Instituto de Microbiología Bioquímica/Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas (CSIC)/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain
Correspondence
Ramón I. Santamaría
santa{at}usal.es
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ABSTRACT |
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INTRODUCTION |
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On the other hand, the variable availability of nutrients (for example the carbon source) may control the expression of a large number of genes, some of which may or may not be directly involved in their metabolism (Hodgson, 2000). To date, sugar-uptake mechanisms have only been described for a limited number of carbohydrates in Streptomyces (Bertram et al., 2004
; Hurtubise et al., 1995
; Schlösser et al., 1999
; van Wezel et al., 1997
, 2005
). Glucose uptake is mediated by two proton symporters encoded by the glcP1 and glcP2 genes (van Wezel et al., 2005
), and the glucose kinase (GlkA) responsible for glucose phosphorylation plays a key role as a global carbon regulator (Angell et al., 1994
). In contrast, the HPr protein of the phosphotransferase system (PTS) does not have a general role in carbon regulation, as it does in other bacteria (Nothaft et al., 2003b
), although the role of PTS proteins has been clearly demonstrated for the transport of N-acetylglucosamine and fructose, but not for glucose (Nothaft et al., 2003a
, b
; Wang et al., 2002
).
Phosphate availability is also important in gene expression and differentiation. In Streptomyces, the production of secondary metabolites, among them antibiotics, is known to be under phosphate control. However, very little is known about the molecular mechanism(s) of this control (Chouayekh & Virolle, 2002; Gil & Campelo-Diez, 2003
; Liras et al., 1990
; Martin & Demain, 1980
; Martin & McDaniel, 1975
; Sola-Landa et al., 2003
). In bacteria, phosphorus is generally obtained as inorganic phosphate, which is captured by two transport systems of high and low affinity, respectively. Escherichia coli and Bacillus subtilis are the organisms most studied in this sense. In both cases, high-affinity transport is implemented by a Pst (phosphate-specific transport) system, similar to ATP-binding cassette (ABC) transporters, and composed of the proteins PstS, PstC, PstA and PstB in E. coli, and the proteins PstS, PstC, PstA, PstB1 and PstB2 in B. subtilis. Their expression responds to the phosphate level in the medium and they are induced by phosphate starvation. A phosphate-regulated promoter located upstream from the most proximal gene (pstS) controls the transcription of the entire operon (Aguena et al., 2002
; Qi et al., 1997
). The pst operon is part of the Pho regulon under the control of a two-component system composed of the proteins PhoB/PhoR in E. coli and PhoP/PhoR in B. subtilis (Hulett, 2002
; Qi & Hulett, 1998
; Torriani, 1990
). The phosphorylated forms of the transcriptional factors PhoB or PhoP are the direct effectors of the activation or repression of a large number of genes through binding to a DNA sequence known as the Pho box, which does not have a canonical sequence for all micro-organisms. The second phosphate transporter system, composed of divalent metal transporters (Hantke, 2001
; Harris et al., 2001
; Hoffer et al., 2001
), is a low-affinity one that is expressed constitutively and is functional at high inorganic phosphate concentrations. In Streptomyces, the functionality of both transport systems has been described (Licha et al., 1997
) and the two-component PhoP-PhoR system has also been elucidated (Sola-Landa et al., 2003
).
In a previous work, we described the morphological changes induced in S. lividans when grown in liquid medium in the presence of high concentrations of glucose and fructose (Santamaría et al., 2002).
The present work reports the differences in the secreted protein pattern of S. lividans 66 under these different culture conditions. The most marked deviation was observed in the expression of the high-affinity phosphate-binding protein (PstS), which was accumulated in the presence of fructose and other carbon sources. Mutation of phoP, the transcriptional regulator of the two-component PhoP-PhoR system involved in the Pho regulon, impaired the expression of pstS, while a mutation in ppk, a polyphosphate kinase-encoding gene, elicited overexpression of the PstS protein. Analysis of the S. coelicolor and S. lividans pstS null mutant phenotypes revealed that spore differentiation was triggered earlier in these mutants (lacking the PstS protein) than in the corresponding wild-type strains.
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METHODS |
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E. coli was grown in Luria Broth (LB) at 37 °C, supplemented with ampicillin (100 µg ml1), apramycin (50 µg ml1), chloramphenicol (25 µg ml1) or kanamycin (2550 µg ml1) when needed.
Microscopic techniques.
Scanning electron microscopy was performed on pieces of agar obtained from the corresponding plates and coated with gold (Trujillo et al., 2005). Samples were examined in a Zeiss DSM 940 electron microscope and all the images were recorded digitally.
DNA manipulations and transformations.
Streptomyces and E. coli total genomic and plasmid DNA, transformation and protoplast collection were accomplished as indicated by Kieser et al. (2000) and Sambrook et al. (1989)
. Transfer of cosmids from E. coli to S. coelicolor or S. lividans was carried out by intergeneric conjugation, as described by Gust et al. (2003)
.
DNA sequencing and analysis.
The DNA sequences were determined in both strands with a Perkin Elmer ABI Prism 377 DNA sequencer, using several oligonucleotides designed from the DNA sequences collected. Manipulation was accomplished with the Gene Construction Kit (GCK) (Textco) and analyses were done with the DNA Strider (Marck, 1988). Comparisons of DNA or protein sequences were carried out online (http://www2.ebi.ac.uk/) with FASTA (Pearson & Lipman, 1988
) and BLAST (Altschul et al., 1997
). CLUSTAL W (Thompson et al., 1994
) was used for sequence alignment. RSA-Tools was used for DNA-pattern analysis (van Helden et al., 2000
).
Construction and verification of pstS mutants in S. coelicolor and S. lividans.
The pstS gene, encoding the PstS phosphate-binding protein, corresponds to SCO4142 from the S. coelicolor genome. The whole ORF was replaced by the apramycin gene-oriT fragment [acc(3)IV-oriT] in E. coli BW25113/pIJ790 harbouring the SCD84 cosmid, using the REDIRECT technology (Gust et al., 2003). The deletion cassette with acc(3)IV-oriT DNA in the middle of the pstS flanking sequences (39 nt each side) was obtained by PCR amplification. Plasmid pIJ773 (see Table 1
) digested with HindIII/EcoRI was used as template and the oligonucleotides pRA1 (TCC TGG AAG GAA CTC CCT CAA GTG AAG CTT CAG CGC ATG ATT CCG GGG ATC CGT CGA CC) and pRA2 (CTG TCG GCT CCG CGC GGA GGC CGG ACC GCA CTC GGG TCA TGT AGG CTG GAG CTG CTT C) were used as primers in the PCR reaction [pstS flanking sequences in italics and the sequence that anneals the template (acc(3)IV-oriT) underlined]. Correct replacement of the pstS gene by the deletion cassette [acc(3)IV-oriT] in the SCD84 cosmid was checked by restriction enzyme digestion. The new recombinant cosmid [SCD84 pstS : : acc(3)IV-oriT] was then introduced into S. lividans 66 and S. coelicolor M145 to obtain the pstS null mutants by intergeneric conjugation (Gust et al., 2003
). The correct replacement of pstS in the S. coelicolor and S. lividans genomes was confirmed by Southern blot experiments.
Cloning of pstS promoters from S. coelicolor and S. lividans.
The intergenic regions between SCO4142 (pstS gene) and SCO4143 (possible MutT-like protein) in S. coelicolor and the equivalent region from S. lividans were cloned by PCR using oligonucleotides based on the S. coelicolor sequence of this region: MRG-27 (reverse, TAA TAA CAT ATG GCG CTG AAG CTT CAC TTG AGG GAG) and MRG-28 (forward, TTT TTA GAT CTC AGC CCC GGG ACC GGG CCC T). The NdeI or BglII (underlined) restriction sites were included in MRG-27 and MRG-28, respectively, for further cloning.
Amplification was carried out in an MJ Research thermocycler as described previously (Fernández-Abalos et al., 2003), using genomic DNA from S. coelicolor M145 and S. lividans 66 as template. Both PCR products (about 350 bp) were purified by agarose gel electrophoresis and digested with NdeI and BglII. The amplified bands were cloned in plasmid pNX24 (Table 1
), digested with the same restriction enzymes and transformed in E. coli DH5
(under kanamycin selection), yielding plasmids pNUF2 and pNUF3 (Table 1
). In these plasmids, the pstS promoter regions control the expression of the xysA xylanase gene from Streptomyces halstedii JM8, used as reporter (Adham et al., 2001
). In a second step (detailed data not shown), the transcriptional terminator mmrt (T1) was introduced upstream from the pstS promoter region to yield the final plasmids pNUF4 and pNUF5 (Table 1
), which were introduced by transformation into S. lividans 66.
Cloning of the pstS ORFs from S. coelicolor and S. lividans.
The complete ORFs of the pstS genes from S. coelicolor and S. lividans were amplified by PCR, using oligonucleotides based on the pstS gene from S. coelicolor: MRG-33 (forward: TTTT CAT ATG AAC CGG CGG GCC CTC GC) and MRG-34 (reverse: TTT TTC TAG ATC AGC TCA GGC CCG AGA TGG TC) including an NdeI or XbaI restriction site (underlined), respectively, for further cloning.
Amplification was carried out in an MJ Research thermocycler as described previously (Fernández-Abalos et al., 2003). The PCR products (1130 bp) were purified by agarose gel electrophoresis and digested with NdeI and XbaI. The amplified ORFs were placed under the control of their corresponding promoters using the plasmids pNUF4 and pNUF5, digested with the same enzymes removing the xylanase ORF. Thus, the amplified band from S. coelicolor was cloned into pNUF4 downstream from the S. coelicolor pstS promoter, yielding plasmid pNUF6, while the band amplified from S. lividans DNA was cloned in pNUF5 downstream from the S. lividans pstS promoter, yielding plasmid pNUF7 (Table 1
). These plasmids were introduced into S. lividans 66 for PstS overproduction analysis.
pstS integrative plasmids.
To perform complementation studies, an integrative plasmid (pINTUF5) containing one copy of the pstS gene was obtained. The pstS gene with its promoter region was isolated from pNUF7 by digestion with PvuII/XbaI and cloned in EcoRV/XbaI sites of a hygromycin-resistance plasmid, called pKC796Hyg (Table 1). These plasmids (pINTUF5 and the empty vector pKC796Hyg) were introduced into S. lividans
pstS by protoplast transformation, and the integrated strains were selected for apramycin and hygromycin resistance.
Phosphate uptake.
The uptake of phosphate was measured as described by Sola-Landa et al. (2003), with slight modifications. S. lividans cultures were grown in liquid YEG medium for 40 h (28 °C, 200 r.p.m.). The cells collected were washed twice with 0·9 % NaCl and transferred to asparagine minimal medium without any inorganic phosphate (Martin & McDaniel, 1975
). After stabilization of the cell suspension for 6 h at 28 °C, 32P-labelled Na2HPO4 (Amersham Biosciences) was added (2x105 c.p.m. ml1). Phosphate uptake was measured after 15 min at 30 °C. Cells were recovered by filtration through Whatman GF/C filters and washed twice with 0·9 % NaCl, and the radioactivity from the filter was quantified in a liquid scintillation counter (Wallac 1409-001).
Protein analysis and enzyme assays.
Electrophoresis in denaturing polyacrylamide gels (SDS-PAGE) was performed as described elsewhere (Ruiz-Arribas et al., 1995). The N-terminal end of the protein was determined using an Applied Biosystems Protein Sequenator.
Enzymes and reagents were purchased from Boehringer Mannheim, Promega, Bethesda Research Laboratories, Pharmacia, Sigma, Merck, Panreac, Bio-Rad, Santa Cruz and Ambion, and were used following the manufacturers' guidelines.
The sequence of the S. lividans pstS gene has been deposited in the EMBL Database with accession number AJ698727.
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RESULTS |
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In other systems, such as E. coli and B. subtilis, expression of this gene responds to the phosphate concentration of the medium, being expressed at low phosphate concentrations (lower than 50 and 160 µM, respectively) and repressed at high phosphate concentrations (Aguena et al., 2002; Antelmann et al., 2000
). We studied the effect of the phosphate concentration on S. lividans PstS supernatant accumulation using different liquid media. The initial phosphate concentration in YE medium (without extra phosphate added) was 2 mM and decreased to lower than 100 µM after 4 days of S. lividans growth. No accumulation of extracellular PstS was observed, even when the culture was maintained for up to 10 days. Furthermore, modifications of two minimal media low in phosphate (200 µM, without extra phosphate added) were used; the phosphate concentration fell to 30 µM after 4 days of S. lividans growth, but no PstS accumulation was observed in the supernatant. The addition of 5 % fructose to these minimal media induced S. lividans extracellular PstS accumulation, although to a lower extent than that obtained in YE supplemented with the same carbon source (data not shown). The addition of different amounts of phosphate (from 5 mM to 50 mM sodium phosphate) to YE medium containing 5 % fructose prevented PstS accumulation, showing that high phosphate concentrations in the medium impair PstS accumulation in the presence of high fructose concentrations (Fig. 1D
). Intracellular protein levels of cells grown in the presence of fructose and phosphate were also studied by SDS-PAGE, but no accumulation of PstS at the Coomassie blue-stained gel level was observed under any of the conditions used (data not shown).
Cloning of the promoter region of pstS from S. coelicolor and S. lividans 66
The pstS promoter regions from S. coelicolor and S. lividans were cloned as described in Methods. The sequences of the regions of both species displayed several differences. The intergenic region of S. lividans was 28 bp longer than that of S. coelicolor (329 versus 301 bp). The sequence ACTCACCCCCGC is repeated three times in the S. coelicolor promoter and, with some discrepancies, is repeated eight times in the S. lividans promoter and six times in S. coelicolor. Comparison of the S. lividans and S. coelicolor pstS promoters with the DNA database revealed that the above sequence was absent in another two pstS promoters sequenced from Streptomyces avermitilis and Streptomyces griseus. Nevertheless, all four promoters maintained several conserved regions that could be involved in their regulation (Fig. 2A).
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PstS is overexpressed in a ppK mutant and its expression is controlled by the PhoR-PhoP system
Since intracellular phosphate results from the transport of extracellular phosphate and from the mobilization of intracellular polyphosphate, we decided to study the expression of PstS in a null mutant in the gene encoding polyphosphate kinase (ppk), which is responsible for polyphosphate accumulation inside the cells (Chouayekh & Virolle, 2002); PstS was overproduced in this mutant (Fig. 3
). This result clearly indicates that the need for phosphate in this mutant is met by the overexpression of other genes involved in phosphate uptake, such as the one encoding the high-affinity phosphate-binding protein PstS.
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Deletion of pstS and phenotypic effects on solid media
pstS null mutants of S. coelicolor M145 and S. lividans 66 were obtained by REDIRECT technology (Gust et al., 2003), as described in Methods. The correct pstS replacement in both genomes was checked by Southern blot (data not shown).
Liquid cultures of these mutants in YE+5 % fructose medium revealed no PstS accumulation in the supernatant (Fig. 4A, lanes 2 and 4). Western blotting with anti-PstS antibodies confirmed the absence of this protein in the mutants (Fig. 4B
, lanes 2 and 4).
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To confirm that the pstS gene indeed encoded a functional high-affinity phosphate-binding protein, PstS, the incorporation of 32P-labelled phosphate was quantified in cells of S. lividans wild-type and in the corresponding pstS null mutant strain. A dramatic reduction in the uptake of inorganic phosphate was observed in the mutant strain in comparison with the uptake in the parental strain, as expected (Fig. 5A). This result clearly demonstrated the functionality of the PstS protein in phosphate transport.
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To check that the pstS null mutant phenotypes were only due to the lack of PstS protein in these mutants, complementation studies were performed. One copy of the pstS gene was introduced into the S. lividans pstS null mutant by transformation with the integrative plasmid pINTUF5. Integration of this copy of the pstS gene occurred at the phage C31 integration site, distant from the pst operon in the genome. This ectopic integration of pstS restored the incorporation of 32P-labelled phosphate to levels slightly lower than those of the S. lividans wild-type strain. As expected, the strain transformed with the integrative empty plasmid used as a control, pKC796Hyg, had a degree of 32P-labelled phosphate incorporation similar to that obtained with the pstS null mutant (Fig. 5A
). Western blot analyses with anti-PstS antibodies confirmed that the production of PstS by the strain transformed with plasmid pINTUF5 was slightly lower than that of the wild-type strain (Fig. 5B
).
With respect to sporulation on solid MSA medium, the S. lividans pstS/pINTUF5 integrated strain behaved like the wild-type strain, the differentiation process in these strains being slower than in the S. lividans pstS null mutant and in the control strain S. lividans
pstS/pKC796Hyg (Fig. 5C
). This result highlighted the ability to restore the mutant phenotypes of the single copy of the pstS gene integrated in the mutant genome and ruled out the possibility of a polar effect.
Overproduction of the PstS protein was studied by cloning the corresponding ORF under the control of S. coelicolor and S. lividans pstS promoters, obtaining the multicopy plasmids pNUF6 and pNUF7. Both plasmids were introduced into S. coelicolor M145 and S. lividans 66 by transformation. No clear phenotype was observed either on solid media or in liquid media when PstS was overexpressed in both strains, in spite of the high amount of protein produced detected in SDS-PAGE gels (data not shown).
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DISCUSSION |
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PstS overexpression has been described in several organisms under different stress conditions (Runyen-Janecky & Payne, 2002; Atalla & Schumann, 2003
; Duwat et al., 1999
; Rallu et al., 2000
). This multiresponse of pstS induction suggests that the gene is one of the many emergency genes able to respond to multistress conditions that permit rapid adaptation of the micro-organism to different habitats, perhaps because these conditions lead to a depletion of the internal phosphate pool and hence to the need for external phosphate to be captured. This phosphate depletion may induce the expression of the pstS promoter more than 100-fold in E. coli, and more than 5000-fold in B. subtilis (Qi et al., 1997
; Wanner, 1993
). In S. lividans, we were unable to find a phosphate-limited medium able to support growth and originate PstS accumulation at the same level that the presence of the mentioned carbon sources does. However, the effect of phosphate on the expression of this gene is clear, because the addition of 5 mM inorganic phosphate impaired PstS production in the presence of the inducer carbon sources. Since PstS was not accumulated in the presence of glucose, even if fructose was present, a catabolite repression response might also be regulating the expression of pstS. This hypothetical repression has been suggested previously for R. sphaeroides (Matsuzaki et al., 2003
).
Analysis of the S. coelicolor and S. lividans pstS promoter sequences allowed us to detect the repetition (with some discrepancies) in these promoters of the sequence ACTCACCCCCGC, which may play a role in regulation. This sequence is not present in any other promoter region of the S. coelicolor genome. Comparison of all known Streptomyces pstS promoters revealed the conservation of several sequences, among which the sequence GTTCAN6GTTCA shows similarity to the consensus Pho boxes' of E. coli and B. subtilis: CT(G/T)TCATA(A/T)A(A/T)CTGTCA(C/T) and TT(A/T/C)ACAN5±2TT(A/T/C)ACA, respectively (Blanco et al., 2002; VanBogelen et al., 1996
; Wanner, 1996
; Qi et al., 1997
; Liu et al., 1998
). Analysis of the S. avermitilis and S. coelicolor genomes with the Regulatory Sequence Analysis Tools (RSAT) (http://rsat.ulb.ac.be/rsat/) permitted us to observe that this sequence is located upstream from 16 and 21 ORFs, respectively. Most of these ORFs encode hypothetical proteins, and at least one-quarter of these ORFs are the same in both organisms. Interestingly, one of them is the putative low-affinity phosphate transport protein that corresponds to SCO1845 in S. coelicolor and SAV6965 in S. avermitilis. The putative role of these sequences in the control of the expression of the pstS gene and the complete pst operon must be demonstrated experimentally in future work.
The deletion of pstS in S. coelicolor led to the overproduction of actinorhodin when grown on solid R2YE media. The overproduction of antibiotics in pstS null mutants has also been described in other organisms, such as Serratia, which displays a high level of prodigiosin and carbapenem production (Slater et al., 2003). A plausible explanation for this induction is that this mutation mimics low-phosphate conditions because, as demonstrated here, this mutation is impaired in phosphate transport. An acceleration of differentiation was also observed in this mutant, indicating that phosphate limitation may be responsible for this phenotype. A phosphate downshift, achieved by adding calcium ions, has been described elsewhere as being responsible for the submerged sporulation of different species of Streptomyces (Daza et al., 1989
).
The S. coelicolor and S. lividans PstS proteins are 100 % identical and share 79 % identity with the PstS protein from S. avermitilis and 68 % with the protein from S. griseus, respectively. At the same time, they share about 4243 % identity with three different putative phosphate receptors PstS-1, PstS-2 and PstS-3 from Mycobacterium tuberculosis (Lefevre et al., 1997). These three proteins share a high degree of similarity among one another, but their expression seems to respond differently to the habitat. One of them, PstS-1, is one of the most important immunodominant antigens of M. tuberculosis (Chang et al., 1994
; D'Souza et al., 2002
). Although there is no clear duplication of the pstS gene in the S. coelicolor genome, a second phosphate-binding protein was identified by sequence similarity. That protein corresponds to SCO2428 and is a putative secreted protein with a length of 522 amino acids that shares 23 % identity with the protein studied in this work, corresponding to ORF SCO4142. It is possible that this protein could somehow mimic or partially complement the function of PstS in the pstS null mutant, permitting the cells to survive. The functionality of this protein will be further studied by obtaining the corresponding single and double null mutants with pstS.
One plausible hypothesis from the present work would be that the activation of carbohydrate metabolism in general, produced by an excess availability of certain carbon sources in the culture medium, elicits a dramatic decrease in inorganic phosphate inside cells and activation of the Pho regulon. The requirement of extra phosphate to phosphorylate the high concentration of internalized sugar would trigger PstS expression in order for inorganic phosphate molecules to be captured from the environment.
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ACKNOWLEDGEMENTS |
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Received 22 February 2005;
revised 27 April 2005;
accepted 29 April 2005.
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