycnKJI operon involved in copper uptake through two transcriptional repressors, YcnK and CsoR, in Bacillus subtilis. J. Bacteriol. 2012;194:5675–5687. [79] Zeng G, Ye S, Larson TJ. Repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K-12: primary structure and identification of the DNA-binding domain. J. Bacteriol. 1996;178:7080–7089. [80] Mortensen L, Dandanell G, Hammer K. Purification and characterization of the deoR repressor of Escherichia coli. EMBO J. 1989;8:325–331. [81] Anantharaman V, Aravind L. Diversification of catalytic activities and ligand interactions in the protein fold shared by the sugar isomerases, eIF2B, DeoR transcription factors, acyl-CoA transferases and methenyltetrahydrofolate synthetase. J. Mol. Biol. 2006;356:823–842. [82] McGuirl MA, Bollinger JA, Cosper N, Scott RA, Dooley DM. Expression, purification, and characterization of NosL, a novel Cu(I) protein of the nitrous oxide reductase (nos) gene cluster. J. Biol. Inorg. Chem. 2001;6:189–195. [83] Uchiyama I. MBGD: microbial genome database for comparative analysis. Nucleic Acids Res. 2003;31:58–62. [84] Rademacher C, Masepohl B. Copper-responsive gene regulation in bacteria. Microbiology. 2012;158:2451–2464. [85] Hirokawa T, Boon-Chieng S, Mitaku S. SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics. 1998;14:378–379.

Transcriptional response machineries of Bacillus subtilis conducive to plant growth promotion.

Bacillus subtilis collectively inhabits the rhizosphere, where it contributes to the promotion of plant growth, although it does not have a direct sym...
535KB Sizes 0 Downloads 4 Views