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1 Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006 León, Spain
2 Área de Microbiología, Fac. CC. Biológicas y Ambientales, Universidad de León, Campus de Vegazana, s/n, 24071 León, Spain
Correspondence
Juan F. Martín
jf.martin{at}unileon.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Streptomyces species have a central role in soil ecology processes, and produce an impressive array of secondary metabolites with interesting biological properties and pharmacological activities (Bérdy, 2005). There are hundreds of genes in the sequenced genomes of Streptomyces that encode enzymes for the biosynthesis of secondary metabolites (Omura et al., 2001; Bentley et al., 2002). The biosynthesis of most secondary metabolites is negatively regulated by phosphate (Martín et al., 1994).
Phosphorus is an essential component of bacterial nutrition and is one of the major constituents of the cell, making up 
1.5–2.1 % of the cell dry weight. Phosphorus plays an important role in cell metabolism and is a constituent of nucleic acids, phospholipids, phosphate-containing lipopolysaccharides, nucleotide cofactors and some post-translationally modified proteins.
Expression of phosphate-regulated genes in Streptomyces species is modulated by the two-component system PhoR–PhoP (Sola-Landa et al., 2003). PhoR is the membrane sensor kinase, and senses phosphate scarcity; PhoP is the response regulator, and binds DNA and controls the transcription of genes belonging to the so-called PHO regulon. PhoP has been shown to modulate the expression of primary and secondary metabolism genes, including the actinorhodin and undecylprodigiosin biosynthesis genes (Sola-Landa et al., 2003; Ghorbel et al., 2006). Binding of PhoP to the promoter regions of three different genes of the PHO regulon, pstS, phoU and phoRP, has been shown in both Streptomyces coelicolor (Sola-Landa et al., 2005) and Streptomyces natalensis (Mendes et al., 2007). The PhoP operator sequences of these genes, as well as those present in the promoter regions of phoA and phoD of S. coelicolor (Apel et al., 2007), are composed of direct repeat units (DRus) of 11 nt. Novel operator sequences have been recently described in S. coelicolor (Sola-Landa et al., 2008). Analysis of the novel and the previously known operators (up to 19) by means of electrophoretic mobility shift assays (EMSAs), footprinting, and information theory studies, has revealed the structure of the PhoP-binding sites. Two or three well-conserved DRus form the core of the binding site, in which each DRu is bound by a protein monomer. Complex sites have adjacent DRus that are bound subsequently (Sola-Landa et al., 2008).
The transport of phosphorus sources is essential for the growth of all living organisms. The preferred source of phosphorus in bacteria is inorganic phosphate (Pi), which can enter the cell with the aid of at least two different transport systems (as described below), although there are other phosphorus-containing compounds that can enter the cell intact, such as organophosphates and phosphonates. However, numerous organic phosphate compounds have to be hydrolysed before being transported into the cell, such as nucleotides, some sugar phosphates, and phospholipids (Martín & Demain, 1977, 1980). S. coelicolor and other Streptomyces species contain at least three phosphatases and a phosphodiesterase system (Apel et al., 2007) to hydrolyse different organic phosphates.
Pi is taken up in bacteria mainly by two different transport systems, the high-affinity phosphate-specific transporter (Pst) and the low-affinity phosphate inorganic transporter (Pit). Pst is an ABC transporter that has an ATP-driven high-affinity Pi uptake. In Escherichia coli this transporter is composed of four proteins. These are the PstS (periplasmic phosphate-binding), PstA and PstC (integral membrane proteins), and PstB (ATP-binding) subunits. The pstSCAB operon belongs to the PHO regulon and has been identified in E. coli and in other bacterial species (Rao & Torriani, 1990; Nikata et al., 1996). The pstS gene has also been studied in S. coelicolor (Sola-Landa et al., 2005; Díaz et al., 2005), in which its expression is subject to a strict phosphate control.
Pit is a low-affinity, high-velocity phosphate-uptake system and is dependent on the proton motive force for energy. The Pit transport system has been studied in E. coli (Hoffer et al., 2001; Harris et al., 2001; van Veen et al., 1994a; van Veen, 1997), Acinetobacter johnsonii (van Veen et al., 1993, 1994b, c; van Veen, 1997) and Sinorhizobium meliloti (Voegele et al., 1997; Bardin & Finan, 1998; Bardin et al., 1998; Yuan et al., 2006a). In E. coli, Pit is the major Pi uptake system when Pi is in excess (Rosenberg et al., 1977, 1979) and consists of a single transmembrane protein (Elvin et al., 1986). Divalent cations such Mg2+ and Ca2+ are essential for Pit activity in both E. coli (Russell & Rosenberg, 1980) and A. johnsonni (van Veen et al., 1994b), since phosphate is symported with a proton, as a soluble and neutral metal complex (MeHPO4; van Veen et al., 1994a, c; van Veen, 1997).
E. coli contains two functional pit genes, pitA and pitB (Hoffer et al., 2001; Harris et al., 2001). Two or more paralogues can also be found in many other genomes. PitA and PitB have similar transport characteristics (Hoffer et al., 2001) and have 90 % similar amino acid sequences. However, the regulation of the two genes appears to be different. Thus, pitA is expressed constitutively, while pitB appears to be repressed under conditions of phosphate limitation (Elvin et al., 1986; Harris et al., 2001).
The pit gene(s) have not been studied in Streptomyces species, but their characterization is important to establish their role in phosphate transport and their effect on the biosynthesis of secondary metabolites and on the ecology of these bacteria in soil. This work describes two paralogous pit genes in S. coelicolor, and elucidates their pattern of regulation by means of both in vivo assays, using wild-type S. coelicolor and 
phoP mutant strains, and DNA-binding PhoP-footprinting analysis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. S. coelicolor strains M145 (Kieser et al., 2000) and INB101 (phoP) (Rodríguez-García et al., 2007) were manipulated according to standard procedures (Kieser et al., 2000). TBO medium (Higgens et al., 1974) was used to obtain spore preparations. E. coli DH5
was the general cloning host. Cloning procedures were performed as described by Sambrook et al. (1989).
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The pstS promoter region was cloned from pGEM-PpstS (Sola-Landa et al., 2005). BamHI–NdeI fragments of 222 bp from pFS-pitH1, 260 bp from pFS-pitH2, and 403 bp from pGEM-PpstS were cloned into the promoter-probe vector pLUXAR+ (Rodríguez-García et al., 2007), yielding pLUX-pitH1, pLUX-pitH2 and pLUX-pstS, respectively. In order to introduce the pLUX plasmids into the S. coelicolor INB101 strain (which is apramycin resistant), the neo gene was inserted to construct derivatives with neomycin resistance (Table 1
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Culture conditions.
S. coelicolor cultures were performed in defined MG (maltose and glutamate) medium containing starch (Scharlau; 50 g l–1) and glutamate (60 mM) (Doull & Vining, 1989). Aliquots (100 ml) of MG medium in 500 ml baffled flasks were inoculated with 106 spores ml–1 and incubated at 30 °C, 300 r.p.m. (25.4 mm orbit diameter) for reproducible and dispersed growth. For the phosphate-replete and the phosphate-limited conditions, cultures contained 18.5 and 3.2 mM potassium phosphate, respectively (MG-18.5 and MG-3.2 media; experimentally determined concentrations). The potassium concentrations in MG-18.5 and MG-3.2 were equalized by adding KOH to MG-3.2, instead of NaOH alone, when the pH was adjusted (sodium ions in both media are in excess, 60–75 mM). Samples were taken after 36, 40, 44, 48, 60, 70 and 90 h of incubation.
Luciferase assay, and growth and phosphate determination.
luxAB gene expression was determined in a Luminoskan luminometer (Labsystems). Culture samples (1 ml) were taken, spun down (4 °C, 10 min) and kept frozen until all samples from an experiment were available to be processed simultaneously; then the cells were resuspended in 1 ml NaCl (0.9 %, w/v), incubated at 25 °C for 15 min and measured in triplicate, as described in Rodríguez-García et al. (2007).
For dry weight determination, culture samples (2 ml) were washed twice with MilliQ water and dried for 4 days at 80 °C.
The phosphate concentration of MG medium and culture supernatants was measured using the malachite green assay (Lanzetta et al., 1979).
RT-PCR.
RNAs from S. coelicolor wild-type and phoP strains were isolated using RNeasy Mini Spin columns (Qiagen). Before RNA isolation, one volume of each culture was treated with two volumes of RNAProtect Bacteria reagent (Qiagen) to provide immediate stabilization of RNA. Cell lysis and phenol pre-purification were carried out prior to column purification as described previously (Rodríguez-García et al., 2007). RNA preparations were treated on-column with DNase I (Qiagen), and the eluted solution with the DNA-free kit (Ambion) to eliminate possible chromosomal DNA contamination. RNA concentration and quality were checked using a NanoDrop ND-1000 (Thermo Fisher Scientific) and RNA nano labchips in a 2100 Bioanalyser (Agilent).
Gene expression analysis by RT-PCR was performed with the SuperScript One-Step RT-PCR system with Platinum Taq (Invitrogen). Primers CAR65 (5'-ATTTCACGAACGGTTTCCAC) and CAR66 (5'-GATGCCCATGGTCTTCTGG) were used to amplify 542 bp of the pitH2 coding region; primers CAR67 (5'-CGTACACCAACGGTTTCCAC) and CAR68 (5'-ATGCCCATCGTCTTCTGC) were used to amplify 541 bp of pitH1. The synthesis of a common transcript pap-pitH1 was detected with the primer pair CAR73 (5'-ACCGCGAGGACATCTACAAC) and CAR74 (5'-ACCAGGTGATGAGGTTCCAG). RT-PCR cycling conditions were as follows: 50 °C for 60 min, 94 °C for 2 min; 11 cycles of 94 °C for 30 s, 65 °C for 30 s with touchdown of 1 °C per cycle, 72 °C for 60 s; 23–29 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 40 s; finally 72 °C for 10 min. Total RNA (200 ng), 1.6 mM MgSO4 (final concentration), and 5 % DMSO were added in all reactions. Platinum Taq was used in the control reactions to check for the absence of DNA contamination.
DNA–protein binding assays.
DNA-binding analyses were performed by EMSA with the GST–PhoPDBD protein (PhoP DNA-binding domain fused to glutathione-S-transferase), as described previously (Sola-Landa et al., 2005). The probes used were the BamHI–KpnI fragments of 222 and 260 bp obtained by PCR of the promoter regions of pap-pitH1 and pitH2 and purified after agarose-gel electrophoresis (GFX columns; GE Healthcare). Probes were labelled at both ends with DIG (DIG Oligonucleotide 3'-End Labeling kit, Roche Applied Science).
DNase I footprinting assays.
DNase I footprinting assays based on the fluorescent label procedure (Rodríguez-García et al., 1997) were performed as described in Sola-Landa et al. (2005). DNA probes were obtained by PCR using pFS-pitH2 as template. For analysis of the coding strand, a fluorescently-labelled universal primer and a non-labelled reverse primer were used; for the complementary strand, the labelled primer was the reverse primer. The labelled probes (424 and 419 bp for the coding and complementary strand, respectively) were purified by agarose electrophoresis (GFX columns). DNase I footprinting was performed by incubating 0.28 pmol of the DNA probe with different concentrations of GST-PhoPDBD protein for 30 min at 30 °C. Nuclease digestions were carried out for 1 min at 30 °C. The reaction products were resolved in an ALF DNA sequencer (GE Healthcare) and analysed with the Fragment Manager program.
Primer extension analysis.
For primer extension analysis, RNA samples were isolated as described above, but with the RNeasy Midi kit (Qiagen) and the on-column DNase treatment only. Total RNA concentration and quality were checked both spectrophotometrically and by denaturing agarose gel electrophoresis. Transcription start sites were determined by the fluorescent primer extension procedure (Altermann et al., 1999; Fekete et al., 2003), modified as follows. Primers LUX-FAM+47 [6-carboxyfluorescein- (6-FAM) 5'-GATAGCTCAGGTGGCTGATAAG] and LUX-FAM+135 (6-FAM 5'-GTGGTGCTCTAGCAACCAAAC), both obtained from MWG-Biotech, were complementary to the coding region of luxAB genes of the pLUXAR+ vector (nucleotides +25 to +47 and +114 to +135 with respect to the translation start, respectively). These primers (20–40 pmol) were hybridized with total RNA (20–30 µg) in a 10 µl final volume by heating to 90 °C for 2 min and cooling to 30 °C at a rate of 2 °C min–1. Then, cDNA synthesis was performed with either Superscript II or Superscript III reverse transcriptase (Invitrogen). The primer extension reaction (30 µl) contained 10 mM DTT, 500 µM dNTPs (NEB), 1.5 µg actinomycin D, 10 U SUPERase-In (RNase inhibitor; Ambion), and 200 U reverse transcriptase in 1x First Strand buffer (Invitrogen). The samples were incubated at 42 °C for 5 or 15 h. Then, 10 µl NaOH (1 M) was added and the RNA was lysed by heat treatment (70 °C, 10 min). A 10 µl volume of HCl (1 M) was added to neutralize the solution; the cDNA samples were phenolized and precipitated with ethanol. The pellets were dissolved in a solution of 9.6 µl Hi-Di formamide (Applied Biosystems) and 0.4 µl GeneScan LIZ-500 internal size standard (Applied Biosystems). Samples were heated to 95 °C for 5 min, placed immediately on ice for 5 min, and loaded onto an ABI PRISM 3130 sequencer (Applied Biosystems). To determine the product size accurately, sequencing reactions were performed with the same primer as the reverse transcription step and with the Thermo Sequenase Primer Cycle Sequencing kit (GE Healthcare). Reactions contained 400 ng plasmid DNA as template, and 2 pmol of the corresponding primer. Cycling conditions were 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, for a total of 60 cycles. The EDTA/ethanol-precipitated products of the four sequencing reactions were combined with the GeneScan LIZ-500 size standard and analysed on the DNA sequencer. Electrophoretograms were aligned according to the size standards using the GeneMapper 3.7 software (Applied Biosystems).
Bioinformatic analysis.
The phylogenetic studies were done using the BLAST tree option on the NCBI Web BLAST service with the Fast Minimum Evolution method (Desper & Gascuel, 2002) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Kanehisa et al., 2006). Comparative genomics was done using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; von Mering et al., 2007). The percentages of sequence similarity between two proteins were calculated using the Needleman-Wunsch global alignment of the European Molecular Biology Open Software Suite (EMBOSS; Rice et al., 2000). The putative transmembrane segments (TMSs) were determined using the TopPred server (Claros & von Heijne, 1994) and the TMpred server (Hofmann & Stoffel, 1993). Prediction of signal peptides was done with the SignalP server (Bendtsen et al., 2004).
To identify and analyse putative binding sites of the response regulator PhoP, the information theory programs delila, makebk, encode, rseq, dalvec, ri, scan and lister (Schneider & Stephens, 1990; Schneider, 1996, 1997) were used. Model 1 of Sola-Landa et al. (2008) provided the information content matrix to assign the information content (Ri) values.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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); they are designated in this work as pitH1 and pitH2, respectively, following the annotation of the homologous genes of Streptomyces avermitilis (Ikeda et al., 2003). The PitH1 and PitH2 proteins show 39 and 33 % amino acid sequence similarity, respectively, to the transporter PitA of E. coli and contain the Pfam motif characteristic of the phosphate transporter family (PF01384 PHO4). Their sizes and similarities with homologous proteins are shown in Fig. 1. Both PitH1 and PitH2 contain putative TMSs.
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). In Sinorhizobium meliloti, the homologous genes of SCO4137-pitH1 are considered to form an operon (orfA-pit). The orfA gene is annotated as pit-accessory, although the possible role of the OrfA protein in Pi transport has not yet been investigated (Voegele et al., 1997; Bardin et al., 1998). In this work the SCO4137 protein is referred as Pit-accessory protein (Pap). On the other hand, downstream of pitH2 there is a gene (SCO1846) that encodes a membrane protein. The amino acid sequences of homologous proteins in other bacteria are poorly conserved, as expected for membrane proteins, but in all cases they contain two putative TMSs (Fig. 1). Comparative genomics showed that whereas the pap-pitH1 genetic organization is widespread in bacteria, the distribution of the pitH2-SCO1846 gene organization is confined to actinobacteria.
The upstream sequences of pitH2 and pap-pitH1 were selected for study of their transcriptional regulation by phosphate-limitation. Interestingly, bioinformatics analysis identified putative PhoP-binding sequences (PHO boxes) in the promoter region of pitH2, but no PHO boxes were found in the upstream region of pap-pitH1. The promoter of the pstS gene, which encodes the phosphate-binding transport protein, was also included in this study as a positive control PhoP-activated gene (Sola-Landa et al., 2005).
Phosphate-limited and phosphate-replete cultures of wild-type and phoP mutant S. coelicolor strains
In order to study phosphate regulation, Pi-limited and Pi-replete conditions were defined in initial experiments. Cultures of S. coelicolor wild-type and phoP mutant strains were carried out in MG medium with initial Pi concentrations of 2.1, 3.2 and 18.5 mM (media designated MG-2.1, MG-3.2 and MG-18.5, respectively). The growth of both cultures was quite similar in MG-18.5 (Fig. 2a). Indeed, in MG-18.5, the Pi concentration in the medium was in excess throughout the time-course of the culture (always higher than 4 mM in both strains; Fig. 2a). For this reason, the MG-18.5 medium was chosen as the Pi-replete condition. On the other hand, an initial Pi concentration of 2.1 mM supported only poor growth of the phoP mutant, which can be explained by the lack of both PhoP-dependent phosphate transport and a nutrient-limitation response; however, the growth of the wild-type strain was also severely reduced (results not shown). In contrast, MG-3.2 medium allowed a substantial biomass increase in the phoP mutant during the first 44 h, although the growth did not follow the diauxic pattern of the wild-type (Fig. 2b). About 85 % of the initial Pi was utilized in the first 40 h (corresponding with the rapid growth), and after 44 h it was mostly spent (coinciding with the end of the rapid growth phase), falling below 0.1 mM in both wild-type and phoP mutant cultures (Fig. 2b). The growth of the wild-type in MG-3.2 was also reduced as compared to the phosphate-replete condition (Fig. 2a, b). Therefore, MG-3.2 was chosen as the Pi-limited condition.
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phoP mutant strains. The reporter activity was quantified in liquid cultures of phosphate-replete (MG-18.5) or phosphate-limited (MG-3.2) media (Fig. 3).
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and 3a). As shown in Fig. 3(a), the maximum activity of pitH2p was reached at 40 h, when the Pi concentration had decreased to 0.5 mM; until 44 h of culture this was the major activity. This time (44 h) marked the end of the transition growth phase, as well as the maximum of pap-pitH1p activity, which showed a small delay with respect to the peak of pitH2p activity. Later, the pstSp activity became increasingly predominant. In fact, this is the only one of these promoter activities that can be detected up to 70 h. This is not surprising, since the Pst transporter is the high-affinity system that accounts for Pi transport under conditions of low concentration (Rao & Torriani, 1990). The highest induction of pstSp correlated with the drop of Pi below 0.1 mM (44–48 h; Figs 2b and 3a).
In MG-18.5, in which Pi is in excess throughout the course of growth (Fig. 2a), the pattern of expression changed dramatically. Firstly, pstS promoter activity remained at very low levels throughout the culture (Fig. 3b). Secondly, the activity of pitH2p was constant until the late growth phase, and lacked the high levels reached in MG-3.2 at 40 and 44 h (compare Fig. 3a, b). The predominant activity in cultures in MG-18.5 was that of pap-pitH1p throughout most of the culture. Indeed, its values increased 1.3–5.8-fold, as compared with that of the MG-3.2 culture, throughout the growth course. In conclusion, the pitH2 and pstS promoters are induced by Pi limitation, while the pap-pitH1 promoter is more active under Pi-replete conditions.
Expression of pitH2, but not that of pap-pitH1, is phoP-dependent
The transcriptional regulator PhoP controls expression of the pstSCAB transporter gene cluster by binding to the PHO box located in the pstS promoter (Sola-Landa et al., 2005). In order to study whether the expression of pap-pitH1 and pitH2 genes depends on the PhoP response regulator, reporter expression studies were performed with the S. coelicolor phoP mutant (Rodríguez-García et al., 2007). Results (Fig. 3c, d) clearly showed that expression of pitH2 was dependent upon the PhoP activator. In contrast, expression from the pap-pitH1 promoter was not affected by the lack of PhoP in the phoP mutant (Fig. 3c, d). Indeed, the pap-pitH1 activity was higher in the mutant than in the wild-type strain in MG-3.2 (1.7 times higher when the maximum values are compared). This result may reflect a mechanism of adaptation of pap-pitH1 to the lack of expression in the phoP mutant of the other PhoP-dependent transporters, Pst and PitH2. In the phoP mutant grown in MG-18.5, the pap-pitH1 promoter showed a similar behaviour to that in the wild-type strain, although the values at 36 and 60 h were higher in the mutant than in the wild-type strain.
Transcriptional analysis of pitH2 and pap-pitH1
In order to confirm the promoter-probe results, RT-PCR was carried out as described in Methods using total RNA isolated from 40 h MG-3.2 cultures of both wild-type and phoP mutant strains. Amplification of the pitH2 transcript was clearly detected in the wild-type RNA; in contrast, only a low-intensity band was present in the phoP mutant reaction, even after 40 cycles of PCR (Fig. 4a). The presence of a low level of pitH2 transcriptin in the phoP mutant correlates well with the very low, but significant, activity of the pitH2 promoter at 40 h in the phoP mutant (see Fig. 3c). On the other hand, both wild-type and mutant RNA gave rise to amplification products corresponding to pitH1 and pap-pitH1 transcripts (Fig. 4a). As expected, these results indicate that pap and pitH1 form a bicistronic transcript and are PhoP-independent.
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). Amplification of the cDNA from pitH2 transcripts is higher at the earlier times (36 and 40 h) than the later ones (44 and 48 h). Interestingly, maximum luciferase activities from the pitH2p-luxAB fusion were observed 4 h later (Fig. 3a).
PhoP binds to the pitH2 promoter but not to the pap-pitH1 promoter
In order to test whether the PhoP protein binds to the pitH2 and pap-pitH1 promoters in vitro, the PhoP DNA-binding domain (PhoPDBD) fused to GST was used in binding assays, as described in Sola-Landa et al. (2005). The promoter DNA–PhoP interaction was studied by EMSA. A protein concentration of 0.25 µM was sufficient to produce up to four complexes with the pitH2p DNA fragment (Fig. 5, arrows). As the protein concentration increased, the amount of unshifted probe decreased. However, no shifted bands were observed with the pap-pitH1 promoter under the same experimental conditions.
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). Extension products with an apparent size of 76 and 166 nt, primed with LUX-FAM+47 and LUX-FAM+135, respectively, were observed clearly (Fig. 6). The same transcription start point (TSP) was deduced from both products when compared with the respective sequencing reactions. This corresponds to a guanine located 32 bp upstream of the ATG codon (Fig. 6). Sequences resembling the –10 and –35 consensus (Strohl, 1992) were located at the proper distance from the TSP, centred at positions –7 and –33, respectively. These –10 and –35 boxes overlap the PhoP-binding site (Fig. 8). Although the distance between the –10 and –35 boxes (20 nt) is longer than usual (16–18 nt), this feature seems to be a characteristic of the PhoP-dependent activation mechanism of this and other genes (see Discussion).
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, the pap-pitH1 upstream sequences TTAGCA-N18-TAGCAT constitute plausible –35 and –10 promoter boxes (located at positions –61 and –37, respectively, from the translation start codon). However, we have failed to obtain clear extension products with the pap-pitH1 promoter, despite exhaustive trials. Retrotranscription reactions were done with five different RNA samples from wild-type and mutant cultures using two different enzymes (Superscript II and Superscript III, both from Invitrogen), two different primers (LUX-FAM+47 and LUX-FAM+135), varying the amounts of RNA (5–200 µg) and primer (5–50 pmol), varying reaction time and temperature (5–15 h; 42–50 °C), and with or without DMSO (5 %) or actinomycin D. The lack of detected extension products also occurs in the Sinorhizobium meliloti wild-type strain carrying only one copy of the orfA-pit gene region (Bardin et al., 1998), and suggests that the pap-pitH1 transcript may be rapidly degraded, thus precluding retrotranscription.
Analysis of the PhoP-binding sequence in the pitH2 promoter
To determine the PhoP-binding sequence, DNase I footprinting of the pitH2 promoter region in the presence and absence of PhoPDBD was performed as described previously (Rodríguez-García et al., 1997; Sola-Landa et al., 2005). PhoPDBD was found to protect a 69 bp region in the pitH2 promoter. The protected sequence was of 53 nt in the pitH2 coding strand and 64 nt in the complementary strand; 48 nt were coincident in both strands (Figs 7 and 8). Full protection of the coding strand was achieved at 0.5 µM GST-PhoPDBD, and the protected region was not enlarged by increasing protein concentration (Fig. 7a). The protection of the complementary strand showed the same requirement for GST-PhoPDBD protein (0.5 µM), and the protected sequence was not enlarged by increasing protein concentration (Fig. 7b). However, when a protein concentration of 0.26 µM was used, the protected region was shortened in the upstream and downstream regions (see the marked traces of the fluorograms in Fig. 7b), suggesting that the core region of the protected region has a higher affinity for PhoP than the adjacent nucleotide sequences (see below).
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, 1997) analysis of this pitH2 region revealed six DRus of 11 nt in length using the published matrix model I (Sola-Landa et al., 2008). The central sequence of the pitH2 protected region contains four consecutive DRus (DRu-1, DRu-2, DRu-3 and DRu-4), and the flanking sequences contained two additional repeats, DRu-A and DRu-B. Both DRu-A and DRu-B are separated from the central repeats by 1 and 2 bp, respectively (Figs 7 and 8). The individual Ri values indicate that three of the DRus in the pitH2 operator (DRu-1, DRu-3 and DRu-4) are well conserved, whereas DRu-2 is poorly conserved (Fig. 8). The DRu-A and DRu-B sequences, in contrast, show a negative Ri that reflects a poor sequence conservation (Fig. 8). The binding of PhoP to these poorly conserved DRus can be explained by a cooperative interaction between consecutive protein monomers at high protein concentration. We have proposed elsewhere (Sola-Landa et al., 2008), for these complex operator structures, that at lower PhoP concentrations only DRu-3 and DRu-4 are bound, and these form the core of the site. Higher PhoP concentrations would allow the sequential binding to DRu-1-DRu-2, DRu-A and DRu-B (Fig. 8; see Discussion). This explains the appearance of up to four retarded complexes in the EMSA assays (Fig. 5). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and four phosphatase genes (Apel et al., 2007; Sola-Landa et al., 2008). As a parallel strategy to survive Pi starvation, Streptomyces cells may consume phosphorous storage material, such as polyphosphates, nucleotides and teichoic acids. The ppk gene, which encodes an enzyme that catalyses the reversible polymerization of the 
phosphate of ATP into polyphosphate, is under the positive control of the PhoR–PhoP system in Streptomyces lividans (Ghorbel et al., 2006). Moreover, two PhoP-binding sites have been found in a cluster of genes putatively involved in the biosynthesis of cell wall polysaccharides, so that phosphate-free polymers could substitute for phosphate-rich polymers in the cell wall (Rodríguez-García et al., 2007).
Inorganic phosphate transport (Pit) proteins are found in all groups of living organisms, including bacteria, archaea, yeast, fungi, plants and animals. pit genes encoding proteins from 195 to 763 aa in length are found in different organisms (Saier et al., 1999). In some bacteria the Pit system has been proposed to have a role in heavy metal tolerance; thus, in the presence of heavy metals, metal phosphates are transported out of the cell by Pit (Keasling, 1997; Beard et al., 2000; Álvarez & Jerez, 2004).
Three Pi transport systems have been identified in the genome of S. coelicolor. Of these three, PitH1 is the major transporter when Pi is in excess, whereas PitH2 and Pst work when Pi is limited. In E. coli there are two pit genes as well, although PitA and PitB share greater sequence similarity (90 %) than PitH1 and PitH2 in S. coelicolor (53 %), and have the same topological structures (10 TMS; Harris et al., 2001). Moreover, the S. coelicolor pit genes are clustered with distinct accessory genes (Fig. 1), which is not the case for E. coli. On the other hand, in Acinetobacter johnsonii, Sinorhizobium meliloti and other bacteria, there is only one pit gene.
The pit genes can also be classified according to their regulation. We have described the regulation of the pap-pitH1 and pitH2 genes above. The pitH2 gene is PhoP-dependent, while the pap-pitH1 one is not. In E. coli the pitA promoter is constitutive, but the pitB promoter appears to be repressed by PhoB (the PhoP orthologue) (Elvin et al., 1986; Harris et al., 2001). Both patterns of regulation were also found in different organisms with a single pit gene. In A. johnsonii, the pit gene is expressed constitutively (van Veen et al., 1993; van Veen, 1997), whereas in Sinorhizobium meliloti it is repressed by PhoB (Bardin & Finan, 1998; Bardin et al., 1998; Yuan et al., 2006a, b). In Corynebacterium glutamicum the pit gene has also been proposed to be repressed by PhoR, the response regulator of the two-component PhoS–PhoR (Schaaf & Bott, 2007). Similarly, the Myxococcus xanthus pit gene is repressed in phosphate-starved cells. This repression is not observed in a mutant in phoP4, which is one of the four response regulators involved in the PHO response in this organism (Whitworth et al., 2008). In contrast, PhoP positively regulates pitH2 of S. coelicolor. To our knowledge, this is the first pit gene to be described as PhoP-upregulated in bacteria, suggesting that the mechanism of action of the phosphate response regulator may be positive in some bacteria and negative in others.
As shown in this work, in the phoP mutant only the pap-pitH1 promoter is active, but both wild-type and mutant strains take up Pi from the medium with similar efficiency (Fig. 2). Gebhard et al. (2006) proposed that the loss of one or two Pi transport systems can be compensated for by increased activity of the remaining systems. It is reasonable to assume that the higher pitH1 expression in the S. coelicolor phoP mutant serves to compensate for the lack of the other two transport systems (Fig. 3). Additionally, another pst operon could exist in S. coelicolor (SCO6816–SCO6814), as suggested by Díaz et al. (2005). Two specific ABC Pi transport systems have been described in various bacteria, such as Sinorhizobium meliloti (Voegele et al., 1997; Yuan et al., 2006a) and some species of mycobacteria (Braibant et al., 1996; Gebhard et al., 2006).
The phoP mutant did not grow after 44 h in phosphate-limited MG-3.2 medium, when the Pi concentration dropped below 0.1 mM (Fig. 2b). This is probably due to the fact that the phoP mutant is defective in the expression of PhoP-dependent genes. The mutant strain has an altered expression of genes involved in central metabolism and protein synthesis, which are PhoP upregulated (Rodríguez-García et al., 2007).
Although transcription from pitH2 and pstS promoters is PhoP-dependent, the two genes showed different patterns of expression. Firstly, sequential activation and decay of the reporter activity at distinct levels of residual Pi are evident from the results. The first gene induced in MG-3.2 medium was pitH2, encoding the low-affinity transporter (Fig. 3a). It makes sense that when the Pi in the medium falls, the bacterium first responds with high induction of a low-affinity transporter (pit), since this system is energetically less expensive to the cell than the pst system. The expression of pst occurs when the Pi in the medium becomes scarce, which is consistent with the higher affinity of this transporter.
In phosphate-replete cultures the pitH2 promoter was constantly active from 36 to 48 h, while pstS promoter activity was nearly absent (Fig. 3b). We propose two mechanisms to explain the differences between pitH2 and pstS expression: (i) the different structure of the PhoP-binding sites, and (ii) the interaction of PhoP with specific sigma factors. These mechanisms, which can act together, are based on the operator structures and might involve the participation of alternative sigma factors.
The differences in structure of the PhoP-binding sites in the promoters of pitH2 and pstS provide mechanisms to explain their expression patterns. The PHO box of pstS is composed of two well-conserved DRus located upstream of the promoter elements (Sola-Landa et al., 2005). In contrast, EMSA, footprinting and information content analysis of the pitH2 operator revealed a complex structure of six DRus. It is proposed, following the model of Sola-Landa et al. (2008), that phosphorylated PhoP (PhoPP) binds first to the most conserved DRu-3 and DRu-4 sequences, which form the core of the binding site. Increasing concentrations of PhoPP would result in the consecutive occupancy of DRu-1 and DRu-2. Since the last DRu shows a negative Ri (Fig. 8), the binding here of a protein monomer can be explained by protein–protein interactions, as proposed previously for the phoRP operator (Sola-Landa et al., 2005). Higher concentrations of PhoPP would allow the binding of DRu-A and DRu-B, although the relevance to the control of gene expression of the binding of PhoP to this sequence may be minor. The functionality of a DRu separated by 2 bp has already been demonstrated for the phoD promoter (Apel et al., 2007) and the SCO1196 operator (Sola-Landa et al., 2008). This stoichiometry matches the number of EMSA complexes observed (Figs 5 and 8).
The position of these DRus in relation to the promoter elements accounts for the dual role, positive and negative, of PhoP, as occurs in the phyC gene of Bacillus amyloliquefaciens (Makarewicz et al., 2006). The main features of the phyC promoter are: (i) one PHO box overlapping the –35 element; (ii) an improper spacing with the –10 element (21 bp); and (iii) a direct repeat overlapping the –10 element. Makarewicz et al. (2006) have proved that while binding of PhoP at –35 is essential for activation of the promoter, binding of PhoP at –10 suppresses promoter activity. Similarly, the PhoP operator core in the pitH2 promoter overlaps the –35 region (Fig. 8); the separation from the –10 element is 20 bp, which is larger than those of standard Streptomyces promoters (16–18 bp; Strohl, 1992), and DRu-B overlaps the –10 region of the pitH2 promoter (Fig. 8). This is a characteristic feature of some promoters regulated by dual activator/repressor proteins (Hidalgo & Demple, 1997; Makarewicz et al., 2006). With this model in mind, PhoPP oligomerization on the DNA up to DRus A, 1, 2, 3 and 4 would result in the activation of the pitH2 promoter. However, the binding of PhoPP to DRu-B would cause repression. The repression at the highest PhoPP concentrations is in agreement with the rapid activity drop of the pitH2 promoter from 40 to 48 h, coincident with the rise in pstSp activity (Fig. 3a).
The homologous pitH2 gene in S. avermitilis (SAV6419) also has a PhoP-binding site in its promoter region with a similar structure, which supports our proposed model. In this species, there are two highly conserved DRus with Ri values of 9.8 and 11.6 bits, respectively. As in S. coelicolor, downstream of these DRus, separated by 2 bp, there is another DRu with an Ri of 0.1 bits. This DRu should be the functional homologue of S. coelicolor DRu-B in the pitH2 promoter.
The earlier activation of the pitH2 gene with respect to the pstS gene can be also explained if its promoter is recognized by a sigma factor, active in the first phase of growth, that interacts with PhoP, whereas pstS is triggered by PhoP in combination with a phosphate-limitation-responsive sigma factor. This mechanism is supported by the finding that the Bacillus subtilis PhoP is able to interact with promoters of at least three different sigma factors 
A, E and M (Paul et al., 2004; Minnig et al., 2005). The sequencing of the S. coelicolor genome has identified a plethora of genes that encode sigma-factors, including four homologues (hrdA, hrdB, hrdC and hrdD) of the principal sigma factors, nine homologues of B. subtilis stress-response B, and 51 extracytoplasmic function sigma factors (Bentley et al., 2002; Hahn et al., 2003). Therefore, it will not be surprising if one or more sigma factors are dedicated to the phosphate-limitation stress response. Moreover, the sigma factor genes hrdB and hrdD of Streptomyces griseus are expressed differentially under Pi-rich or Pi-starved conditions, respectively (Marcos et al., 1995).
Finally, the pitH2 promoter activity in phosphate-replete cultures (Fig. 3b) indicates that unphosphorylated PhoP can bind and activate this promoter. In vitro DNA binding of the unphosphorylated PhoP (the PhoP form existing under high-phosphate conditions) proteins of S. coelicolor (Sola-Landa et al., 2005) and B. subtilis has been reported, although in B. subtilis, the phosphorylated protein is required for full activation (Liu et al., 1998; Eder et al., 1999). Also, in S. coelicolor, the full pitH2 promoter activity is achieved under conditions of phosphate limitation, i.e. when PhoP is phosphorylated.
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ACKNOWLEDGEMENTS |
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Edited by: J.-H. Roe
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REFERENCES |
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Received 11 April 2008;
accepted 27 April 2008.
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) and residual Pi concentration (
) for S. coelicolor M145 (wild-type) and INB101 (
), pLUX-pitH1 (
) and pLUX-pstS (
) derived from the wild-type M145 grown in MG-3.2 (a) and MG-18.5 (b), and from the 
