1 Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
2 Génétique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France
Correspondence
François Denizot
denizot{at}ibsm.cnrs-mrs.fr
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ABSTRACT |
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Present address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
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INTRODUCTION |
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Genomic sequencing of several micro-organisms has revealed great diversity of the TCS repertory in many species. This fact reflects the capability of some organisms to respond to a wide range of environmental changes. Bacillus subtilis, a Gram-positive spore-forming soil bacterium, possesses more than 30 such TCSs (36 sensor kinases and 35 response regulators; Fabret et al., 1999). The largest group, the IIIA/OmpR family, comprises 14 systems, to only four of which have known functions been attributed. The PhoP/PhoR and ResD/ResE systems participate in the response to phosphate starvation (Hulett et al., 1994
), the latter system playing a central role in aerobic and anaerobic respiration (Sun et al., 1996
). The YycF/YycG system is an essential two-component regulator of B. subtilis growth that modulates ftsAZ operon expression (Fukuchi et al., 2000
), and the bceRS (formerly ytsAB) system plays a role in resistance to bacitracin (Bernard et al., 2003
; Mascher et al., 2003
; Ohki et al., 2003
).
As exporters or importers of a wide variety of compounds across the membrane (Ames, 1986; Higgins et al., 1986
), ABC (ATP-binding cassette) transporters play a key role in the response of bacteria to environmental changes. The prototypic ABC transporter comprises two membrane-spanning domains and two cytoplasmic nucleotide-binding domains (NBDs) that bind and hydrolyse ATP to provide energy for the transport. The inventory and classification of B. subtilis ABC transporters indicated that among the 59 systems predicted as ABC transporters more than 60 % are of unknown function (Quentin et al., 1999
).
We recently demonstrated genetic and functional relationship between some members of the IIIA/OmpR family of TCSs and of subfamily 9 of ABC transporters in B. subtilis (Joseph et al., 2002), the TCS structural genes, yxdJK, yvcPQ and bceRS (formerly ytsAB), controlling the expression of the cognate ABC transporter genes yxdLM, yvcRS and bceAB (formerly ytsCD), respectively. In addition, the BceR/BceS TCS, together with the BceA/BceB ABC transporter, were shown to participate in bacitracin resistance of this bacterium (Bernard et al., 2003
; Mascher et al., 2003
; Ohki et al., 2003
).
We have focused our study on the yxd locus. The operon encoding these ABC transporter structural genes contains an additional gene, yxeA, which encodes an 80 aa peptide conserved in several bacteria of the Bacillus/Clostridium group. The goal of the present work was to characterize the promoter region of the yxdLMyxeA operon and to identify other genes regulated by YxdJ. We showed that YxdJ directly interacts with DNA upstream of the yxdL gene, but this is the only strongly regulated transcript which we detected.
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METHODS |
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Primer extension analysis.
Primer extension reactions were carried out with 550 µg of total RNA, 10 pmol of one of the 33P end-labelled primers YxdL_EA1 or YxdL_EA2, and 200 units of SuperScript II RNaseH reverse transcriptase (Gibco-BRL) in 20 µl of 1x first-strand buffer (Gibco-BRL) containing 10 mM dithiothreitol, 0·5 mM each deoxyribonucleotide triphosphate (Amersham Pharmacia Biotech). 5' end labelling of oligonucleotide was done using T4 polynucleotide kinase (Amersham Pharmacia Biotech) and [-33P]ATP (Amersham Pharmacia Biotech). Primer extension products were analysed by electrophoresis on a 6 % polyacrylamide/6 M urea gel, alongside a sequencing ladder (lanes C, T, G and A) obtained with the same end-labelled primer and the yxdL promoter PCR fragment as template. Sequencing reactions were done with Sequenase (United States Biochemical).
Deletion analysis of the yxdLMyxeA promoter.
DNA fragments were PCR-amplified using genomic B. subtilis DNA as template and were cloned upstream of lacZ in plasmid pGE593 (Eraso & Weinstock, 1992). The PCR products were generated using a common primer (YxdKL_right) and one of the following specific primers: for fragment A (281, 159), YxdKL_left_A; for fragment B (94, 159), YxdKL_left_B; for fragment C (69, 159), YxdKL_left_C; and for fragment D (30, 159), YxdKL_left_D. yxdJ was amplified with Yxdj_dir and Yxdj_rev and then cloned in pBAD33 (Guzman et al., 1995
) under the control of an arabinose-inducible promoter. Both recombinant plasmids were introduced into E. coli.
-Galactosidase activities were then measured in the recombinant bacteria grown in the presence of either glucose (repression of yxdJ expression) or arabinose (induction of yxdJ expression).
Overexpression and purification of His-tagged YxdJ protein from B. subtilis.
yxdJ was amplified from B. subtilis genomic DNA as template using Yxdj_dir and Yxdj_rev primers. The DNA fragment obtained was purified and cut with EagI. It was then cloned in a modified version of pET22b+ (Novagen) linearized with PmlI. To get the modified pET22b+, an adapter, obtained by hybridization of the two primers pET_his_1 and pET_his_2, was cloned between the NdeI/EcoRI sites of pET22b+. This construction introduces a PmlI cloning site into this vector and also allows addition of several codons at the 5' end of the cloned gene, creating a His-tag at the N-terminus of the protein to be produced. The resulting plasmid was introduced into E. coli BL21/DE3. The recombinant His-tagged YxdJ protein was thus overproduced and purified on Ni-NTA resin (Qiagen). Resin-bound His-tagged YxdJ was washed with buffer A (HEPES 10 mM, NaCl 150 mM, pH 7·4) containing 30 mM imidazole. The protein was eluted with buffer A containing 300 mM imidazole. After dialysis against buffer A containing 10 % (v/v) glycerol, the tagged protein was stored at 80 °C.
Gel mobility shift assay.
DNA fragments were obtained by PCR amplification using one of the YxdKL_left primers (A, B, C or D) and the YxdKL_right primer with B. subtilis genomic DNA as template. A nonspecific DNA fragment, chosen within the yxdL gene, was PCR-amplified with primers Yxdl1 and Yxdl2. Fifty nanograms each of DNA fragment (A, B, C or D) and nonspecific DNA fragment were mixed together with variable amount of purified His-tagged YxdJ protein in 50 mM Tris/HCl (pH 8), 1·25 mM EDTA, 0·25 M sucrose and 0·025 % bromophenol blue. The mixture (4 µl final volume) was incubated for 30 min at room temperature and loaded on a native 12·5 % acrylamide Phast gel (PhastSystem from Amersham Pharmacia Biotech). After migration the gel was soaked in an aqueous ethidium bromide solution (0·5 µg ml1) for 5 min and then observed on a UV transilluminator.
DNase I protection assay.
The DNA fragment obtained by PCR amplification of B. subtilis genomic DNA with primers (YxdKL_right_1 and YxdKL_F) was cloned into SmaI-linearized pBluescript KS (Promega) and the sequences of the recombinant clones were verified. Labelling of the DNA fragment used for DNAse I footprinting was done as follows. PCR-amplified fragment obtained with primers YxdKL_F and PBS-X was 5'-end-labelled with [-32P]ATP (4000 Ci mmol1, 150 TBq mmol1; NEN) and T4 polynucleotide kinase (Promega). Unincorporated nucleotides were removed using Nucleotide Removal Kit (Qiagen) following the recommendation of the manufacturer. Once purified, the labelled DNA fragment was digested with BamHI and subjected to treatment with the Qiaquick PCR purification kit (Qiagen). Then 5x104 c.p.m. purified labelled DNA fragment diluted to a concentration of 1·5 nM was incubated with His-tagged YxdJ for 30 min at room temperature in 50 µl binding buffer containing 10 mM Tris/HCl pH 7·5, 50 mM NaCl, 2·5 mM MgCl2, 0·5 mM dithiothreithol, 4 % glycerol and 1·5 µg poly(dI-dC).poly(dI-dC). The DNAprotein complexes were treated with 1 unit DNase I (Amersham Pharmacia Biotech) for 1 min at room temperature. The reaction was stopped by addition of a solution containing 192 mM sodium acetate, 32 mM EDTA, 0·14 % SDS and 64 µg yeast RNA ml1. The samples were extracted by a phenol/chloroform treatment and, after ethanol precipitation, they were resuspended in conventional loading buffer. After denaturation, the samples were loaded on a 6 M urea/8 % polyacrylamide gel together with a G+A Maxam and Gilbert reaction done on the same labelled DNA fragment.
Global transcriptional analysis.
Fluorescently labelled cDNA was synthesized during reverse transcription of BSmrs112 and BSmrs139 RNA (10 µg) using Cyanine-modified dCTP. The reaction mixture contained (in 40 µl): 20 µg random primers (GibcoBRL); 1x first-strand buffer (GibcoBRL); 10 mM dithiothreitol (Amersham Pharmacia Biotech); 100 µM (each) dATP, dTTP and dGTP; 50 µM dCTP; 25 µM Cy (3 or 5)-dCTP (Amersham Pharmacia Biotech) and 200 units Superscript II (GibcoBRL). cDNA synthesis was carried out at 42 °C for 1 h and continued for a further 1 h after another addition of 200 units Superscript II. RNA was subjected to alkaline hydrolysis by adding NaOH to a final concentration of 0·05 M, and incubating at 70 °C for 10 min. The mixture was neutralized with HCl (0·05 M final concentration). Unincorporated Cy (3 or 5)-dCTP was removed using Microcon-30 (Millipore). The labelled cDNA was diluted in 450 µl water, and then loaded on the Microcon-30 filter. The filter was washed three times by adding 540 µl water followed by centrifugation (8 min at 12 000 r.p.m.). cDNA was recovered by inverting the filter and centrifuging it for 3 min at 12 000 r.p.m.
Fluorescently labelled cDNAs from the two strains were mixed and used to hydridize to the same microarray (Eurogentec). Microarrays were first pre-hybridized for 1 h at 42 °C in 25 µl Dig Easy buffer (Roche) containing 10 µg salmon sperm DNA (Sigma) previously denatured for 5 min at 95 °C. Hybridizations were done in a final volume of 25 µl containing a mixture of the labelled cDNA at a final concentration of 1 µg ml1. Each slide was incubated in water-bath using a waterproof hybridization chamber (Corning). Slides were scanned on a ScanArray 4000 (Packard Bioscience) and hybridization signals were quantified with QuantArray software version 2.1 (Packard Bioscience).
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RESULTS |
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We therefore used the strain BSmrs112, in which the overproduction of the YxdJ response regulator mimics the unknown stimulus of the YxdJ/YxdK TCS and triggers the expression of the yxdLMyxeA operon (Joseph et al., 2002). Using an RNA preparation from this strain, the yxdLMyxeA transcription start site was identified; it corresponds to an adenine located 87 bp upstream from the putative translation initiation codon of yxdL (Fig. 1
). Upstream of this transcription start, the sequences corresponding to an extended
A-binding site (TGXTAATAT), and a 35 region (Helmann, 1995
; Jarmer et al., 2001
) were found.
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Characterization of the YxdJ binding site by DNase I protection assay
To define the YxdJ binding site more precisely, DNase I footprint experiments were performed using a fragment corresponding to positions 178 to +160 with respect to the yxdL transcriptional start site. This fragment encompasses fragment B and a part of fragment A (Fig. 2). As shown in Fig. 3
, a 38 bp region of the minus-strand, extending from base 41 to 78, was efficiently protected by YxdJ from DNase I digestion. Analysis of the protected region sequence revealed a 9 nucleotide direct repeat, TTAMRAAAA. Spacing of 21 nucleotides between the repeats indicates that they lie on the same side of the DNA helix, a feature that is expected from regulatory regions controlled by the OmpR-subfamily members acting as multimers (Makino et al., 1988
; Rampersaud et al., 1989
; Tsung et al., 1989
). Thus, the YxdJ binding site extends between nucleotides 41 to 78 in the regulatory region of the yxdLMyxeA operon, and contains two direct repeats.
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Previous experiments done by real-time PCR (Joseph et al., 2002) showed that neither the yxdK gene, encoding the histidine kinase partner of YxdJ, nor the yvcR gene, encoding an ABC transporter NBD, was induced upon YxdJ overexpression. These results are totally different from those obtained using the microarray approach and we believe that the latter are artefactual. First, to construct the recombinant plasmid for YxdJ overproduction we used a DNA fragment containing 29 bp that overlap with the 5' end of the yxdK gene. Thus, a large amount of an mRNA containing this overlap is produced when overproducing YxdJ. After reverse transcription and labelling, hybridization of the 29 bp fragment to the yxdK probe might easily occur on the microarray and produce the artefactual signal. Second, among the NBD-encoding genes, yvcR is by far the best yxdL homologue in B. subtilis (62·1 % identity at the DNA sequence level). In addition, the yvcR and yxdL DNA sequences contain long stretches of identical nucleotides. Thus, in the conditions of high-level yxdJ mRNA production, cross-hybridization between the yxdL labelled target and the yvcP probe might occur.
The dltA and dltD genes, involved in alanination of lipoteichoic and teichoic acids, are also induced. Using longer IPTG induction times, we have observed that dltB, dltC and dltE were also induced (data not shown) as well as the ywaA gene, which is predicted to encode a putative branched-chain amino acid aminotransferase. All these genes (dltABCDE and ywaA) are predicted to constitute an operon.
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DISCUSSION |
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Data collected from microarray experiments indicate a very restricted regulon of YxdJ: only four genes show significant change in their transcription level upon conditions mimicking TCS induction. Indeed, after 30 min of IPTG-mediated YxdJ overproduction, we detected only the expression of the cognate ABC transporter genes yxdLM, confirming our previous observation (Joseph et al., 2002), and the induction of the dltA and dltD genes.
The experimental conditions we used were slightly different from those of Kobayashi et al. (2001) in a similar approach. We used a wild-type strain rather than a yxdK-deleted mutant and a more efficient expression system (Joseph et al., 2001
) giving a yxdJ overexpression ratio that reaches 122 (Table 3
) whereas Kobayashi et al. (2001)
used a 30-fold increase in yxdJ expression (data available at ftp://ftp.genome.ad.jp/pub/kegg/expression/ex0000286.dat). A very restricted gene expression pattern change was obtained in both cases with a common characteristic, a strong positive control of the yxdLM cognate ABC transporter gene expression by the YxdJ response regulator.
Using longer IPTG induction time, we have seen that all the genes of the dlt operon were induced, including the ywaA gene encoding a putative branched-chain amino acid aminotransferase (data not shown). The five dlt gene products are involved in teichoic acid polyalanylation (Perego et al., 1995). The promoter region of the dlt operons did not show detectable DNA binding of His-YxdJ in the gel mobility shift assays (data not shown). This probably indicates indirect regulation of expression of these genes by YxdJ and presumably explains the lack of detection of any YxdJ binding site sequence using bioinformatic approaches. Interestingly, expression of the dlt operon from Streptococcus agalactiae is also controlled by a TCS belonging to the OmpR family, the DltS/DltR system (Poyart et al., 2001
). The exact physiological role of the yxd gene cluster remains to be elucidated. From our results it appears that the YxdJ regulon is limited to a set of genes encoding systems responsible for compound efflux, such as the membrane pump of the ABC family, and cell wall biosynthesis/modification. YxdL shows strong similarity to several NBD ABC transporters, such as SalX, involved in salivaricin resistance in Streptococcus salivarius and Streptococcus pyogenes (Upton et al., 2001
), VraD and VraF from Staphylococcus aureus (Kuroda et al., 2000
), and MbrA, responsible for bacitracin resistance in Streptococcus mutans (Tsuda et al., 2002
). It was also shown recently that the bce system (formerly yts), which is paralogous to the yxd system, is involved in bacitracin resistance (Bernard et al., 2003
; Mascher et al., 2003
; Ohki et al., 2003
). The increase in D-alanyl esterification of teichoic acids caused by activation of dlt transcription should result in a neutralization of the negative charge of adjacent phosphoryl residues of this anionic polymer, eventually leading to an increased resistance of the cells to some antibiotics. In fact, mutants lacking D-alanine on teichoic acids displayed an increased sensitivity: of B. subtilis to methicillin (Wecke et al., 1997
), of S. agalactiae to several cationic antimicrobial peptides (Poyart et al., 2003
) and of S. aureus to gallidermin or nisin (Peschel et al., 1999
).
We therefore suggest that the yxdLMyxeA operon gene products might be involved in resistance to an as yet unknown group of antibiotics, and that yxdJK encodes the corresponding signal detector/transducer system.
Related to yxdLM, the gene yxeA encodes a long peptide which is conserved in several Gram-positive bacteria: Bacillus anthracis, Enterococcus faecalis, Lactococcus lactis, Listeria innocua, Listeria monocytogenes and Staphylococcus aureus. This peptide might participate in the proposed antibiotic resistance mechanism as an immunity peptide interacting with and neutralizing the antibiotic. It is predicted to be processed (Nielsen et al., 1997) and exported via the general secretion pathway. In that case the YxdLM ABC transporter might work in conjunction with YxeA as an antibiotic efflux pump. However, one cannot exclude that YxeA might be exported by the ABC transporter YxdLM to protect the cell as indicated above. We are currently studying these different possibilities.
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ACKNOWLEDGEMENTS |
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Received 10 March 2004;
revised 21 May 2004;
accepted 27 May 2004.