Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions

[EN] Three putative alkaline phosphatase genes, phoA, phoC and phoD, were identified in the genome of Streptomyces coelicolor by homology with the amino acid sequence obtained from the PhoA protein of Streptomyces griseus. PhoA and PhoC correspond to broad-spectrum alkaline phosphatases whereas PhoD is similar to a Ca2+-dependent phospholipase D of Streptomyces chromofuscus. The phoA and phoD genes were efficiently expressed in R5 medium under phosphate-limited conditions, as shown by studies using the xylE reporter gene, whereas phoC was poorly transcribed under the same conditions. Expression of phoA was clearly PhoP-dependent since it was not transcribed in the S. coelicolor ΔphoP mutant and was strongly activated under low phosphate concentrations. Similarly, expression of phoD was PhoP-dependent and highly sensitive to phosphate availability. By contrast, expression of phoC was not PhoP-dependent. Electrophoretic mobility shift assays showed that PhoP binds to the phoA and phoD promoters, but not to that of phoC. Footprinting studies with GST-PhoP revealed the presence of a PHO box (two direct 11 nt repeats) in the phoA promoter and two PHO boxes in the promoter of phoD. The transcription start points of the three promoters were identified by primer extension. The transcription start point of phoD coincides with the G of its translation start codon, indicating that this gene is transcribed as a leaderless mRNA. The deduced -10 and -35 regions of phoD (but not those of phoA) overlapped with the PHO boxes in this promoter, suggesting that an excess of PhoP interferes with binding of the RNA polymerase to this promoter. In summary, the three promoters showed clear differences in the modulation of their expression by PhoPSIThis work was supported by grants of the CICYT (BIO2003-01489), Madrid (Spain) and the European Union (Project ACTINOGEN, LSHM-CT-2004-005224) Brussels. A. K. Apel received a F.P.U. fellowship of the Ministry of Education and Science. We thank J. Merino, B. Martín, B. Aguado and A. Casenave for their excellent technical assistance

Target genes and structure of the direct repeats in the DNA-binding sequences of the response regulator PhoP in Streptomyces coelicolor

[EN] Expression of genes belonging to the pho regulon in Streptomyces coelicolor is positively regulated (as shown by comparing the wild-type and a Δ phoP mutant) by binding of the response regulator PhoP to 11-nt direct repeats (DRus). These sequences have been found in over 100 genes of Streptomyces coelicolor ; 20 of them were cloned and the binding of PhoP DBD to most of their promoters has been shown by electrophoretic mobility shift assays. Deletion experiments showed that at least two DRus are required for proper binding of PhoP DBD . Deletion of 1 nt leaving a 10-nt direct repeat reduced drastically binding of PhoP DBD . Three different types of operators have been identified. Complex operators (class III) contain up to six DRus, some of them with poor conservation of the 11-nt consensus sequence, which however were protected by PhoP DBD in footprinting analyses. A cooperative binding of PhoP DBD molecules initiated at conserved core DRus appears to be the mechanism involved in binding of several PhoP DBD monomers to those complex operators. The information theory-based model that incorporates the positive or negative contribution to the binding of PhoP DBD of adjacent sequences has been used to deduce the structure of PHO boxes and the relevance of each DRuSIA.K.A. received a fellowship of the FPU Program of the Ministry of Education and Science, Madrid, Spain. We acknowledge the excellent technical help of B. Martín, J. Merino, A. Casenave and B. Aguado. This work was funded by ‘Comisión Interministerial de Ciencia y Tecnología’ (BIO2003-01489, BIO2006-14853-C02-01); ‘Ministerio de Educación, Ciencia y Tecnología’ (GEN2003-20245-C09-01) and ‘European Union’ (ACTINOGEN LSHM-CT-2004-005224). Funding to pay the Open Access publication charges for this article was provided by the ACTINOGEN project

Phosphate control over nitrogen metabolism in Streptomyces coelicolor: direct and indirect negative control of glnR, glnA, glnII and amtB expression by the response regulator PhoP

[EN] Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxescomposed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB - glnK - glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoP-binding sequence overlaps the GlnR boxes in both glnA and glnII. 'Information theory' quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussedSIFunding by Comisión Interministerial de Ciencia y Tecnología [BIO2003-01489, BIO2006-14853-C02-01]; the ‘Ministerio de Ciencia e Innovación’, Madrid [GEN2003-20245-C09-01]; the AECID (Agencia Española de Cooperación Internacional para el Desarrollo), ‘Ministerio de Asuntos Exteriores y de Cooperación’, Madrid [A/010257/07]; the ERA-NET SySMO Project [GEN2006-27745-E/SYS]; and the European Union (ACTINOGEN LSHM-CT-2004-005224). F.P.U. fellowship of the Ministerio de Ciencia e Innovación (Spain) (to K.A.); fellowship of the F.P.I. program (Ministerio de Ciencia e Innovación, Spain) (to F.S.B.). Funding for open access charge: Institute of Biotechnology of Leó

Target genes and structure of the direct repeats in the DNA-binding sequences of the response regulator PhoP in Streptomyces coelicolor

Expression of genes belonging to the pho regulon in Streptomyces coelicolor is positively regulated (as shown by comparing the wild-type and a ΔphoP mutant) by binding of the response regulator PhoP to 11-nt direct repeats (DRus). These sequences have been found in over 100 genes of Streptomyces coelicolor; 20 of them were cloned and the binding of PhoPDBD to most of their promoters has been shown by electrophoretic mobility shift assays. Deletion experiments showed that at least two DRus are required for proper binding of PhoPDBD. Deletion of 1 nt leaving a 10-nt direct repeat reduced drastically binding of PhoPDBD. Three different types of operators have been identified. Complex operators (class III) contain up to six DRus, some of them with poor conservation of the 11-nt consensus sequence, which however were protected by PhoPDBD in footprinting analyses. A cooperative binding of PhoPDBD molecules initiated at conserved core DRus appears to be the mechanism involved in binding of several PhoPDBD monomers to those complex operators. The information theory-based model that incorporates the positive or negative contribution to the binding of PhoPDBD of adjacent sequences has been used to deduce the structure of PHO boxes and the relevance of each DRu

Characterization of DNA Binding Sites of RokB, a ROK-Family Regulator from Streptomyces coelicolor Reveals the RokB Regulon

ROK-family proteins have been described to act either as sugar kinases or as transcriptional regulators. Few ROK-family regulators have been characterized so far and most of them are involved in carbon catabolite repression. RokB (Sco6115) has originally been identified in a DNA-affinity capturing approach as a possible regulator of the heterologously expressed novobiocin biosynthetic gene cluster in Streptomyces coelicolor M512. Interestingly, both, the rokB deletion mutants as well as its overexpressing mutants showed significantly reduced novobiocin production in the host strain S.coelicolor M512. We identified the DNA-binding site for RokB in the promoter region of the novobiocin biosynthetic genes novH-novW. It overlaps with the novH start codon which may explain the reduction of novobiocin production caused by overexpression of rokB. Bioinformatic screening coupled with surface plasmon resonance based interaction studies resulted in the discovery of five RokB binding sites within the genome of S. coelicolor. Using the genomic binding sites, a consensus motif for RokB was calculated, which differs slightly from previously determined binding motifs for ROK-family regulators. The annotations of the possible members of the so defined RokB regulon gave hints that RokB might be involved in amino acid metabolism and transport. This hypothesis was supported by feeding experiments with casamino acids and L-tyrosine, which could also explain the reduced novobiocin production in the deletion mutants

Draft Genome Sequence of Streptomyces niveus NCIMB 11891, Producer of the Aminocoumarin Antibiotic Novobiocin

Flinspach K, Rückert C, Kalinowski J, Heide L, Apel AK. Draft Genome Sequence of Streptomyces niveus NCIMB 11891, Producer of the Aminocoumarin Antibiotic Novobiocin. Genome announcements. 2014;2(1):e01146-13.Streptomyces niveus NCIMB 11891 is the producer of the gyrase inhibitor novobiocin, which belongs to the aminocoumarin class of antibiotics. The genome sequence of this strain was found to contain, besides the gene cluster for novobiocin, a putative gene cluster for the macrolactam antibiotic BE-14106 and further secondary metabolite gene clusters

SPR experiments with RokB and PnovH.

<p>a Scheme of the promoter of <i>novH</i> with localisation of the tested DNA fragments (oligo 1–18) on PnovH and binding curves of RokB to the tested oligonucleotides. b Binding site of RokB on PnovH. Boundaries determined by SPR spectroscopy are indicated by vertical lines. Start codon of <i>novH</i> in grey (TTG). c SPR binding curves of RokB to 42 bp starting sequence shortened by 2 bp each for determination of the left-hand and right hand boundary of the RokB binding site on PnovH.</p

RokB binding sites on the genome of <i>S</i>. <i>coelicolor</i>.

<p>a SPR binding curves with RokB and ProkB/sco6114-BS1, ProkB/sco6114-BS2, Psco3215/3216-BS, Psco6108-BS, Psco0938-BS. b Binding motif of RokB on ProkB/sco6114-BS1, ProkB/sco6114-BS2, Psco3215/3216-BS, Psco6108-BS, Psco0938-BS, calculated by MEME and binding site of RokB on PnovH. Inverted repeat is underlined, TTG Startcodon of <i>novH</i> depicted in grey.</p