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Institute of Oceanic Research and Development, Tokai University, 3-20-1 Orido-Shimizu, Shizuoka 424-8610, Japan
Correspondence
Mitsuo Ogura
oguram{at}scc.u-tokai.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary figure showing the structure of plasmid pDG-N17 is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The catalytic part of the kinase autophosphorylates a histidine residue in response to an environmental signal. The phosphoryl group is then transferred to a conserved aspartate residue on the cognate response regulator, which acts as a transcription factor in most cases.
In the Bacillus subtilis genome, 35 response regulators, and 30 operonic kinase-regulator pairs, have been identified (Kunst et al., 1997). Following the completion of genomic sequencing, several newly identified operons that encode two-component systems have been characterized, e.g. the BceRS, CitST, CssSR, DesKR, GlnKL, LiaRS, MalKR, YdbFG, YdfHI, YxdJK and YycFG systems (Aguilar et al., 2001; Asai et al., 2000; Doan et al., 2003; Fukuchi et al., 2000; Hyyrylainen et al., 2001; Joseph et al., 2004; Mascher et al., 2004; Ohki et al., 2003; Satomura et al., 2005; Serizawa & Sekiguchi, 2005; Tanaka et al., 2003; Yamamoto et al., 2000). In addition, close relationships have been identified between some operons encoding two-component systems and genes for ATP-binding cassette (ABC) transporter homologues, e.g. YccGH, YclJK, YfiJK, YvcPQ, YvfTU and YxdJK (Joseph et al., 2002).
Comprehensive DNA microarray analyses of response regulators, using gene amplification in a sensor-deficient strain, have contributed to the identification of the target genes for these systems (Kobayashi et al., 2001; Ogura et al., 2001). In those studies, the NatR (YccH) response regulator was identified as an activator for the natAB operon encoding a two-gene ABC transporter involved in Na+ extrusion (Cheng et al., 1997; Kobayashi et al., 2001). This ABC transporter is a member of a small subfamily named ABC-2, which includes the two-component daunomycin- and doxorubicin-efflux system in Streptomyces peucetius Drr (Guilfoile & Hutchinson, 1991; Reizer et al., 1992). It has been reported that NatAB has a physiological role in Na+ resistance at alkaline pH, since a high concentration of cellular Na+ is toxic to the cell, and plays a role in ethanol resistance at neutral pH (Cheng et al., 1997; Padan & Schuldiner, 1994).
In this paper, we investigated whether or not NatK–NatR (formerly YccG–YccH) positively regulates the natAB operon. Gel retardation and footprint assays revealed direct binding of NatR to the promoter of natAB. Furthermore, we investigated the role of a direct repeat sequence, located within the natA promoter, in NatR recognition.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. One-step competence medium (MC) was used for the determination of 
-galactosidase activity (Ogura & Tanaka, 1996; Ogura et al., 1997). TKM medium was used for growth tests in the presence of NaCl or ethanol (Cheng et al., 1997). Escherichia coli JM103 cells for DNA manipulation were grown in Luria–Bertani (LB) medium. Concentrations of antibiotics used have been described previously (Ogura & Tanaka, 1996).
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, respectively. The vector pMutInIII (Vagner et al., 1998), and a PCR product spanning the 310 bp upstream region from the first nucleotide of the 24th codon of natA, prepared using the natA-FH and natA-RB primers, were digested with HindIII and BglII, and ligated to form pMutIn-natA. To construct pDG-N17 (see supplementary Fig. S1, available with the online version of this paper), pDG-His-degU (Shimane & Ogura, 2004) was digested with BamHI, and the large fragment was ligated with a double-stranded oligonucleotide, made by annealing single-stranded NotI-I and NotI-II oligonucleotides, after which the reaction mixture was used to transform E. coli. The presence of the 6x His tag, followed by the BamHI–NotI sites, on pDG-N17 was confirmed by sequencing using a 377 DNA Sequencer (Perkin-Elmer), and a Dye Terminator Cycle Sequencing Kit (Applied Biosystems). In order to construct pDG-N17-natR, which has a histidine tag at the N-terminus of NatR, pDG-N17 and a PCR product, prepared using the NatR-Bam and NatR-NotI primers, were digested with BamHI and NotI, and ligated. Sequencing of the cloned fragment in pDG-N17-natR confirmed the absence of PCR amplification errors. The plasmid pPhl2 (Ogura et al., 1997), and a PCR product prepared using the natK-E and natK-Sa primers, were digested with EcoRI and SalI, and ligated to form pPhl2-natK. PCR products were prepared using the primer pairs NatA-pISB1 and NatA-pISH, NatA-pISB2 and NatA-pISH, NatA-pISB2M and NatA-pISH, NatA-pISB3 and NatA-pISH, and YccK-pISB and YccK-pISH. The fragments were digested with BamHI and HindIII, and ligated into the similarly digested pIS284, in order to construct pIS-natA1, pIS-natA2, pIS-natA2M, pIS-natA3 and pIS-yccK, respectively. The plasmid pIS-natA1M was constructed by site-directed mutagenesis of pIS-natA1 using the oligonucleotide-based PCR method described previously (NatA-pISB1, NatA-M1, NatA-M2 and NatA-pISH; Ogura & Tanaka, 1996). The sequences cloned into pIS284 were confirmed by sequencing.
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Primer-extension analysis.
The isolation of total RNA, and primer-extension analysis, were performed as described previously (Yoshida et al., 2001; Ogura & Tanaka, 1996; respectively). Sequencing was performed using a Cycle sequencing kit (Toyobo), and detection of biotinylated DNA was performed using a chemiluminescence DNA detection kit (Toyobo).
Protein purification.
The induction and purification procedures for preparation of protein expressed in E. coli have been described previously (Ogura et al., 2003; Shimane & Ogura, 2004). His-tagged NatR was expressed as a soluble protein in E. coli M15 cells carrying pRep4, and step-wise elution with imidazole from a Ni-affinity column was used for the purification. Following SDS-PAGE analysis of fractions, the purified protein was dialysed against TEDG buffer (Mukai et al., 1990).
Gel shift and DNase I footprinting assays.
We used biotinylated DNA probes for the gel shift assay (Ogura et al., 2003); the DNase I footprinting assay was carried out as described previously (Hayashi et al., 2005).
-Galactosidase assays.
Samples were taken at hourly intervals, and the level of -galactosidase activity was determined as described previously (Ogura & Tanaka, 1996).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Northern analysis has indicated that natA forms an operon with natB, but not with yccK located immediately downstream of natAB (Cheng et al., 1997). There is no ORF immediate downstream of yccK on the same DNA strand, and this strongly suggests that yccK is monocistronic. This notion was reinforced by two observations. (1) The presence of a putative rho-independent terminator downstream of natB (Fig. 1a; Cheng et al., 1997). (2) The promoter activity of the yccK upstream region at an ectopic amyE locus; yccK shows a temporal expression pattern that is different from that of natA–lacZ (data not shown). YccK is predicted to encode a soluble protein with similarity to members of the aldo/keto reductase family (BSORF; http://bacillus.genome.jp/).
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)] expressing natR was introduced into the natK strain carrying a transcriptional natA–lacZ or yccK–lacZ fusion. Each of the fusions was generated by Campbell-type recombination of derivatives of the plasmid pMutIn (Table 1). Next, -galactosidase activities were examined in the presence and absence of IPTG. The induction of natR by the addition of IPTG resulted in 35- and 5-fold increases in the levels of expression of natA–lacZ (Fig. 1b) and yccK–lacZ (data not shown), respectively. Thus, the observations obtained with the lacZ fusions confirmed the results of DNA microarray analysis. However, the promoter of yccK was not a target of NatR, because induction of natR on pDG148-natR did not enhance the expression of yccK–lacZ at the amyE locus (data not shown). Thus, the apparent induction of yccK expression detected by microarray analysis was most probably caused by artificial read-through of the yccK gene resulting from the increased activity of the natAB promoter under the condition of natR overexpression. In addition, in the presence of an intact natK gene, induction of natR on pDG148-natR resulted in a ninefold increase in the expression of natA–lacZ (Fig. 1b).
natAB is located adjacent to natRK, but on the complementary strand (Fig. 1a). The chromosomal structure of the region that contains the transcription control elements for natRK and natAB raised the possibility that natR and natK genes might be part of an autoregulatory loop under the control of NatR. To examine this, we introduced pDG148-natR into cells carrying natR–lacZ, and measured -galactosidase activities in the presence and absence of IPTG. We did not observe any change in the expression of natR–lacZ when the natR gene was induced by IPTG (data not shown). This indicates that natR expression itself is not regulated by NatR.
Expression of natA was abolished in the mutants natK and natR
To examine the notion that natR is a positive regulator of natAB, we measured the -galactosidase activities of the fusions natA–lacZ in wild-type, natK and natR strains. The expression level of natA–lacZ in the wild-type cells was significantly high compared with that in cells carrying the same fusion and pDG148-natR, without addition of IPTG (Fig. 1b, c). The reason for this is unknown; however, it is possible that addition of kanamycin affects the expression level of the fusion. The expression of natA–lacZ was almost abolished in both natK and natR backgrounds (Fig. 1c). Thus, we concluded that natK and natR are required for natA expression. In addition, the expression of yccK–lacZ at the yccK locus was not affected by the disruption of natR, reinforcing the notion that yccK is not a member of the NatR regulon (data not shown).
Functional analysis of the natR strain
It was expected that introduction of the natR disruption mutation would decrease Na+ resistance at alkaline pH, and ethanol resistance at neutral pH, because NatR positively regulates the natAB operon, whose product is responsible for the resistant phenotypes (Cheng et al., 1997). Thus, we examined the growth properties of the natR and natA strains in the presence or absence of 200 mM NaCl in TKM medium. The medium composition, pH, concentration of NaCl or ethanol, and culture conditions, including temperature and culture volume, were the same as those used by Cheng et al. (1997), with the exception that we used B. subtilis 168 as a control strain, and not the BD99 strain, which is a derivative of 168. As shown in Fig. 2(b, d), the natA and natR strains showed a delay in growth, and a low cell density at the end of the exponential phase, compared with the control strain (Fig. 2a, c), indicating that NaCl sensitivity at alkaline pH, and ethanol sensitivity at neutral pH, were higher in both strains. These observations confirmed our prediction regarding the phenotypes of the natR strain. The natK strain showed similar phenotypes to the natR strain under both alkaline and neutral conditions (data not shown). We used strain 168, which was more resistant to NaCl at alkaline pH, and ethanol at neutral pH, than BD99. Subtle genetic differences between our strain and the one used by Cheng et al. (1997) may explain these differences.
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), which was grown in MC medium with and without 1 mM IPTG. According to the above results, natAB expression should be enhanced by the addition of IPTG. The transcription start was clearly indicated by a band that was detected using a natA-specific primer in the reaction with RNA from the cells grown with 1 mM IPTG, and not from those grown without IPTG (Fig. 3). Using the RNA from the wild-type cells without multicopy natR, we observed a faint band of the same size as that in Fig. 3, indicating that natA transcription starts at the same nucleotide position, irrespective of the presence of multicopy natR (data not shown).
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). The binding was specific, because the reaction contained a 500-fold excess amount of poly dI-dC.
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). These results are consistent with the observations made using gel retardation. The commonly protected region of both strands contains a direct repeat of 5'-TTCRCGACA-3', separated by 12 bp. This relatively long space between directly repeated motifs is characteristic of the LytTR family of DNA-binding proteins (see Discussion).
Confirmation that the direct repeat in the natA promoter region acts as a positive cis-element for NatR
Transcriptional lacZ fusions were constructed using three distinct promoter regions, with different 5' ends of natA, at the amyE locus. The common 3' end of the promoter regions was 1 nt upstream from the initiation codon. Since the fusion expression levels were very low, we introduced multicopy natR on pDG148-natR, driven by an IPTG-inducible promoter. The -galactosidase activities of the strains were examined in the presence and absence of IPTG. As expected, a high level of expression of the fusions carrying the direct repeat recognized by NatR, i.e. natA1–lacZ (–116 to +27) and natA2–lacZ (–79 to +27), was induced by the addition of IPTG (Fig. 5). The deletion of the –116 to –79 region resulted in a 2.5-fold increase in fusion expression when natR was overexpressed, suggesting that there is a negative regulatory region in this region. However, the expression of natA3–lacZ (–57 to +27), which carries the downstream repeat only, was not induced by IPTG. This indicates that the complete direct repeat is required for expression of natA. Further, we mutagenized both of the direct repeat sequences in the fusions, and examined their -galactosidase activities. The expression of both fusions carrying disruptions in the direct repeat was severely decreased in the presence of IPTG compared with the reference strains. These results demonstrate that the direct repeat, recognized by NatR, is important for the regulation of natA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In the several B. subtilis two-component regulatory systems, i.e. DegS–DegU, ComP–ComA and PhoP–PhoR, multicopy of the response regulator gene can only induce the expression of the target genes when the cognate sensor kinase gene is absent (Ogura et al., 2001). In the NatK–NatR system, however, the multicopy natR gene was able to enhance the expression of natA–lacZ, even in the presence of the cognate natK gene, with a slightly decreased level compared with that obtained in the absence of natK (Fig. 1b). The discrepancy might be due to the nature of the sensor kinase. For example, it is known that some sensor kinases have both kinase and phosphatase activities for the cognate response regulator, while, on the other hand, other kinases lack the phosphatase activity (Stock et al., 2000).
The NatK sensor kinase has three transmembrane domains, and is classified as a group IIIA kinase (Fabret et al., 1999). Generally, the kinases in this group are paired with group IIIA regulators. However, NatR is distinct from the other members of the group because it has characteristics that are specific to the LytTR family; these characteristics include response regulators involved in the biosynthesis of extracellular polysaccharides, fimbriation, expression of exoproteins (including toxins), and quorum sensing (Nikolskaya & Galperin, 2002). The regulators in this family have been reported to bind to imperfect direct repeat sequence patterns composed of nine nucleotides [T/A] [A/C] [C/A]GTTN[A/G] [T/G] separated by a 12–13 bp spacer. Although the NatR-binding sequence that was identified, i.e. TTCACGACA-N12-TTCGCGACA, does not contain this sequence, it does maintain the 9-12-9 bp structure. This conforms to the idea that regulators within the same protein family may have evolved to recognize different sequence motifs.
In the footprint analysis, NatR protected not only the upstream region, but also the core promoter region of natA on the top strand, from the DNase I digestion. Since this region does not contain the NatR-recognition sequence, protection might be generated by the spatial proximity of DNA-bound NatR to the core promoter region. This raised the possibility that NatR may interact with RNA polymerase in the natA promoter, leading to activation of natAB transcription, although how NatR activates natAB expression remains unknown. In the gel retardation assay, at the highest concentration of the protein (1.2 µM), a significant amount of the probe was not bound by NatR. Similarly, in the footprint analysis, a large amount of NatR (2.0 µM) was required for the clear footprint pattern. These results suggested that a significant portion of purified protein was not active.
To examine the possibility that other genes might be directly regulated by NatR, we searched the NatR-recognition sequence in the B. subtilis genome using GRASP-DNA sequence software (Schilling et al., 2000). However, we did not find a candidate target of NatR.
It has been reported that the expression of natAB–lacZ is induced fourfold by the addition of 2 % ethanol (Cheng et al., 1997). The expression of the fusion was measured in the natB background (Cheng et al., 1997). We also observed the induction of natA–lacZ expression by ethanol in natA cells; however, there was no ethanol induction in the wild-type cells (unpublished results). It is speculated that the lack of NatAB may perturb cellular ionic homeostasis, which affects the activity of the natAB promoter.
The basal expression of the natA1–lacZ fusion at the amyE locus was very low compared with the natA–lacZ fusion in the natural context. There could be several possible reasons for this: (1) some regulatory cis-element might be lost in natA1–lacZ, since –116 relative to the transcription start site is the 5' end of the fusion; (2) a local topology of the natA promoter region might be changed in the natA fusions at the amyE locus, leading to low expression; (3) different mRNA structures of the two fusions might result in altered mRNA stability, leading to different expression levels. However, the reason for the discrepancy is yet to be determined.
Response regulators demonstrating close similarities to NatR have been found in many bacterial genomes, including those of E. coli, Bacillus halodurans, Shigella flexneri and Streptococcus thermophilus. However, we investigated over 20 genomes, among which only the Str. thermophilus genome has conserved the genomic structure as shown in Fig. 1(a) (Bolotin et al., 2004). In the case of Str. thermophilus LMG18311, the LytTR-type response regulator (stu0543) is associated with a putative ABC-transporter for extrusion of an unknown substrate.
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ACKNOWLEDGEMENTS |
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Edited by: J. M. van Dijl
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REFERENCES |
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Received 23 October 2006;
revised 13 December 2006;
accepted 14 December 2006.
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