Identified members of the Streptomyces lividans AdpA regulon involved in differentiation and secondary metabolism

  • Aurélie Guyet1,

    Affiliated with

    • Nadia Benaroudj2,

      Affiliated with

      • Caroline Proux3,

        Affiliated with

        • Myriam Gominet1,

          Affiliated with

          • Jean-Yves Coppée3 and

            Affiliated with

            • Philippe Mazodier1Email author

              Affiliated with

              BMC Microbiology201414:81

              DOI: 10.1186/1471-2180-14-81

              Received: 8 November 2013

              Accepted: 27 March 2014

              Published: 3 April 2014

              Abstract

              Background

              AdpA is a key transcriptional regulator involved in the complex growth cycle of Streptomyces. Streptomyces are Gram-positive bacteria well-known for their production of secondary metabolites and antibiotics. Most work on AdpA has been in S. griseus, and little is known about the pathways it controls in other Streptomyces spp. We recently discovered interplay between ClpP peptidases and AdpA in S. lividans. Here, we report the identification of genes directly regulated by AdpA in S. lividans.

              Results

              Microarray experiments revealed that the expression of hundreds of genes was affected in a S. lividans adpA mutant during early stationary phase cultures in YEME liquid medium. We studied the expression of the S. lividans AdpA-regulated genes by quantitative real-time PCR analysis after various times of growth. In silico analysis revealed the presence of potential AdpA-binding sites upstream from these genes; electrophoretic mobility shift assays indicated that AdpA binds directly to their promoter regions. This work identifies new pathways directly controlled by AdpA and that are involved in S. lividans development (ramR, SLI7885 also known as hyaS and SLI6586), and primary (SLI0755-SLI0754 encoding CYP105D5 and Fdx4) or secondary (cchA, cchB, and hyaS) metabolism.

              Conclusions

              We characterised six S. lividans AdpA-dependent genes whose expression is directly activated by this pleiotropic regulator. Several of these genes are orthologous to bldA-dependent genes in S. coelicolor. Furthermore, in silico analysis suggests that over hundred genes may be directly activated or repressed by S. lividans AdpA, although few have been described as being part of any Streptomyces AdpA regulons. This study increases the number of known AdpA-regulated pathways in Streptomyces spp.

              Keywords

              Streptomyces lividans Microarrays AdpA bldA ramR hyaS CYP105D5 cchB Coelichelin

              Background

              Streptomycetes are Gram-positive soil bacteria that display a complex morphological and metabolic differentiation. Streptomyces develop branched hyphae that expand by tip extension to form a vegetative mycelium meshwork. In response to as yet unidentified signals and to nutritient depletion, aerial branches emerge from the surface of colonies and may produce spores. As the aerial mycelium develops, Streptomyces colonies produce diverse secondary metabolites and synthesise antibiotics [1]. This differentiation cycle can be reproduced in laboratory conditions by growing Streptomyces cells on solid media. Most Streptomyces species do not form aerial mycelium or spores when in liquid media (e.g. S. coelicolor and S. lividans), and antibiotic production occurs in submerged cultures [2].

              AdpA, also known as BldH, has been identified as a conserved major transcriptional regulator involved in the formation of aerial mycelia in various Streptomyces species [36]. AdpA is a member of the family of AraC/XylS regulator proteins that contain a C-terminal domain with two helix-turn-helix DNA-binding motifs; these features are strictly conserved in all Streptomyces AdpAs in the StrepDB database [7]. The N-terminal domain of AdpA is responsible for its dimerization and regulation [8, 9]. Protein/DNA interaction experiments identified the following consensus AdpA-binding site in S. griseus: 5′-TGGCSNGWWY-3′ (with S: G or C; W: A or T; Y: T or C; N: any nucleotide) [10].

              AdpA was discovered and has mostly been studied in S. griseus, in which it was first shown to activate expression of about thirty genes directly. They include genes encoding secreted proteins (e.g. proteases), a sigma factor (AdsA), a subtilisin inhibitor (SgiA), SsgA which is essential for spore septum formation and the AmfR transcriptional regulator involved in production of AmfS (known as SapB in S. coelicolor), a small hydrophobic peptide involved in the emergence of aerial hyphae [11, 12]. AdpA also plays a role in secondary metabolism and directly activates streptomycin biosynthesis [3].

              Proteomic, transcriptomic and ChIP-sequencing analyses revealed that, in fact, several hundred genes are under the control of S. griseus AdpA and that AdpA acts as transcriptional activator as well as repressor [1215]. In S. coelicolor, few genes have been identified as being directly regulated by AdpA: sti1 (sgiA orthologs), ramR (amfR orthologs), clpP1 (encoding a peptidase) [16] and wblA (encoding a transcriptional regulator) [15].

              The regulation of adpA gene expression is complex and various mechanisms have been described [17]. AdpA represses its own gene expression in S. griseus[18] whereas it activates its own transcription in S. coelicolor[16]. In several Streptomyces species, the binding of γ-butyrolactones to a γ-butyrolactone receptor represses the adpA promoter [19, 20]. In S. coelicolor, BldD represses adpA expression [21]. At the translational level, a feedback-control loop regulates levels of AdpA and AbsB (a RNAse III) in S. coelicolor[22, 23]. A positive feedback loop between AdpA and BldA, the only tRNA able to read the UUA codon present in all adpA mRNA, has been demonstrated in S. griseus[22, 23]. In S. coelicolor, adpA expression is constant during growth in liquid media [4] whereas on solid media, adpA is strongly expressed before aerial hyphae formation and AdpA is most abundant during the early aerial mycelium stage [4, 16].

              Even though AdpA plays a major role in development of Streptomyces spp., little is known about the pathways it controls in S. lividans, a species closely related to S. coelicolor and whose genome has recently been sequenced [24]. We have recently shown that in S. lividans AdpA directly controls sti1 and the clpP1clpP2 operon, encoding important factors for Streptomyces differentiation; we also found interplay between AdpA and ClpP1 [25]. Here, we report microarray experiments, quantitative real-time PCR (qRT-PCR), in silico analysis and protein/DNA interaction studies that identify other genes directly regulated by AdpA in S. lividans. Finally, in silico genome analysis allowed the identification of over hundred genes that are probably directly activated or repressed by AdpA in S. lividans. These findings and observations reveal new AdpA-dependent pathways in S. lividans.

              Methods

              Bacterial strains, growth conditions and media

              S. lividans 1326 was obtained from the John Innes Culture Collection. In this S. lividans background, we constructed an adpA mutant in which adpA was replaced with an apramycin-resistance cassette [25].

              Streptomyces was grown on NE plates [26] and in YEME liquid medium [27] in baffled flasks. MS medium was used for sporulation experiments [27]. Apramycin was added to final concentrations of 25 μg mL-1 to solid media and 20 μg mL-1 to liquid media as appropriate.

              Microarray experiments

              S. lividans microarrays were not available, so S. coelicolor oligonucleotide arrays covering most open reading frames (ORFs) of the genome (for array coverage and design, see [28, 29]) were used. Aliquots of 60 mL of liquid YEME medium were inoculated with about 108 spores and incubated at 30°C with shaking at 200 rpm until early stationary phase (about 30 h of growth). Samples of 12 mL of culture (at OD450nm = 2.3, corresponding to time point T on Figure 1a) were then collected and RNA extracted as previously described [30]. RNA quality was assessed with an Agilent 2100 Bioanalyser (Agilent Technologies). RNA indirect labelling and array hybridization were performed as described [31] and hybridized microarrays were scanned with a Genepix 4000A scanner (Molecular Devices).
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-14-81/MediaObjects/12866_2013_2232_Fig1_HTML.jpg
              Figure 1

              Effects of S. lividans adpA mutation on expression of selected genes. a. Growth curve of wild-type S. lividans (dashed line) and adpA mutant (solid line) in YEME liquid medium at 30°C with shaking at 200 rpm as followed by measuring absorbance at 450 nm. A, B, C, D and T indicate the time points when cultures were harvested for RNA extraction. Microarray experiments were performed on RNA samples extracted at time T. b. Change in gene expression S. lividans adpA mutant compared to the wild-type at each time point of growth. RNA was extracted from S. lividans wild-type 1326 and adpA mutant cells cultivated in liquid YEME medium after various times of growth (OD450nm of 0.3, 0.8, 1.5, 1.9 and 2.3, respectively, at time points A, B, C, D and T). Relative amounts of SLI0755, SLI6586, hyaS, cchA, cchB, ramR PCR product were measured by qRT-PCR. At each time point of growth, gene expression levels were normalized using hrdB as an internal reference and are indicated in this figure as the n-fold change in adpA mutant compared to the wild type. Results are expressed as means and standard deviations of at least three replicates. Data are representative of at least two independent experiments for each strain at each growth time. Note that a different scale is used for hyaS.

              Statistical analysis of array data

              R software [32] was used for normalization and differential analysis. A Loess normalization [33] was performed on a slide-by-slide basis (BioConductor package marray; [34]). A paired t-test was used for differential analysis. Variance estimates for each gene were computed under the hypothesis of homoscedasticity, together with the Benjamini and Yekutieli P-value adjustment method [35]. Only genes with a significant (P-value < 0.05) fold change (Fc) were taken into consideration. Empty and flagged spots were excluded, and only genes with no missing values were analysed. A few genes which displayed excessive variation were analysed using the Vmixt method from the VarMixt package [36]. We defined our cut-off for microarray data acquisition as Fc <0.625 or Fc > 1.6 with P-value < 0.05. The genome of S. lividans 1326 was sequenced only recently [24], so we used the StrepDB database [7], and in some cases a basic local alignment search tool (Blast), to identify S. lividans orthologs (SLI gene number) of S. coelicolor genes. We also used the protein classification scheme for the S. coelicolor genome available on the Welcome Trust Sanger Institute database [37].

              qRT-PCR analysis

              Oligonucleotide pairs specific for cchA (SLI0459), cchB (SLI0458), SLI0755, SLI6586, ramR (SLI7029), hyaS (SLI7885) and hrdB (SLI6088, MG16-17) (Additional file 1: Table S1) were designed using the BEACON Designer software (Premier BioSoft). RNA samples were extracted from cultures in YEME liquid medium at OD450nm values of about 0.3, 0.8, 1.5, 1.9 and 2.3 (time points A, B, C, D and T, respectively). Aliquots of 20 μg of RNA were treated twice with 2 Units of DNase I with the TURBO DNA-free reagent (Ambion) for 30 min at 37°C. Reverse transcription and quantitative real-time PCR were performed as previously described [25]. PCRs involved a hybridization step of 55°C, except for ramR, SLI0755 and cchB where a temperature of 58°C was used. Each assay was performed in triplicate and repeated with at least two independent RNA samples. The critical threshold cycle (C T ) was defined for each sample. The relative amounts of cDNA for the tested genes were normalized to that of the hrdB gene transcript which did not vary under our experimental conditions (and thus served as an internal standard). The change (n-fold) in a transcript level was calculated using the following equations: ΔC T  = C T(test DNA) - C T(reference cDNA), ΔΔC T  = ΔC T(target gene) - ΔC T(hrdB), and ratio =  http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-14-81/MediaObjects/12866_2013_2232_IEq1_HTML.gif [38]. Student’s t test was used to evaluate the significance of differences between the expression level of tested genes and that of a reference gene. A P-value < 0.05 was considered significant.

              In silico analysis and electrophoretic mobility shift assays (EMSA)

              Several AdpA-binding site sequences, identified in S. griseus by DNase I footprinting experiments [10, 13, 18, 23], were used with the PREDetector software (version 1.2.3.0) [39] to generate a S. griseus matrix [25]. This matrix was used with the S. coelicolor genome sequence (the S. lividans genome sequence was not available during the course of this study and is still not available on PREDetector software) to identify putative AdpA-binding sites upstream from S. lividans AdpA-dependent genes (scores > 3). The StrepDB database [7] and Blast were used to identify S. lividans, S. coelicolor and S. griseus ortholog gene names.

              Radioactively labelled DNA fragments (180 bp to 496 bp) corresponding to promoter regions of putative S. lividans AdpA-regulated genes were obtained by PCR. Primers (named GSgene in Additional file 1: Table S1) were used to amplify the promoter regions of cchA (opposite orientation to cchB), SLI0755, SLI6586 (opposite orientation to SLI6587), ramR and hyaS as described elsewhere [25]. Purified radiolabelled fragments (10,000 cpm) were then used with purified AdpA histidine-tagged protein (AdpA-His6) in EMSA as previously described [25, 40].

              Results

              Deletion of adpA affects the expression of hundreds of genes during early stationary phase

              We had previously inactivated adpA in S. lividans and found that this adpA mutant failed to produce aerial mycelium on rich media and that its growth was comparable to that of the parental strain 1326 in liquid YEME medium at 30°C [25]. Expression studies with this S. lividans adpA mutant cultivated in liquid medium identified two differentiation-regulating factors (STI1 and the ClpP1ClpP2 peptidases) whose ORFs were under the direct control of AdpA [25]. We used transcriptome analysis of this adpA mutant to identify other AdpA-dependent pathways in S. lividans; however, this analysis was performed using S. coelicolor microarrays [29] because the S. lividans genome sequence was not yet available [24] and the two species are very closely related [41]. Total RNA was isolated from S. lividans 1326 and adpA cells during early stationary phase (time point T in Figure 1a) because at this growth phase, S. coelicolor adpA is expressed [4]; also the expression of genes involved in secondary metabolism in a S. coelicolor bldA mutant [42], a strain defective for AdpA translation, starts to diverge from that in the wild-type.

              Global gene expression in the mutant was compared to that in the parental strain. The expression of more than 300 genes was affected in the adpA mutant at early stationary phase (Table 1 and Additional file 2: Table S2): 193 genes were significantly down-regulated (1.6-to 30-fold i.e. 0.033 < Fc < 0.625), and 138 were up-regulated (1.6-to 3.6-fold) with a P-value < 0.05 (see Additional file 2: Table S2 for the complete data set). Theses genes encode proteins of several different classes according to the Welcome Trust Sanger Institute S. coelicolor genome database [37]: 72 of the genes are involved in metabolism of small molecules, including seven playing a role in electron transport (e.g. SLI0755-SLI0754, cydAB operons) (Table 1); 18 encode proteins involved in secondary metabolism, for example the cchA-cchF gene cluster (SLI0459-0454) involved in coelichelin biosynthesis [43] and the SLI0339-0359 cluster encoding the putative deoxysugar synthase/glycosyltransferase. Deletion of adpA in S. lividans also affected the expression of 32 genes involved in regulation including ramR (SLI7029), wblA (SLI3822), bldN (SLI3667), hrdD (SLI3556) and cutRS (SLI6134-35) [1, 6]. Sixty-two genes involved in the cell envelope [37] were differentially expressed in the adpA mutant; they include hyaS (SLI7885) [44], chpE, chpH[1], SLI6586 and SLI6587 which were strongly down-regulated in the adpA mutant (Table 1). Thirty-nine genes encoding proteins involved in various cellular processes (osmotic adaptation, transport/binding proteins, chaperones, and detoxification) [37] were also deregulated in the absence of AdpA (Additional file 2: Table S2). The expression of 111 genes coding for proteins with unidentified or unclassified function was altered in the adpA mutant. Thus, deletion of adpA influenced the expression of a large number of genes involved in a broad range of metabolic pathways, and indeed other functions, in S. lividans.
              Table 1

              Genes differentially expressed in S. lividans adpA mutant at early stationary phase in YEME medium a

              S. coelicolor geneb

              S. lividans genec

              Other gene namesd

              Annotated functionb

              Fce

              Class or metabolismf

              SCO0382

              SLI0340

               

              UDP-glucose/GDP-mannose family dehydrogenase

              0.491

              Secondary (s. m.)

              SCO0383

              SLI0341

               

              Hypothetical protein SCF62.09

              0.527

              Secondary (s. m.)

              SCO0384

              SLI0342

               

              Putative membrane protein

              0.611

              Secondary (s. m.)

              SCO0391

              SLI0349

               

              Putative transferase

              0.613

              Secondary (s. m.)

              SCO0392

              SLI0350

               

              Putative methyltransferase

              0.606

              Secondary (s. m.)

              SCO0394

              SLI0352

               

              Hypothetical protein SCF62.20

              0.518

              Secondary (s. m.)

              SCO0396

              SLI0354

               

              Hypothetical protein SCF62.22

              0.454

              Secondary (s. m.)

              SCO0397

              SLI0355

               

              Putative integral membrane protein

              0.312

              Secondary (s. m.)

              SCO0399

              SLI0357

               

              Putative membrane protein

              0.532

              Secondary (s. m.)

              SCO0494

              SLI0454

              cchF

              Putative iron-siderophore binding lipoprotein

              0.615

              Secondary (s. m.)

              SCO0496

              SLI0456

              cchD

              Putative iron-siderophore permease transmembrane protein

              0.505

              Secondary (s. m.)

              SCO0497

              SLI0457

              cchC

              Putative iron-siderophore permease transmembrane protein

              0.492

              Secondary (s. m.)

              SCO0498

              SLI0458*

              cchB

              Putative peptide monooxygenase

              0.336

              Secondary (s. m.)

              SCO0499

              SLI0459*

              cchA

              Putative formyltransferase

              0.374

              Secondary (s. m.)

              SCO0762

              SLI0743

              sti1, sgiA

              Protease inhibitor precursor

              0.124

              (m. m.)

              SCO0773

              SLI0754

              soyB2

              Putative ferredoxin, Fdx4

              0.098

              Electron transport (s. m.)

              SCO0774

              SLI0755*

               

              Putative cytochrome P450, CYP105D5

              0.075

              Electron transport (s. m.)

              SCO0775

              SLI0756*

               

              Conserved hypothetical protein

              0.424

              Unknown function

              SCO1630-28

              SLI1934-32

              rarABC, cvnABC9

              Putative integral membrane protein

              ± 0.43

              Cell envelope

              SCO1674

              SLI1979

              chpC

              Putative secreted protein

              0.564

              Cell envelope

              SCO1675

              SLI1980

              chpH

              Putative small membrane protein

              0.237

              Cell envelope

              SCO1800

              SLI2108

              chpE

              Putative small secreted protein

              0.256

              Cell envelope

              SCO2780

              SLI3127

              desE

              Putative secreted protein

              1.757

              Cell envelope

              SCO2792

              SLI3139

              bldH, adpA

              araC-family transcriptional regulator

              0.383

              Regulation

              SCO2793

              SLI3140

              ornA

              Oligoribonuclease

              1.966

              (m. m.)

              SCO3202

              SLI3556

              hrdD

              RNA polymerase principal sigma factor

              2.499

              Regulation

              SCO3323

              SLI3667

              bldN, adsA

              Putative RNA polymerase sigma factor

              0.389

              Regulation

              SCO3579

              SLI3822

              wblA

              Putative regulatory protein

              0.310

              Regulation

              SCO3945

              SLI4193

              cydA

              Putative cytochrome oxidase subunit I

              3.386

              Electron transport (s. m.)

              SCO3946

              SLI4194

              cydB

              Putative cytochrome oxidase subunit II

              3.594

              Electron transport (s. m.)

              SCO4114

              SLI4345

               

              Sporulation associated protein

              0.487

              Cell envelope

              SCO5240

              SLI5531

              wblE

              Hypothetical protein

              2.246

              Unknown function

              SCO5862-63

              SLI6134-35

              cutRS

              Two-component regulator/sensor

              ± 1.82

              Regulation

              SCO6197

              SLI6586*

               

              Putative secreted protein

              0.147

              Cell envelope

              SCO6198

              SLI6587*

               

              Putative secreted protein

              0.618

              Cell envelope

              SCO6685

              SLI7029*

              ramR, amfR

              Putative two-component system response regulator

              0.624

              Regulation

              SCO7400-398

              SLI7619-17

              cdtCBA

              Putative ABC-transport protein

              ± 1.75

              Cell process

              SCO7657

              SLI7885*

              hyaS

              Putative secreted protein

              0.033

              Cell envelope

              SCO7658

              detected

               

              Hypothetical protein SC10F4.31

              0.103

              Unknown function

              aGene expression in the S. lividans adpA mutant was compared to that in the wild-type, using S. coelicolor microarrays. Table 1 shows a selected subset of the genes (see Additional file 2: Table S2 for the complete list). The genes presented here were further studied or are discussed in the text because of their role in Streptomyces primary or secondary metabolism [1, 6, 17].

              bGene names for S. coelicolor (SCO) and S. lividans (SLI) and annotated function are from the StrepDB database [7].

              c S. coelicolor microarrays were used for transcriptome analysis of the S. lividans adpA mutant (the complete microarray data set is presented in Additional file 2: Table S2). The S. lividans genome sequence was recently made available [24] and SLI ortholog gene numbers were identified as SCO gene orthologs with StrepDB database [7]. The expression of genes shown in bold was analysed by qRT-PCR. Intergenic DNA regions between genes labelled with asterisks were analyzed by EMSA (Figure 2). A SCO7658-orthologous sequence (98% nucleotide identity according to BLAST) was detected in S. lividans, downstream from hyaS, but it was not annotated as a S. lividans coding DNA sequence (CDS). However our microarray data suggest that this sequence is indeed a CDS or alternatively that the S. lividans hyaS CDS is longer than annotated.

              dSCO genes and their S. griseus orthologs studied and described under another name found on StrepDB database [7] or see “References”.

              eFold change (Fc) in gene expression in the S. lividans adpA mutant with respect to the parental strain with P-value < 0.05, as calculated by Student’s t-test applying the Benjamini and Hochberg multiple testing correction. ± indicates average Fc of some gene operons (see Additional file 2: Table S2 for details).

              fFrom a protein classification scheme for the S. coelicolor genome available from the Welcome Trust Sanger Institute database [37]: macromolecule metabolism (m. m.), small molecule metabolism (s. m.).

              Identification of new AdpA-controlled genes

              To confirm that S. lividans AdpA controls the expression of genes identified as differentially expressed in microarray experiments, six genes were studied in more detail by qRT-PCR. The six genes were selected as having biological functions related to Streptomyces development or the cell envelope (ramR[1], hyaS[44] and SLI6586 [37]) or primary or secondary metabolism (SLI0755, cchA, and cchB[43]), and for having very large fold-change values (Table 1). The genes in S. coelicolor and griseus orthologous to SLI6586 and SLI6587 encode secreted proteins [12, 42]. The expression levels of these genes in S. lividans wild-type and adpA strains were measured after various times of growth in liquid YEME media (Figure 1b), as shown in Figure 1a.

              The S. lividans hyaS gene was strongly down-regulated in the adpA mutant compared to the wild-type (Fc < 0.03) (Figure 1b) as previously observed for the SCO0762 homolog also known as sti1[25]. This suggests that hyaS expression is strongly dependent on S. lividans AdpA or an AdpA-dependent regulator. SLI0755, SLI6586 and ramR, were also expressed at a lower level in the adpA mutant than wild-type, particularly after mid-exponential phase (Figure 1b, times C, D and T); cchB seemed to be mostly affected by AdpA during stationary phase (Figure 1b, time T). The expression of cchA was strongly down-regulated by the absence of AdpA at times D and T (Figure 1b): note that despite repeated efforts, cchA expression could not be detected in samples corresponding to times A to C for unknown reasons. The findings for gene expression as determined by microarrays and by qRT-PCR were consistent, with the exception of those for ramR. The expression of ramR observed by qRT-PCR at time T differed from that determined in microarray experiments (Table 1), suggesting that some of our microarray data are flattened. Nevertheless, these qRT-PCR experiments confirmed that the expression of the six selected genes is indeed AdpA-dependent in S. lividans at every growth time studied.

              Direct binding of AdpA to the promoter regions of S. lividans AdpA regulon members

              To determine whether S. lividans AdpA directly controls these genes, we searched for potential AdpA-binding sites in their promoter regions in silico. A consensus AdpA-binding sequence (5′TGGCSNGWWY3′) has been established in S. griseus, and AdpA can bind up to five sites between positions -260 bp and +60 bp with respect to the transcriptional start point of the target gene [10]. BLAST analysis revealed that the S. griseus AdpA DNA-binding domain is conserved in S. coelicolor and S. lividans AdpAs (data not shown) suggesting that all three species share the same AdpA-binding consensus sequence.

              The DNA sequences upstream from the S. coelicolor ramR and hyaS genes and the intergenic region between the divergently transcribed genes cchA/cchB, SCO0774/SCO0775 and SCO6197/SCO6198 were analyzed using PREDetector software [39] and a matrix was generated with identified S. griseus AdpA-binding sequences [10, 23, 25]. Between three and nine putative AdpA-binding sites were detected within the promoter region of the S. coelicolor genes and by analogy in orthologous S. lividans AdpA-dependent genes (Table 2, location with respect to translation start point). During the course of this study, the S. lividans 1326 genome sequence became available [24] (but not in a form suitable for analysis with PREDetector (version 1.2.3.0) [39]) and its analysis suggested that the position and composition of AdpA-binding sites were different from those predicted. The putative AdpA-binding sites of S. lividans cchA/cchB at -101 nt and -86 nt are GGGCCGGTTC and TGGCTGGAAC, respectively. The AdpA-binding sites located upstream of SLI0755, SLI6586, and hyaS differ from their S. coelicolor orthologs (see Table 2, changes in the location from translation start site are indicated in bracket).
              Table 2

              AdpA-binding sites identified in silico in the promoter regions of S. lividans AdpA-dependent genes a

              S. coelicolor gene (S. lividans gene)b

              Putative AdpA-binding sitec

              Position (bp) with respect to translation start sitec

              Strand locationd

              Scorese

              Sites in EMSA probesf

              cchA/cchB*

              TGGCCGGATT#

              -425#

               

              CS

              9.30

              +

               

              TGGCGACATT#

              -254#

               

              CS

              5.19

              +

               

              GGGCCGATTC (G7th)

              -101

               

              CS

              4.99

              +

               

              TGGCTCGAAT (C10th)

              -86

               

              NCS

              6.91

              +

              ramR

              GTGCCGGTTC

              -464

               

              NCS

              3.37

              -

               

              TGGCGCGAAA

              -384

               

              NCS

              6.42

              +

               

              CGGCCGAAAA

              -358

               

              NCS

              5.85

              +

               

              GGGCGGGTTC

              -280

               

              NCS

              5.08

              +

               

              TGGCCAGGAC

              -279

               

              CS

              3.86

              +

               

              GGGCGGATAA

              -184

               

              NCS

              3.87

              +

               

              TGTCGTGTTC

              -95

               

              CS

              4.83

              -

               

              CGGCGGAACA

              -81

               

              NCS

              3.15

              -

               

              TGGCCCGAAC

              -30

               

              CS

              7.23

              -

              SCO0774/SCO0775*

              CGGCGCGTTC

              -268

              (-226)

              CS

              4.25

              -

              (i.e. SLI0755/SLI0756)

              GGACGGGAAC

              -253

              (-211)

              NCS

              3.37

              +

               

              GGGCGCGATC

              -207

              (-165)

              CS

              4.53

              +

               

              TGGCGCGATC

              -170

              (-128)

              NCS

              6.90

              +

               

              CGGCCAGTCT

              -110

              (-68)

              CS

              3.06

              +

               

              TGGCCGAACT

              -84

              (-42)

              CS

              6.20

              -

               

              CGGCCAGATC

              -79

              (-37)

              NCS

              5.84

              -

              SCO6197/SCO6198*

              GGTCCGGACA

              -499

              (-547~)

              CS

              4.98

              -

              (i.e. SLI6586/SLI6587)

              TGACCAGAAG

              -414

              (-462~)

              CS

              3.82

              +

               

              TGGCCGAGTT

              -362

              (-410~)

              CS

              5.06

              +

               

              GTTCCTGCAA

              -297

              (-345~)

              NCS

              3.50

              +

               

              GGGCTGAAAC

              -271

              (-319~)

              NCS

              4.77

              +

               

              TGGCTGAATT

              -116

              (-164)

              CS

              7.85

              +

              hyaS

              TGGCCGGATC

              -130

              (-129)

              NCS

              8.90

              +

               

              CGGCCATTTC

              -124

              (-123)

              CS

              3.05

              +

               

              TGTCCAGAAG

              -101

              (-100)

              NCS

              4.48

              +

              a In silico analysis of the S. coelicolor genome using PREDetector software (version 1.2.3.0, the S. lividans database was not available at the time this analysis was performed) [39] to analyse orthologs of S. lividans AdpA-dependent genes. The S. coelicolor AdpA-binding sites identified were checked for their conservation and location using the S. lividans genome StrepDB database [7] (see legend c).

              bGenes are named according to the StrepDB database [7]. *binding sites located between S. coelicolor genes transcribed in the opposite orientation.

              cPutative S. coelicolor AdpA-binding sites were found in silico with PREDetector [39]; #putative site located in the upstream from the CDS of cchB. The site location given corresponds to the position of first nucleotide most distant from the translation start point of the first gene named. The positions of some sites are not the same for the S. lividans orthologs as indicated in brackets (S. lividans StrepDB database [7]). ~ putative sites are in the CDS of SLI6587. Predicted CDS diverge between SLI6586 and SLI6587 locus and their orthologs SCO6197 and SCO6198, resulting in a smaller intergenic region in S. lividans.

              dCS, coding strand; NCS, non coding strand with reference to the first gene named in the S. coelicolor gene column.

              eScores given by PREDetector software for S. coelicolor genes [39].

              fSites present (+) or absent (-) in the S. lividans DNA probes used in EMSA experiments.

              We used EMSA to test whether S. lividans AdpA binds to predicted S. lividans AdpA-binding sequence. Recombinant purified AdpA-His6 bound to the promoter region of S. lividans sti1 (SCO0762 homolog), an AdpA-dependent gene, whereas an excess of AdpA-His6 (up to 34 pmoles) did not bind to the promoter of SLI4380 (SCO4141 homolog), a gene that is not controlled by S. lividans AdpA. This suggests that the binding of AdpA with the promoter of genes tested in our previous study was specific [25]. AdpA-His6 was able to bind to the promoter regions of all S. lividans AdpA-dependent genes tested (Table 2, Figure 2), although with different affinities. For SLI6586/SLI6587, ramR and hyaS, displacement of the DNA fragment to the slower migrating protein-DNA complex was nearly complete with amounts of AdpA of less than 11 pmoles (Figure 2, lane 2). For cchA/cchB and SLI0755/SLI0756, larger amounts of AdpA were necessary for near complete displacement of the DNA probe to a protein-DNA complex. In a competition EMSA performed on SLI6586/6587 with an excess of the corresponding unlabelled probe, AdpA-binding to the labelled probe decreased (data not shown). We also tested a hyaS promoter in which one (highest score) of the three putative AdpA-binding sites was mutated (at position -134 to -129, see Additional file 3: Figure S1a): the affinity of AdpA for this promoter region was reduced and one protein-DNA complex disappeared (Additional file 3: Figure S1b). These results suggest that one dimer of AdpA binds the adjacent sites -129 and -123 of S. lividans hyaS promoter and another dimer binds the -100 site resulting in the formation of the two DNA-AdpA complexes depicted in Figure 2.
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-14-81/MediaObjects/12866_2013_2232_Fig2_HTML.jpg
              Figure 2

              AdpA binds in vitro to promoter DNA regions of S. lividans AdpA-dependent genes. Electrophoretic mobility shift assays performed with 0 (lane 1), 5.7 (lane 2), 11.4 (lane 3) or 17.1 (lane 4) pmoles of purified AdpA-His6 and 32P-labelled probes (10,000 cpm) corresponding to the regions upstream of the S. lividans genes indicated, in the presence of competitor DNA (1 μg poly dI-dC).

              These EMSA experiments demonstrated that S. lividans AdpA directly binds to five intergenic regions and confirmed the in silico prediction presented in Table 2. S. lividans AdpA directly regulates at least the six AdpA-dependent genes listed above and identified by microarrays and qRT-PCR analysis. These newly identified targets highlight the pleiotropic role of S. lividans AdpA: it is involved in primary (SLI0755) and secondary (cchA, cchB and hyaS) metabolisms, in regulation (ramR), and in cell development (hyaS, ramR and SLI6586).

              Discussion

              AdpA, a transcriptional regulator of the AraC/XylS family, is involved in the development and differentiation of various Streptomyces[35, 25]. We report here the first identification of several pathways directly regulated by AdpA in S. lividans cultivated in liquid rich medium.

              Inactivation of adpA in S. lividans affected the expression of approximately 300 genes. This large number was expected in the light of the size of the S. griseus AdpA regulon [14]. Although adpA mutant growth was comparable to that of the parental strain in YEME liquid medium, the expression of around 200 genes involved in primary metabolism was influenced by adpA deletion. These genes encode proteins involved in the major biosynthesis pathways for amino acids (class 3.1. in Additional file 2: Table S2) [37], and in energy metabolism (class 3.5.) including glycolysis, pentose phosphate, pyruvate dehydrogenase pathways, as well as in electron transport (e.g. CydAB cytochrome oxidase, CYP105D5 and Fdx4 involved in fatty acid hydroxylation and encoded by SLI0755-0754 [45]). Other S. lividans AdpA-regulated genes influence Streptomyces development on solid media (e.g. those for RamR, chaplins Chp, BldN, WblA, WblE, HyaS and ClpP1ClpP2 peptidases) (Table 1) [1, 6, 16, 25, 44]. S. lividans AdpA also influences the expression of 18 genes involved in secondary metabolism such as coelichelin biosynthesis (cch genes in Table 1) [43] and also genes described to affect metabolic differentiation (HyaS, CutRS, WblA, DesE, and CdtCBA) (Table 1) [15, 17, 42, 44]. Consistently with transcriptomic studies in S. griseus, these observations suggest that AdpA is a pleiotropic transcriptional regulator in S. lividans.

              We demonstrate that S. lividans AdpA directly activates cchB, SLI0755 and hyaS. As a result of their co-transcription with these genes, the expression of cchCD, SLI0754 and SCO7658-ortholog genes is AdpA-dependent in S. lividans (Table 1). SLI0756 is probably a directly AdpA-regulated gene because its promoter DNA region is shared with SLI0755-SLI0754 operon, which is transcribed in the opposite direction and directly regulated by AdpA (Table 1, Figure 2).

              AdpA directly regulates the genes ramR and sti1 in S. lividans (this study) [25] and in the closely related species S. coelicolor[16]. In an S. coelicolor adpA mutant, levels of sti1 and ramR expression were lower than in the wild-type strain following growth for 48 h in a minimal agar medium [16]. In vitro experiments showed a high affinity of AdpA with a S. coelicolor sti1 probe [16], consistent with our results with S. lividans sti1[25]. However, AdpA had a lower affinity to S. coelicolor ramR (with promoter region -302 nt to +73 nt with respect to the translation start site) than S. lividans ramR (Figure 2, with the promoter region -440 nt to -181 nt). When we used a S. lividans ramR probe carrying the promoter region from -201 nt to +66 nt, we observed that less than half the probe was shifted (data not shown). Therefore, the predicted sites for ramR promoter at positions -384 and -358 (Table 2) may have the greatest affinity for AdpA (Figure 2). Of the genes analysed by qRT-PCR, the ramR gene was that for which the observed expression was the least consistent with the microarray findings, even through the same sample was used for these analyses. This suggests that the expression of genes close to the cut-off we applied to the microarray data will need further investigation by qRT-PCR.

              Among the 28 genes identified as direct targets of AdpA in S. griseus, 13 have no orthologous gene in S. lividans and the orthologous genes of six are not under the control of S. lividans AdpA in our conditions. In addition to ramR (amfR) and sti1 (sgiA), hyaS (SGR3840) is also a directly AdpA-regulated gene that is conserved in the S. lividans and S. griseus AdpA regulons [12, 25]. In S. lividans, hyaS affects hypha aggregation and the amount of mycelium-associated undecylprodigiosin [44]; its function in S. griseus is unknown. The expression of all of bldN, SLI6392, SLI1868 and the SCO2921 ortholog (gene detected in S. lividans genome but not named in StrepDB [7]) is influenced by adpA deletion in S. lividans. It remains to be determined whether AdpA directly controls S. lividans adpA and bldA as described in S. coelicolor and griseus[16, 23].

              S. coelicolor adpA is one of 145 identified TTA-containing genes; the production of the proteins encoded by these genes is dependent on bldA, encoding the only tRNA for the rare leucine codon TTA [46]. Our study has revealed that expression of 11 TTA-containing genes and of 24 genes regulated by S. coelicolor bldA[42, 47, 48] was affected by adpA deletion in S. lividans (Additional files 4: Table S3). We show that cchA, cchB, sti1, hyaS, SLI6586 and SLI6587, previously identified in S. coelicolor as bldA-dependent genes, are direct targets of S. lividans AdpA [25]. Of the 29 other bldA-dependent genes, 19 are probable direct S. lividans AdpA targets: in silico analysis indicated the presence of putative AdpA-binding sites upstream from these genes (most of them with score above 4, see Additional file 5: Table S4). By analogy, this suggests that the deregulation of certain genes observed in the S. coelicolor bldA mutant may have been the consequence of S. coelicolor AdpA down-regulation, as previously suggested [49].

              To predict probable direct targets of AdpA in S. lividans and contribute to knowledge of the AdpA regulon, we carried out in silico analysis of the entire S. coelicolor genome using PREDetector [39], and also restricted to the S. lividans genes identified as being AdpA-dependent (see Additional file 5: Table S4 and Table 3). We identified 95 genes probably directly activated by S. lividans AdpA and 67 genes that could be directly repressed (Additional file 5: Table S4). Most of the putative AdpA-binding sites identified by this analysis are coherent with the findings of Yao et al., demonstrating the importance of G and C nucleotides at positions 2 and 4, respectively [50]. Six genes have been identified as directly regulated by AdpA in other species (adpA, bldN, wblA, SLI6392, SCO2921 orthologs, and glpQ1, as indicated in Table 3 in bold) [10, 12, 15, 16, 18], and 27 more in S. griseus are also probable AdpA-direct targets (e.g. cchB, SLI0755-0754 operon, rarA operon, scoF4, groEL1, SLI6587, SLI4345, cydAB, and ectABD, as indicated in Table 3 and Additional file 2: Table S2, underlined) [7, 1214]. Sixty-three of the 162 probable direct targets of AdpA in S. lividans have no ortholog in the S. griseus genome (Additional file 5: Table S4).
              Table 3

              Genes putatively directly regulated by S. lividans AdpA in liquid rich medium a

              Geneb

              Geneb

              Geneb

              Gene nameb

              cis-elementc

              Scorec

              Positionc

              Fcd

              Classe

              Probably directly activated by S. lividans AdpA:

              SCO2921*

              Detected

              SGR4618

              adbS3-orfa

              tttgcggaca

              4.62

              -260

              0.196

              c. e.

              SCO0494

              SLI0454

              SGR6714

              cchF

              tgtcgcgcca

              4.36

              -28

              0.615

              s. m.

              SCO0929

              SLI1160

              SGR710

               

              tggccggacg

              5.19

              -201

              0.419

              u. f.

              SCO1565

              SLI1668

              SGR5973

              glpQ1

              cggccggaac

              6.75

              -82

              0.531

              c. e.

              SCO1630

              SLI1934

              SGR1063

              cvn9, rarA

              tgtcgggatc

              6.71

              -74

              0.505

              c. e.

              SCO1674

              SLI1979

              SGR5829

              chpC

              cggcggaatc

              5.69

              -154

              0.564

              c. e.

              SCO1800

              SLI2108

              SGR5696

              chpE

              cggccggacc

              4.69

              -65

              0.256

              c. e.

              SCO1968

              SLI2284

              SGR5556

              glpQ2

              cattcagcct

              3.75

              -92

              0.537

              m. m.

              SCO2792

              SLI3139

              SGR4742

              adpA bldH

              gaaccggcca

              8.09

              -148

              0.383

              r.

              SCO3323

              SLI3667

              SGR4151

              bldN, adsA

              gttccggtca

              6.38

              -469

              0.389

              r.

              SCO3579*

              SLI3822

              SGR3340

              wblA

              tggcccgaac

              7.23

              -135

              0.31

              r.

              SCO3917*

              SLI4175

              SGR3663

               

              ctttcggcca

              6.52

              -72

              0.504

              u. f.

              SCO4113

              SLI4344

              SGR3901

               

              aaacccgtca

              5.64

              -52

              0.568

              u. f.

              SCO4114*

              SLI4345

              SGR3902

               

              tggcgggatt

              8.66

              -117

              0.487

              c. p.

              SCO4164

              SLI4405

              SGR3965

              cysA

              gttgccgcca

              5.70

              -170

              0.483

              s. m.

              SCO4295*

              SLI4532

              SGR3226

              scoF4

              attctcgcca

              7.13

              -193

              0.217

              c. p.

              SCO4761

              SLI5031

              SGR2770

              groES

              aaccccgccg

              3.31

              -197

              0.401

              c. p.

              SCO4762

              SLI5032

              SGR2769

              groEL1

              ttgccgtata

              4.40

              -44

              0.44

              c. p.

              SCO4768

              SLI5039

              SGR2759

              bldM

              aatctagccg

              5.52

              -292

              0.586

              r.

              SCO5101

              SLI5379

              SGR2456

               

              cggcgggaac

              6.11

              -28

              0.584

              u. f.

              SCO6004

              SLI6392

              SGR1503

               

              cggccgcatt

              5.21

              -292

              0.603

              c. e.

              SCO6096*

              SLI6490

              SGR1397

               

              catcgcgcca

              5.56

              -147

              0.557

              c. e.

              SCO7550

              SLI7772

              -

              glpQ3

              gaaccggtca

              5.88

              -117

              0.334

              c. e.

              Probably directly repressed by S. lividans AdpA:

              SCO1684

              SLI1989

              SGR5819

               

              gaatgcgcca

              5.36

              -161

              1.626

              u. f.

              SCO1776*

              SLI2080

              SGR5721

              pyrG

              cttccggcca

              7.25

              -170

              1.744

              s. m.

              SCO1821

              SLI2130

              SGR5674

              moaA

              cggcccgaac

              5.39

              -61

              1.679

              s. m.

              SCO1864

              SLI2175

              SGR5635

              ectA

              atttcggaca

              6.71

              -203

              2.903

              c. p.

              SCO1865

              SLI2176

              SGR5634

              ectB

              cggccgggac

              3.24

              -78

              3.154

              c. p.

              SCO1867

              SLI2178

              SGR5632

              ectD

              gaagtggcca

              4.62

              -3

              3.029

              n. c.

              SCO3123

              SLI3480

              SGR4383

              prsA2

              tgaccggaaa

              6.21

              #

              1.891

              s. m.

              SCO3202

              SLI3556

              SGR4276

              hrdD

              aatccggaca

              7.75

              -145

              2.499

              r.

              SCO3811

              SLI4062

              SGR3768

              dacA

              tatccggacg

              5.34

              -175

              1.628

              c. e.

              SCO3945

              SLI4193

              SGR3646

              cydA

              tgtcccgatt

              6.39

              -88

              3.386

              s. m.

              SCO3947

              SLI4195

              SGR3644

              cydCD

              catcccgccg

              5.08

              -30

              2.653

              s. m.

              SCO3971

              SLI4220

              SGR3620

               

              tggccggtac

              7.78

              -465

              1.631

              u. f.

              SCO4215

              SLI4452

              -

              xlnR

              gatgaggccg

              3.74

              -294

              1.964

              r.

              SCO5240

              SLI5531

              SGR2274

              wblE

              tgtcccgatc

              5.99

              -170

              2.246

              u. f.

              SCO5862

              SLI6134

              SGR1670

              cutR

              tggccgaaaa

              7.69

              -99

              1.927

              r.

              SCO6009

              SLI6398

              SGR1498

               

              cttccagcca

              6.53

              -52

              1.736

              c. p.

              aOrthologs of S. lividans AdpA-dependent genes (listed in Additional file 2: Table S2) were analysed in silico using the S. coelicolor genome database (version 1.2.3.0 of PREDetector software [39]). AdpA-binding sites upstream from S. coelicolor genes were identified and are presented in Additional file 5: Table S4. Table 3 presents a selected subset of this complete compilation.

              bGene names for S. griseus (SGR) and annotated function are from the StrepDB database [7]. Ortholog gene names were identified using StrepDB. Genes identified in other Streptomyces as being directly AdpA-regulated are in bold, and those described as being AdpA-dependent are italicized [1215, 22]. * Binding sites in the promoters of these genes were identified in silico[22]. The SCO2921-ortholog was not annotated as a S. lividans CDS; however, our microarray data suggest that this CDS exists.

              ccis-element, score, and binding site position as determined by analysing S. coelicolor genes with PREDetector [39]. When more than one putative AdpA-binding site was detected, only the one with the highest score was shown here. Other genes putatively directly regulated by S. lividans AdpA are listed in Additional file 5: Table S4. # site found in the SCO3122 CDS at position 1447 (total gene length 1449 nt).

              dFold change (Fc) in gene expression in S. lividans adpA mutant relative to the parental strain with P-value < 0.05, as determined by Student’s t-test applying the Benjamini and Hochberg multiple testing correction (details in Additional file 2: Table S2).

              eFrom a protein classification scheme for the S. coelicolor genome available on the Welcome Trust Sanger Institute database [37]: unknown function (u. f.), cell process (c. p.), macromolecule metabolism (m. m.), small molecule metabolism (s. m.), cell envelope (c. e.), extrachromosomal (e.), regulation (r.) and not classified (n. c.).

              Conclusions

              In conclusion, this study has extended our knowledge of the S. lividans AdpA regulon. We identified S. lividans AdpA-regulated genes by transcriptomic analysis, and used in silico analysis to identify over a hundred probable direct targets of AdpA in S. lividans. Most of them are absent from the current predicted S. griseus AdpA regulon. Discovering new S. lividans genes directly regulated by AdpA and that are involved in primary and secondary metabolism will provide valuable information about Streptomyces development and differentiation in liquid culture.

              Availability of supporting data

              Microarray data are available in the ArrayExpress database [51, 52] under accession number A-MEXP-2383.

              Authors’ information

              AG performed qRT-PCR and EMSA experiments while working at Pasteur Institute. Her current address is Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle-upon-Tyne NE2 4HH, UK.

              Abbreviations

              qRT-PCR: 

              Quantitative real-time PCR

              ORF: 

              Open reading frame

              Fc: 

              Fold change

              C T

              Critical threshold cycle

              BLAST: 

              Basic local alignment search tool

              EMSA: 

              Electrophoretic mobility shift assay

              AdpA-His6

              Recombinant AdpA protein with a six-histine tag at the C-terminus

              CDS: 

              Coding DNA sequence

              CS: 

              Coding strand

              NCS: 

              Non coding strand

              u. f.: 

              Unknown function

              c. p.: 

              Cell process

              m. m.: 

              Macromolecule metabolism

              s. m.: 

              Small molecule metabolism

              c. e.: 

              Cell envelope

              e.: 

              Extrachromosomal

              r.: 

              Regulation

              n. c.: 

              Not classified.

              Declarations

              Acknowledgements

              We thank T. Msadek, S. Dubrac, E. Johnson and J.-L. Pernodet for helpful discussion and critical reading of the manuscript, and O. Poupel for assistance with qRT-PCR analysis. We are grateful to G. Bucca for her advice and help with microarray handling. We thank Alex Edelman & Associates for correcting the manuscript.

              This work was supported by research funds from the Institut Pasteur and Centre National de Recherche Scientifique. A. Guyet was the recipient of fellowships from the Ministère de l’Education Nationale, de la Recherche et de la Technologie, the Pasteur-Weizmann foundation and the ERA-IB European grant. AG thanks BBSRC and R. Daniel for his constant support during the preparation of this manuscript.

              Authors’ Affiliations

              (1)
              Unité de Biologie des Bactéries Pathogènes à Gram-Positif, Institut Pasteur, CNRS URA 2172
              (2)
              Unité Biologie des Spirochètes, Institut Pasteur
              (3)
              Plateforme Transcriptome et Epigenome (PF2)

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