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Journal of Bacteriology, February 2007, p. 741-749, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.00891-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Phosphoinositides Are Involved in Control of the Glucose-Dependent Growth Resumption That Follows the Transition Phase in Streptomyces lividans{triangledown}

H. Chouayekh,1 H. Nothaft,2 S. Delaunay,3 M. Linder,3 B. Payrastre,4 N. Seghezzi,5 F. Titgemeyer,2 and M. J. Virolle5*

Laboratoire d'Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax, Route de Sidi Mansour Km6, BP K, 3038 Sfax, Tunisia,1 Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany,2 Laboratoire des Sciences du Génie Chimique, Ecole Nationale Supérieure d'Agronomie et des Industries Alimentaires BP 172, 54505 Vandoeuvre les Nancy, France,3 Département d'Oncogénèse et Signalisation dans les Cellules Hématopoiétiques, 31024 Toulouse, France,4 Laboratoire de Métabolisme Energétique des Streptomyces, Institut de Génétique et Microbiologie, Université Paris XI, 91405 Orsay, France5

Received 21 June 2006/ Accepted 10 November 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The interruption of the sblA gene of Streptomyces lividans was previously shown to lead to relief of glucose repression of the normally strongly glucose-repressed {alpha}-amylase gene. In addition to this relief, an early entry into stationary phase was observed when cells were grown in a minimal medium containing glucose as the main carbon source. In this study, we established that this mutant does not resume growth after the transition phase when cultured in the complex glucose-rich liquid medium R2YE and sporulates much earlier than the wild-type strain when plated on solid R2YE. These phenotypic differences, which were abolished when glucose was omitted from the R2YE medium, correlated with a reduced glucose uptake ability of the sblA mutant strain. sblA was shown to encode a bifunctional enzyme possessing phospholipase C-like and phosphoinositide phosphatase activities. The cleavage of phosphoinositides by SblA seems necessary to trigger the glucose-dependent renewed growth that follows the transition phase. The transient expression of sblA that takes place just before the transition phase is consistent with a regulatory role for this gene during the late stages of growth. The tight temporal control of sblA expression was shown to depend on two operator sites. One, located just upstream of the –35 promoter region, likely constitutes a repressor binding site. The other, located 170 bp downstream of the GTG sblA translational start codon, may be involved in the regulation of the degradation of the sblA transcript. This study suggests that phosphoinositides constitute important regulatory molecules in Streptomyces, as they do in eukaryotes.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In spite of a large number of studies, the molecular basis of the phenomenon of glucose catabolite repression in the genus Streptomyces has yet to be established. The mutants isolated as resistant to glucose repression mainly affect the uptake of glucose via the DasABC transporter in Streptomyces griseus (39) or via the major facilitator GlcP1 in Streptomyces coelicolor (39, 49) or affect the first step of glucose metabolism by the glucose kinase encoded by glkA (2) or, perhaps, affect the regulation of both glucose uptake and glucose kinase activity by the product of SCO2127 (16). However, other mutants whose default in relation to glucose repression was less obvious were also characterized (8, 17, 20, 33). Our scientific interest for this subject led us to isolate mutants of Streptomyces lividans TK24 resistant to glucose catabolite repression by using transposon mutagenesis. We isolated and characterized a mutant that showed the expected relief of glucose repression of the expression of a normally strongly glucose-repressed alpha-amylase gene (14). In this mutant, the gene sblA (equivalent to SCO0479 of S. coelicolor) was interrupted. This gene was predicted to encode a protein of 274 amino acids bearing some similarities to phosphatases of the inositol phosphate monophosphatase family (14). Furthermore, the sblA mutant strain was shown to enter prematurely into stationary phase when grown in a minimal medium containing glucose as a main carbon source. In contrast, very little or no difference in the growth profile of the wild-type and mutant strains was observed when cells were grown in the presence of glycerol (14). In order to get a better understanding of the role of sblA in the phenomenon of glucose repression, we assessed the growth characteristics and glucose uptake abilities of the wild-type and the sblA mutant strains of S. lividans TK24 grown in the complex liquid or solid medium R2YE, rich or poor in glucose. We determined the enzymatic function and the substrate specificity of SblA, using in vitro and in vivo approaches. In addition, we identified some cis-acting regulatory sequences that play a role in the tight temporal control of sblA expression, using appropriate transcriptional fusions of its 5' region with the reporter genes luxA and luxB from Vibrio harveyi.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and plasmids. The Streptomyces strains used were the wild-type strain S. lividans TK24 (19), the S. lividans TK24 sblA::{Omega}aac strain (14) and the S. lividans TK24 EcoRI::{Omega}aac sblA (this study) strain derived from it as well as S. lividans TK24 carrying the replicative vector pHC1002 or the integrative vectors pHC1013, pHC1023, pHC1053, pHC1063, and pHC1073 (Fig. 1).


Figure 1
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  		FIG. 1. Schematic representation of the inserts of the various plasmids used in this study. The black arrow represents sblA, the hatched arrow represents the {Omega}aac cassette conferring resistance to apramycin, and the gray arrow represents the reporter genes luxA and luxB from V. harveyi. Useful restriction sites are indicated. The EcoRI site with an asterisk (EcoRI*) was created by PCR as described in Material and Methods.

 
(i) Construction of the S. lividans EcoRI::{Omega}aacsblA strain. To construct S. lividans EcoRI::{Omega}aacsblA, seven of the nine direct repeats (DRs) of the sequence 5'-C(C/G)GGAGG(C/T)-3', located upstream of the –35 promoter sequence, were moved away from the sblA –35 promoter region using the following strategy. The EcoRI site of the polylinker of pHC1000 (Table 1) was filled in with the Klenow fragment, yielding pHC1019. Then, a novel EcoRI site was introduced 11 bp upstream of the –35 promoter region of sblA, in the seventh DR, using the following strategy. Two PCRs using pHC1000 as a matrix were performed: in reaction 1 the universal reverse primer (–48) and primer L harboring an EcoRI site (Table 2) were used; in reaction 2 the universal primer (–47) and primer R harboring an EcoRI site (Table 2) were used. PCR fragment 1 was cleaved by SphI (site located upstream of the nine DRs) (Fig. 1) and EcoRI, and PCR fragment 2 was cleaved by EcoRI and BamHI (internal to sblA) (Fig. 1). Both of the resulting 193-bp SphI-EcoRI and 328-bp EcoRI-BamHI fragments were cloned into pHC1019, replacing the original 521-bp SphI-BamHI fragment (Fig. 1) and yielding pHC1020. The {Omega}aac cassette from pHP45{Omega}aac conferring resistance to apramycin was then cloned into the EcoRI site of pHC1020, yielding pHC1021. Then, the SphI-Asp718 (site of the polylinker) fragment of pHC1021 and the EcoRI-SphI fragment from pHC1030 (Fig. 1) were cloned into pIJ2925 cut by EcoRI and Asp718, yielding pHC1031 (Fig. 1). The BglII insert of pHC1031 was then transferred into the thermo-sensitive vector pGM160{Delta} (29) cut by BamHI to yield pHC1032. This plasmid was used to replace the wild-type sblA gene by its disrupted version using the usual procedure (29). The chromosomal structure of the recombinants originating from double crossover events was verified by Southern analysis.


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  		TABLE 1. Phage and plasmids from E. coli and Streptomyces used in this study

 

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  		TABLE 2. Sequence of the PCR primers used in this study and their utilization

 
(ii) Introduction of various mutations in the stem-loop structure containing the sequence AGGAGG present 170 bp downstream of the SblA translational start codon. The different primers used are listed in Table 2. In one case, the stem-loop was destroyed and an XbaI site (TCTAGA) was created as described below. The following two PCRs were performed using pHC1000 as the matrix. In reaction 1 primer D and primer I harboring an XbaI site were used. In reaction 2, primer J and primer K harboring an XbaI site were used PCR fragment 1 was cleaved by AgeI (internal to sblA) (Fig. 1) and XbaI, and PCR fragment 2 was cleaved by XbaI and BamHI (internal to sblA) (Fig. 1). Both the resulting 156-bp AgeI-XbaI and 95-bp XbaI-BamHI fragments were cloned into pHC1000 in place of the original 251-bp AgeI-BamHI DNA fragment, yielding pHC1050 (in which the stem-loop was destroyed [SL–] and the AGGAGG sequence was intact).

In another case, the sequence AGGAGG was replaced by the sequence CTCGAG constituting an XhoI site, and compensatory mutations were introduced in the right part of the stem of the stem-loop in order to preserve the possibility of formation of the secondary structure. Two PCRs were performed using pHC1000 as the matrix: in reaction 1 primer D and primer M harboring an XhoI site were used; in reaction 2, primer J and primer N harboring an XhoI site were used. The PCR fragment 1 was cleaved by AgeI (internal to sblA) and XhoI, and PCR fragment 2 was cleaved by XhoI and BamHI (internal to sblA). Both the resulting 145-bp AgeI-XhoI and 106-bp XhoI-BamHI fragments were cloned into pHC1000 in place of the original 251-bp AgeI-BamHI DNA fragment yielding pHC1060 (in which the sequence of the stem-loop [SL*] and the AGGAGG sequence were changed).

In the last case, the sequence AGGAGG was replaced by the sequence CTCGAG constituting an XhoI site, but no compensatory mutations were introduced in the right part of the stem; thus the formation of the stem-loop was impaired. The following two PCRs were performed using pHC1000 as the matrix: in reaction 1 primer D and primer M harboring an XhoI site were used; in reaction 2, primer J and primer O harboring an XhoI site were used. PCR fragment 1 was cleaved by AgeI (internal to sblA) and XhoI, and PCR fragment 2 was cleaved by XhoI and BamHI (internal to sblA). Both the resulting 145-bp AgeI-XhoI and 106-bp XhoI-BamHI fragments were cloned into pHC1000 in place of the original 251-bp AgeI-BamHI DNA fragment yielding pHC1070 (in which the stem-loop was destroyed [SL–] and the AGGAGG sequence was changed to CTCGAG).

(iii) Construction of the transcriptional fusions of the wild-type and the mutant sblA 5' regions with the reporter genes luxA and luxB. First, the 2,794-bp partial BamHI-SacI DNA fragment from M13luxAB12 carrying the V. harveyi luxA and luxB reporter genes optimized for the Streptomyces codon usage (generous gift of K. Chater) was cloned into pIJ2925 (22) cut by BamHI and SacI, yielding pHC1010. Subsequently, the 2,809-bp BamHI-BglII luxA and luxB insert of pHC1010 was cloned into pHC1000 cut by BamHI to yield pHC1011. In this construct, the expression of luxA and luxB is under the control of the native sblA promoter region. The {Omega}aac cassette (6) conferring resistance to apramycin was cloned, as a BamHI blunt-ended fragment, in a region 3' of the luxA and luxB reporter genes, into the blunt-ended SacI site of pHC1011, yielding pHC1012. The 5,777-bp BglII insert of pHC1012 was then cloned into pTO1 (a {Phi}C31-derived integrative vector) (42) cut by BamHI to give pHC1013. The presence of the {Omega}aac cassette confers better stability to pTO1-derived plasmids.

In order to put the expression of luxA and luxB under the control of the "distanced operator" sblA promoter region, the original 270-bp SphI-AgeI DNA fragment from pHC1011 was replaced by the "equivalent" 2,053-bp SphI-AgeI DNA fragment from pHC1021, yielding pHC1022. The 5,797-bp BglII insert of pHC1022 was then cloned into pTO1 (42) cut by BamHI to give pHC1023.

In order to put the expression of luxA and luxB under the control of each of the three different sblA 5' regions carrying various mutations in the stem-loop structure and/or in the AGGAGG sequence, the original 521-bp SphI-BamHI DNA fragment from pHC1011 was replaced by the equivalent 521-bp SphI-BamHI DNA fragment from pHC1050, pHC1060, and pHC1070, yielding pHC1051, pHC1061, and pHC1071, respectively. Then the original 2,171-bp SphI-NsiI (site present in luxA and luxB) fragment from pHC1012 was replaced by the equivalent 2,171-bp SphI-NsiI fragment from pHC1051, pHC1061, and pHC1071 to yield pHC1052, pHC1062, and pHC1072, respectively. Then the 5,777-bp BglII inserts from pHC1052, pHC1062, and pHC1072 were cloned into pTO1 (42) cut by BamHI to give pHC1053, pHC1063, and pHC1073, respectively.

The various pTO1 constructs were inserted as one copy into the chromosome of S. lividans TK24, resulting in the S. lividans TK24 strains carrying the following plasmids with the indicated stem-loop structures and sequences: S. lividans TK24(pHC1013) (SL+; AGGAGG), S. lividans TK24(pHC1023), S. lividans TK24(pHC1053) (SL–; AGGAGG), S. lividans TK24(pHC1063) (SL*; CTCGAG, and S. lividans TK24(pHC1073) (SL–; CTCGAG).

(iv) Construction of the expression plasmid pHC1002. In order to overexpress and purify the protein SblA fused to an His tag, the expression of sblA was placed under the control of the nosiheptide-inducible tipA promoter (28). To do so, sblA was amplified by PCR using primer D harboring an NdeI site and primer E harboring an EcoRI site. The PCR fragment was cleaved with NdeI and EcoRI and cloned into pIJ4123 in place of the red D gene (43) to yield pHC1002.

Media. The growth media used in this study were either the classical R2YE in which 1% (wt/vol) glucose was added before autoclaving (18) or the same medium with no glucose added. The assay of the concentration of glucose in the glucose-containing R2YE medium used in this study was surprisingly found to be 2.7% (150 mM instead of the 55 mM expected). We believe that the extra glucose found originates from the hydrolysis of sucrose (present at 103 g liter–1) during autoclaving in acidic conditions. This medium is called the glucose-rich R2YE medium in the text. On the other hand, in the R2YE medium prepared without any added glucose, approximately 0.7% glucose was found whereas 1.7% derived from sucrose was expected. This discrepancy suggests that the hydrolysis of sucrose might vary under slightly different autoclaving conditions (different positions of the flasks in the autoclave and/or different cooling times, e.g.) or that the presence of glucose promotes sucrose hydrolysis during autoclaving. The latter medium is called the glucose-poor R2YE medium in the text.

Determination of growth, glucose consumption, and glucose transport abilities. In order to follow growth and glucose consumption of S. lividans TK24 and of the sblA mutant (see Fig. 2A), the sblA mutant was inoculated (106 spores/ml) in liquid glucose-rich R2YE medium and grown with shaking at 30°C. One-milliliter samples were taken every 3 h, centrifuged at 13,000 rpm for 5 min, and assayed for glucose (GAGO20 kit; Sigma). Mycelial pellets, resuspended in 1 ml of sterile water, were broken in the presence of glass beads, using a Bio 101 Savant Fast Prep system, and used for protein quantification with the Bradford reagent.


Figure 2
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  		FIG. 2. Growth curves of S. lividans TK24 (black diamonds and continuous line) and TK24 sblA::{Omega}aac (gray squares and continuous line) and glucose concentration of the growth medium related to protein concentration of S. lividans TK24 (black diamonds and dotted line) and TK24 sblA::{Omega}aac (gray squares, dotted line) grown in glucose-rich liquid R2YE medium (A) or in glucose-poor liquid R2YE medium (B).

 
For growth curve and glucose transport assays (see Fig. 2A and B), spores of S. lividans TK24 and of the sblA mutant were pregerminated and used at 7.5 x 107 ml–1 to inoculate 50 ml of R2YE medium without sucrose and grown for 15 h at 30°C. Cells were then washed twice in R2YE medium, and 100 mg of mycelia was inoculated in 1 liter of R2YE medium without sucrose in Erlenmeyer flasks supplemented with 1% (wt/vol) glucose. Cells were grown with vigorous shaking at 30°C, and aliquots of 10 ml were taken every 4 h and centrifuged at 13,000 rpm for 5 min. Pellets were weighed after drying in a Speed Vac concentrator for 30 min at 50°C using an analytical scale (Sartorius). Transport assays were performed following a method described previously (37). Mycelia were harvested every 4 h by centrifugation, washed twice in ice-cold transport buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 10 mM KCl), and adjusted to 1.0 to 1.5 mg of dry weight ml–1 (for determination of dry weight ml–1, see above). Cell suspensions were prewarmed to 30°C for 10 min prior to initiation of uptake assays. Uptake was initiated by the addition of [14C]glucose (Amersham) at a final concentration of 20 µM and a specific radioactivity of 6.1 mCi mmol–1. Samples were taken every 20 s up to 120 s in order to determine the optimal length of the incubation. Incubation of 1 min was shown to correspond to a steady state; thus 0.5-ml samples were taken after 1 min of incubation at 30°C, rapidly filtered through nitrocellulose filters (NC45; Schleicher & Schüll), and promptly washed twice with 3 ml of chilled 0.1 M LiCl. Radioactivity was determined by scintillation oscillography with 3 ml of scintillation solution per filter, and uptake rates were calculated in nmol mg of dry weight–1 min–1 from 24 to 40 h of growth.


Figure 3
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  		FIG. 3. Lawns of S. lividans TK24 and TK24 sblA::{Omega}aac grown on glucose-rich or glucose-poor R2YE medium. Plates were incubated for 72 h at 30°C. Brown and smooth aspect, vegetative mycelium; white and fluffy aspect, nonsporulating aerial mycelium; gray and fluffy aspect, sporulated aerial mycelium.

 
Overexpression and purification of His-tagged SblA. S. lividans TK24 (pHC1002) was cultivated for 40 h at 30°C in 600 ml of yeast extract-malt extract medium with kanamycin (10 µg ml–1); thiostrepton was then added at a concentration of 10 µg ml–1 to induce protein production, and growth was continued for a further 10 h. Mycelium was centrifuged for 15 min at 8,000 rpm, resuspended in 80 ml of buffer A (60 mM Tris-HCl, pH 7.5, 250 mM KCl, 15 mM imidazole, 10% glycerol) and broken with a French press. Soluble fractions obtained after centrifugation for 20 min at 12,000 rpm were filtered with a 0.45-µm-pore-size filter and loaded on an XK 16/20 chromatography column (Pharmacia) packed with chelating Sepharose Fast Flow, charged with 100 mM NiCl2 (pH 7.2), and equilibrated with buffer A. Purification was automated with an ÁKTA FPLC purifier (Pharmacia). The column was washed with buffer A, and His-tagged SblA was eluted with an imidazole gradient of 15 mM (buffer A) to 500 mM (buffer B). Fractions containing purified SblA were concentrated using a Millipore ultrafree-15 centrifuge filter with a Biomax-10K membrane (Sigma) and desalted by passage through an Econo-Pac 10 DG disposable chromatography column (Bio-Rad), preequilibrated with 30 ml of buffer C (30 mM Tris-HCl, pH 8, 50 mM KCl, 5 mM MgCl2, 20% glycerol). Purified SblA was recovered in 3 ml of buffer C and stored at –70°C in 50-µl aliquots.

Determination of SblA enzymatic activity and substrate specificity. The enzymatic activity of purified SblA was determined by incubating SblA (1 µg, or 33 pmol) for 20 min at 37°C under shaking with 1.5 µg of fluorescent di-C6-NBD {D(+)-sn-1-O-[1-[6'-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl]amino]-hexanoyl]-2-O-hexanoylglyceryl} phosphoinositides (Echelon Research Laboratories) in a buffer containing 50 mM ammonium acetate (pH 7) and 2 mM dithiothreitol (45). The reaction products were separated by thin-layer chromatography using CHCl3-CH3OH-CH3COCH3-CH3COOH-H2O (70/50/20/20/20, by volume) as the solvent and visualized under UV light (see Fig. 4A).


Figure 4
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  		FIG. 4. (A) Growth curves of S. lividans TK24 (black diamonds) and TK24 sblA::{Omega}aac (gray squares) in liquid R2YE medium without sucrose and supplemented with 1% (wt/vol) glucose, and [14C]glucose uptake of mycelia of TK24 (black bars) and TK24 sblA::{Omega}aac (gray bars). Uptake rates were determined every 4 h in two independent experiments from 24 h to 40 h of growth. (B) Detailed kinetics of glucose uptake of mycelia of S. lividans TK24 (black diamonds) and TK24 sblA::{Omega}aac (gray squares), grown for 40 h in R2YE medium without sucrose and supplemented with 50 mM glucose.

 
Determination of the in vivo phosphoinositides content. A total of 200 ml of a preculture of each strain, grown for 24 h at 28°C in shake flasks, was used to inoculate 4.8 liters of medium containing 4.05 mM MgSO4 · 7H2O, 0.05 mM ZnSO4 · 7H2O, 1.4 mM KH2PO4, 342 mM NaCl, 0.001 mM CoCl2 · 6H2O, 30 g liter–1 of white dextrin, 3.7 mM NH4Cl, and 6.8 mM CaCl2 · 2H2O in a bioreactor (Applikon, Schiedam, The Netherlands). The precultures were carried out in the same medium except that CaCl2 was replaced by 5 g liter–1 of CaCO3 The cultures were grown at 28°C with aeration at 1 vvm (air volume/working volume/minute), and the pH was maintained at 7 with KOH. Membrane lipids were extracted as previously described (5, 35).

In order to determine the phosphoinositide content by thin-layer chromatography, coupled to a flame ionization detector, extracted lipids were suspended in 500 µl of methanol-chloroform (2:1, vol/vol). An aliquot (1 to 3 µl) of lipids was spotted on a quartz rod coated with silica gel (Chromarod-SIII; Iatron Laboratories, Tokyo, Japan). After 15 min in an NaCl-saturated atmosphere, migrations were performed in chloroform-methanol-32% ammonium (170:30:10) for 45 min. After drying for 1 min at 100°C, the rods were scanned using a thin-layer chromatography-flame ionization detector system (Iatroscan MK-5; Iatron Laboratories) at 30 s per scan, a hydrogen flow rate of 160 ml min–1, and an airflow rate of 2,000 ml min–1. At least six analyses were performed for each sample. The phosphatidylinositol peak was identified by simultaneous injection of a standard. Peak areas were quantified using ChromStar integrator software (version 4.14; Iatron Laboratories). These areas were found to be proportional to the spotted quantity of phosphatidylinositol.

Determination of the luciferase activity. In order to assay the luciferase activity resulting from the sblA/luxA and sblA/luxB transcriptional fusions, 106 spores of the various S. lividans TK24 strains were spread on the surface of cellophane disks (Cannings Packaging Limited, United Kingdom) laid down on the surface of glucose-rich R2YE agar plates (18). Squares of mycelium of a defined area were cut out at regular intervals during growth, suspended in 250 µl of H2O containing a mixture of protease inhibitors (Sigma), and thoroughly homogenized. Luciferase activity was measured with a Berthold LUMAT LB 95501 luminometer by mixing instantaneously 100 µl of the homogenized mycelium and 100 µl of 0.1% N-decyl aldehyde emulsified by sonication. After extensive sonication of the mycelial samples, protein concentration, as a measure of growth, was determined with the Bradford reagent (Bio-Rad). Luciferase activity was then expressed in arbitrary units of luciferase (AUL) mg of protein–1.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth characteristics, glucose consumption, and phenotype of S. lividans TK24 and of the sblA mutant strain. The early growth arrest that characterizes the sblA mutant strain was previously established in a liquid minimal medium containing glucose as main carbon source (14). To further explore the phenotype of the sblA mutant, we grew cultures of S. lividans TK24 and TK24 sblA::{Omega}aac in the more complex and rich R2YE liquid medium with or without added glucose. Proteins and glucose consumption were assessed during growth. Results shown in Fig. 2A indicate that growth of S. lividans TK24 showed the expected profile (15): a first phase of rapid growth up to 28 h, a transition phase between 28 h and 31 h, and then a second phase of renewed growth continuing for a few hours before entry into stationary phase. Surprisingly, the sblA mutant strain did not resume growth after the transition phase. Figure 2A shows that the wild-type and the sblA mutant strains consume glucose at approximately the same rates, up to the 30th hour of growth. Then, from the 30th hour of growth, when the concentration of glucose in the growth medium approximates 10 g liter–1 (approximately 2/3 of initial glucose was consumed), glucose consumption slowed down considerably in the wild type and completely stopped in the sblA mutant strain. In the glucose-poor R2YE medium, both strains showed a similar growth pattern and consumed glucose at the same rates (Fig. 2B). Curiously, both strains entered prematurely into stationary phase when grown in this amino acid-rich but glucose-poor R2YE medium. They stopped growing when they reached a level of biomass comparable to that of the wild-type strain grown in the glucose-rich R2YE medium, at the transition phase (Fig. 2B). These observations suggest that the growth resumption that follows the transition phase relies on the presence of glucose since it is not observed in the glucose-poor R2YE medium (Fig. 2B). This growth resumption is also clearly SblA dependent since it is not observed in the sblA mutant strain (Fig. 2A).

Similarly, when both strains were plated on glucose-rich solid R2YE medium, the sblA mutant sporulated much earlier than the original strain S. lividans TK24 (Fig. 3). The early growth arrest of the sblA mutant strain observed in glucose-rich liquid R2YE medium obviously correlates with an early triggering of the sporulation process on glucose-rich solid R2YE. The "omission" of glucose from the R2YE medium almost completely abolished the phenotypic differences existing between the two strains, with both sporulating early. These observations suggest that a high glucose level represses or delays the sporulation process and that SblA plays a role in this repression. Since the phenotypes of the sblA mutant strain might be explained by a default in glucose uptake, the glucose uptake abilities of the two strains were assessed during growth.

Glucose uptake abilities of S. lividans TK24 and of the sblA mutant strain. The relative abilities to take up glucose (see Materials and Methods) of cultures of each strain grown for 24, 28, 32, 36, and 40 h are shown in Fig. 4A, and the detailed kinetics of glucose uptake of cultures grown for 40 h in glucose is given in Fig. 4B. Figure 4A shows that the kinetics of glucose uptake of both strains are comparable at 24, 28, and 32 h, whereas at 36 and 40 h (time points where sblA expression is switched on in the wild-type strain), the ability of the sblA mutant strain to transport glucose is approximately 1.5-fold lower than that of the wild-type strain (Fig. 4B). Altogether, these observations suggest that SblA has a positive effect on glucose uptake. In order to clarify the regulatory role that SblA might exert on glucose uptake, SblA was overproduced as a histidine-tagged fusion protein in S. lividans TK24; the protein was purified, and its enzymatic function and substrate specificity were determined.

Enzymatic function and substrate specificity of SblA. Since sblA was predicted to encode a protein bearing similarities to phosphatases of the inositol phosphate monophosphatase family (14), we assayed its putative phosphatase activity using the chromogenic substrate para-nitro-phenyl phosphate and then different phosphorylated substrates, including various phosphoinositides (phosphatidylinositol, phosphatidylinositol-4-P, and phosphatidylinositol-4,5-biP), in addition to other phospholipids, such as phosphatidylethanolamine and phosphatidylglycerol (data not shown). SblA was shown to liberate phosphate specifically from phosphoinositides, and this activity was confirmed using fluorescent di-C6-NBD phosphoinositides (Echelon Research Laboratories) in which the fluorescent dye is linked to the acyl chain of the molecule. Figure 5A shows that SblA is able to hydrolyze the phosphodiester bond between the diacylglycerol (DAG) moiety and the inositol moiety, leading to the production of a compound migrating at the front of the solvent of the thin-layer chromatography at the expected position of DAG. Thus, SblA appears to possess a phospholipase C-like activity. Moreover, the detection of phosphoinositides containing less phosphate than the original substrates used (Fig. 5A, PI and PIP) and migrating slightly faster than the latter strongly suggested that SblA also possesses a phosphoinositide phosphatase activity. This enzyme, therefore, can be considered a bifunctional enzyme with phospholipase C-like and phosphoinositide phosphatase activities.


Figure 5
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  		FIG. 5. (A) In vitro assay of SblA activity. Fluorescent di-C6-NBD phosphoinositides, phosphatidylinositol-3-P and -4-P [PI(3)P and PI(4)P, respectively], and phosphatidylinositol-4,5-biP [PI(4,5)P2] (1.5 µg each) from Echelon Research Laboratories were incubated for 20 min at 37°C under shaking, with 30 picomoles of purified SblA (10 µl) in 50 mM ammonium acetate (pH 7), plus 2 mM dithiothreitol. The reaction products were separated by thin-layer chromatography, using CHCl3-CH3OH-CH3COCH3-CH3COOH-H2O (70/50/20/20/20, by volume) as a solvent and visualized under UV light. PI, phosphatidylinositol; PIP, phosphatidylinositol monophosphate. (B) Indirect in vivo assay of SblA activity. The amount of PIs was followed throughout the growth of batch cultures of the wild-type strain S. lividans TK24 (gray circles), TK24 sblA::{Omega}aac (crosses), and TK24 EcoRI::{Omega}aac sblA (black rectangles). The PIs are expressed as percentages of total extracted phospholipids.

 
In order to confirm, in vivo, the enzymatic function of SblA, we checked the effect of the interruption or overexpression of sblA on the phosphoinositide (PI) content of the bacteria. PIs were prepared as described in Materials and Methods from S. lividans TK24, the sblA mutant strain (14), and S. lividans TK24 EcoRI::{Omega}aac sblA. The presence of SblA was detected in TK24 EcoRI::{Omega}aac sblA using polyclonal antibodies against SblA but was not detected in the wild-type strain (data not shown), indicating that, as expected from results shown in Fig. 6, S. lividans TK24 EcoRI::{Omega}aac sblA overproduces SblA. The important decrease in PI shown in the three strains around the 40th hour of growth was also found for other phospholipids (data not shown) and coincides with the transition phase. During the transition phase, the bacteria stops growing, and an extensive degradation of its macromolecules, obviously including phospholipids, is triggered (15). Results shown in Fig. 5B confirmed that PIs were more abundant in the wild-type and the sblA mutant strains than in the strain where SblA was overexpressed. These results confirmed that SblA acts as a phosphoinositide hydrolase in vivo.


Figure 6
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  		FIG. 6. (A) Schematic representation of the transcriptional fusions of the wild-type sblA (pHC1013) and the EcoRI::{Omega}aac sblA (pHC1023) promoter regions with the reporter system luxA and luxB from V. harveyi. The two putative operator regions are represented by nine arrowheads upstream of the sblA promoter region and by two inverted arrowheads downstream of the transcriptional start site, represented by an arrow. (B) Growth curves of S. lividans TK24 (pHC1013) (black diamonds) and TK24 (pHC1023) (gray squares) and luciferase activity (expressed as AUL mg of protein–1) from samples of S. lividans TK24 (pHC1013) (black bars) and TK24 (pHC1023) (gray bars) grown on glucose-rich R2YE solid medium.

 
Temporal pattern of sblA expression. In order to determine the temporality of the expression of this gene during growth, an appropriate transcriptional fusion of the sblA 5' region with the reporter genes luxA and luxB from V. harveyi was constructed and integrated as one copy in the chromosome of S. lividans TK24 via the attachment site of PhiC31. Growth of this strain, TK24(pHC1013), and luciferase activity that reflects the expression of sblA were followed throughout the developmental cycle by cutting out squares of mycelium of a defined area. Figure 6B clearly shows that when TK24(pHC1013) (black bars) is grown on solid glucose-rich R2YE medium, the expression of sblA is transitory (4 to 5 h), taking place mainly toward the middle of the first growth phase and switched off just before the transition phase. Since the expression of sblA is tightly temporally regulated, we inspected the 5' region of sblA for putative operator-like sequences likely to play a role in sblA regulation. A putative operator-like structure was found upstream, and another was found within the sblA coding sequence (Fig. 6A). The former, upstream of the –35 sblA promoter region, was constituted by nine DRs of the sequence 5'-C(C/G)GGAGG(C/T), and the other operator-like sequence, located 170 bp downstream of the sblA translational start codon, could form a stem-loop structure containing the sequence AGGAGG, resembling a ribosome binding site (RBS). The putative regulatory role of these operator-like sequences was then tested as described below.

Role of putative operator sites in the regulation of sblA expression. In order to assess the role of the upstream operator-like structure, the latter was moved away from the promoter region by cloning a cassette conferring resistance to apramycin after the seventh repetition (plasmid pHC1023). Growth of S. lividans TK24(pHC1013) and S. lividans TK24(pHC1023), as well as the luciferase specific activity, was followed throughout the developmental cycle as described above. The growth profiles of the two strains were comparable and showed the expected pattern. Figure 6B (gray bars), clearly indicates that in S. lividans TK24(pHC1023), the displacement further upstream from the –35 promoter of the putative operator sequence led to a two- to threefold enhancement of the expression of sblA, which also lasted longer (10 to 12 h) compared to the wild-type situation. This site is thus likely to constitute a transcriptional repressor binding site. However, the higher and longer expression of sblA in S. lividans TK24(pHC1023) was still strongly reduced before the start of the second growth phase (Fig. 6B), suggesting the existence of another regulatory mechanism that might be specifically involved in the switch off of sblA expression.

Therefore, we investigated a possible regulation of sblA through the operator-like sequence located within the sblA coding region. We constructed three plasmids (pHC1053, pHC1063, and pHC1073) in which the putative internal operator site was altered (Fig. 7A). In pHC1053 (dark gray bars) the stem-loop was destroyed and in pHC1063 (light gray bars), the sequence AGGAGG was replaced by the sequence CTCGAG, and compensatory changes were introduced in the right-hand part of the stem in order to preserve the possibility of the formation of a stem-loop. In pHC1073 (white bars), the sequence AGGAGG was replaced by the sequence CTCGAG, but since no compensatory changes were introduced in the right-hand part of the stem, the possibility of formation of a stem-loop was impaired. S. lividans TK24 strains containing either pHC1013, pHC1053, pHC1063, or pHC1073 were grown on glucose-rich R2YE agar plates, and luciferase measurements were carried out during growth. The results presented in Fig. 7B demonstrate that the different alterations led to an enhancement of the expression of sblA during the first phase of growth and to the subsequent persistence of sblA expression during the transition phase and the second phase of growth. The combination of both types of alterations seems to have a cumulative effect on sblA expression. These results suggested that both the stem-loop and the putative RBS were important for the negative control of sblA expression and, more precisely, for the switch off of sblA expression toward the end of the first growth phase.


Figure 7
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  		FIG. 7. (A) Schematic representation of the modifications introduced by site-directed mutagenesis into the stem-loop structure (SL+, SL–, and SL*) including the AGGAGG sequence present 170 bp downstream of the sblA GTG translational start codon. (B) Growth curves of S. lividans TK24 containing either pHC1013 (black squares), pHC1053 (dark gray squares), pHC1063 (light gray squares), or pHC1073 (white squares) grown on glucose-rich R2YE solid medium and luciferase activity (expressed in AUL mg of protein–1) from samples of S. lividans TK24 containing either pHC1013 (black bars), pHC1053 (dark gray bars), pHC1063 (light gray bars), or pHC1073 (white bars) grown on glucose-rich R2YE solid medium.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Streptomycetes are a group of high-GC DNA, gram-positive, filamentous bacteria living in the superficial layers of the soil. In liquid, as in solid medium, the growth of Streptomyces was reported to be multiphasic (15, 52), starting with a phase of active growth that ends with a transitory growth arrest called either the "transition phase" (15) or the "diauxic lag" (32, 50, 51). Then growth resumes for a few hours before entry of the bacteria into stationary phase. The transitory growth arrest is thought to result from some nutritional limitations (52) and is characterized by extensive degradation of cellular macromolecules (15) and by the induction of the expression of proteins associated with specific stress-induced regulons (32). This short pause in growth is considered a key regulatory transition since it precedes a phase of renewed growth and the triggering of secondary metabolite production (51). However, very little is known about the regulatory mechanisms underlying the complex changes that are taking place during this transition phase. This study suggested that the previously characterized sblA gene of S. lividans (14) might be involved in the regulation of this process since the sblA mutant strain was shown to be unable to pursue growth beyond the transition phase in a minimal medium containing glucose as a main carbon source (14) and even in the complex medium R2YE rich in glucose (this study). The second phase of renewed growth is not observed when the wild-type strain is grown in a minimal medium containing glycerol as the main carbon source (14) or in the rich and complex medium R2YE poor in glucose (this study), suggesting a key role for glucose in this growth resumption.

On the glucose-rich solid R2YE medium, the sblA mutant develops aerial mycelium and sporulates much earlier than the wild-type strain, whereas the omission of glucose from the R2YE medium abolishes the phenotypic differences between the two strains that both sporulate early. An acceleration of aerial mycelium development and sporulation was previously reported to be conferred by the interruption of the gene cvnD9 of S. coelicolor encoding a G protein of the Ras family usually found in eukaryotes (25) or by the overexpression of the ram genes (24, 27) or of the mutant alleles Y21A and F75A of the bldB gene of S. coelicolor (12), which has been described as involved in carbon source sensing and/or utilization (33).

The early growth arrest that characterizes the sblA mutant strain grown in the glucose-rich R2YE medium was shown to correlate with a reduction in its capacity to take up glucose when the concentration of glucose reaches approximately 10 g liter–1. However, it is difficult to know whether the reduction in the glucose uptake ability is the cause or the consequence of the observed growth arrest of the sblA mutant strain. It was recently reported that in S. coelicolor, a close relative of S. lividans, glucose is mainly transported via the major facilitator GlcP1 (49). We can thus propose that during the first phase of growth, when the concentration of glucose is still high, glucose would be transported by GlcP1 (49) whereas when the concentration falls below a certain threshold, this uptake (and thus the continuation of growth) may necessitate either an increase in the abundance and/or in the affinity of GlcP1 for glucose (perhaps via a posttranslational modification of the latter?) or the induced synthesis of an alternative glucose transporter with a higher affinity for glucose than GlcP1. These hypothetical processes, aimed at stimulating glucose uptake, might be under the indirect control of sblA. However, since the sblA mutant was obviously shown to be able to take up glucose when the latter was present at a low concentration (less than 10 g liter–1) in the growth medium (Fig. 2B), it seems more reasonable to propose that SblA governs a complex regulatory cascade controlling, in addition to glucose uptake, many other processes necessary for growth resumption after the transition phase. The ability of SblA, to cleave or dephosphorylate signaling phospholipids of the phosphoinositide family suggests that it may play a rather complex regulatory role. In eukaryotes, phosphoinositides are very important signaling and regulatory molecules (40). Their cleavage by enzymes of the phospholipase C family leads to the production of secondary messengers, inositol-phosphate and DAG, involved in the regulation of various cellular processes including the modulation of actin cytoskeletal organization (38, 44), calcium flux (4, 23, 41), and phosphorylation of regulatory proteins (9, 34) as well as glucose transport (13, 26). The topology prediction for SblA using the TMPRED software (http://www.ch.embnet.org/software/TMPRED_form.html) suggests that this protein is indeed likely to be a membrane protein with three predicted transmembrane helices. A membrane location for SblA is consistent with its enzymatic activity since phosphoinositides are membrane components.

The temporal expression of sblA is consistent with a role of this gene during the late stages of growth since we established that the transient expression of sblA mainly occurred toward the middle of the first phase of growth and was switched off just before the transition phase. Tight temporal control of transitory sblA expression was shown to involve at least two cis-acting negative operator sequences: one constituted by nine DRs of the sequence 5'-C(C/G)GGAGG(C/T)-3' present just upstream of the –35 promoter region (the last copy overlapping the putative –35 sequence) and the other constituted by a 23-nucleotide-long putative stem-loop structure containing AGGAGG, resembling an RBS, present 170 bp downstream of the GTG start codon. The nine DRs are likely to constitute a transcriptional repressor binding site, but the precise molecular mechanisms by which the stem-loop-RBS structure achieves its negative regulatory role remain mysterious. However, it is noteworthy that this structure strongly resembles the binding site of the CsrA protein of Escherichia coli (36). CsrA is a small, RNA binding, regulatory protein that interacts with RBS-like sequences, inhibiting the binding of ribosomes and thus translation of the target transcript (10). The "naked" untranslated transcripts are thus more vulnerable to endo- and exo-ribonucleolytic attack and are more rapidly degraded (10, 11, 53). By analogy with this system, we may speculate that the abrupt switch off of sblA expression just before the transition phase might involve the specific degradation of the sblA transcript indirectly promoted by a CsrA-like RNA-binding protein interacting with the stem-loop-RBS structure.

In conclusion, this study strongly suggests that the cleavage of the signaling lipids, phosphoinositides, by SblA is necessary to trigger the glucose-dependent phase of renewed growth that follows the transition phase in S. lividans TK24. The previous characterization of genes encoding phospholipase C-like activities in Streptomyces species (21), as well as the production by several Streptomyces species of inhibitors of phosphatidylinositol turnover in eukaryotes (1, 30, 31, 46, 48), already suggested the likely existence of a eukaryotic-like phosphoinositide signaling pathway in Streptomyces. However, this study constitutes the first evidence that phosphoinositides are indeed playing important signaling and regulatory roles in Streptomyces, as they do in eukaryotes. Nevertheless, more work is obviously needed to clarify the regulatory role of phosphoinositides and of SblA in Streptomyces. Attempts will be made to elucidate the possible relationships of sblA with other genes playing a role in the regulation of the morphological differentiation process by glucose. It is noteworthy that some of these genes encode, similar to sblA, "eukaryotic-like" functions, such as cvnD9 mentioned above (25) or asfK encoding a eukaryotic-like protein serine/threonine kinase essential for morphogenesis in the presence of glucose (47). Altogether, these observations suggest that eukaryotic-like signaling and regulatory networks might be involved in the regulation by glucose of the late growth stages and of the morphological differentiation process in Streptomyces.


   ACKNOWLEDGMENTS
 
This work received support from France through UMR 8621 CNRS/University Paris XI and the "Institut National Polytechnique de Lorraine" and from Germany through grant SFB473 of the Deutsche Forschungsgemeinschaft.

We are most grateful to Barry Holland and Emmanuelle Darbon-Rongère for critical reading of the manuscript and stimulating discussions.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institut de Génétique et Microbiologie, UMR CNRS 8621, Université Paris XI, 91405 Orsay, France. Phone: 33 (0) 1 69 15 46 42. Fax: 33 (0) 1 69 15 46 29. E mail: marie-joelle.virolle{at}igmors.u-psud.fr. Back

{triangledown} Published ahead of print on 22 November 2006. Back


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Journal of Bacteriology, February 2007, p. 741-749, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.00891-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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