Complex Transcriptional Control of the Antibiotic Regulator
            <i>afsS</i>
            in Streptomyces: PhoP and AfsR Are Overlapping, Competitive Activators

Santos-Beneit, Fernando; Rodrı́guez-Garcı́a, Antonio; Martı́n, Juan F.

doi:10.1128/jb.01462-10

Cited by 34 publications

(33 citation statements)

References 42 publications

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“…PhoP also has been shown to function as an antiactivator (third mechanism). PhoP represses the glnA and afsS genes competing with the binding sites of the transcriptional activators GlnR and AfsR (38,40,42). In this study, we also describe PhoP acting as a transcriptional repressor according to the second mechanism.…”

Section: Discussionmentioning

confidence: 90%

The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

Santos-Beneit

Barriuso-Iglesias

Fernández-Martínez

et al. 2011

Appl Environ Microbiol

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The RNA polymerase (RNAP) omega factor () forms a complex with the ␣ 2 ␤␤ core of this enzyme in bacteria. We have characterized the rpoZ gene of Streptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of the rpoZ gene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ⌬rpoZ strain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ⌬rpoZ mutant with the wild-type rpoZ allele restored both phenotype and antibiotic production. Interestingly, the rpoZ gene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed that rpoZ promoter activity was increased in a ⌬phoP background, it can be concluded that rpoZ is controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation in Streptomyces.In bacteria, the RNA polymerase (RNAP) complex plays a central role in transcription and is a target for regulation of primary metabolism (6,7,44). The rpoZ gene encodes the RNAP omega () subunit, which forms a complex with the ␣ 2 ␤␤Ј core of this enzyme. The subunit has been identified in the RNAPs of most free-living bacteria. This protein is functionally homologous to the RpoK subunit of the archaeal RNA polymerase complex and the RPB6 subunit of the eukaryotic RNA polymerases I, II, and III (32). In Escherichia coli the subunit interacts with the ␤Ј subunit and promotes assembly of the RNA polymerase complex (14, 32), although it is not essential for survival in this bacterium (13).Streptomyces spp. are soil-dwelling bacteria that are notorious for their ability to produce thousands of antibiotics, pigments, antitumor agents, immunomodulators, and a variety of other bioactive secondary metabolites (1, 2, 8). Differential expression of secondary metabolism genes occurs following nutrient depletion (34), but the transcriptional control mechanisms that govern the onset of secondary metabolites are still obscure (29). The rpoZ gene of Streptomyces kasugaensis has been shown to be required for antibiotic production and morphological differentiation but is not essential for growth (21). A DNA fragment containing the rpoZ gene was shown to complement an S. kasugaensis pleiotropic mutant deficient in aerial mycelium formation and kasugamycin biosynthesis. Although sigma factors in Streptomyces have received considerable attention in relation to the expression of antibiotic biosynthetic genes (9,19,20), the role of the RNAP subunit is still obscure.The expression of many genes involved in antibiotic biosynthesis is negatively controlled by the phosphate concentration in the medium (reviewed in references 28 and 30). Limitati...

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Section: Discussionmentioning

confidence: 90%

The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

Santos-Beneit

Barriuso-Iglesias

Fernández-Martínez

et al. 2011

Appl Environ Microbiol

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“…have developed complicated mechanisms to adapt their metabolism to the change of the extracellular environments (16). Among these mechanisms, cross talk between different regulators may integrate different signal inputs through finetuning the expression of key target genes (10,15). A regulatory link between nitrogen metabolism and phosphate metabolism was proposed in S. coelicolor, which was coordinated by PhoP through directly binding to the promoters of the nitrogen metabolism-associated genes, including glnA, glnII, and amtB (14).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB

et al. 2012

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The transcription of amtB in Streptomyces coelicolor has been proposed to be counter-regulated by GlnR (a global regulator for nitrogen metabolism) and PhoP (a global regulator for phosphate metabolism). However, the GlnR-protected region, which was deduced to be two 22-bp GlnR binding boxes (gTnAc-n6-GaAAc-n6-GtnAC-n6-GAAAc-n6, abbreviated as a1-b1 and a2-b2), was separated from the PhoP-protected region in the promoter of amtB, leaving the mechanism for this regulation undefined. In this study, another 22-bp GlnR binding box, which consisted of a3-site-n6-b3-site (a3-b3) overlapping with the PhoP-binding sequences, was identified in the promoter region of amtB by a DNase I footprinting assay. An electrophoretic mobility shift assay (EMSA) using purified recombinant GlnR and the synthetic amtB promoter fragments with the three GlnR binding boxes individually mutated demonstrated that every box was involved in GlnR binding in vitro. Further in vivo assays using the egfp reporter gene fused to various kinds of mutated promoter regions of amtB demonstrated that all of the three GlnR binding boxes were required for GlnR-mediated activation of amtB transcription under the nitrogen-limited condition. The results of EMSA using the amtB promoter with mixtures of recombinant His-tagged GlnR and Trx-His-S-tagged PhoP inferred that PhoP might compete against GlnR from binding at the a3-b3 site, attributable to the PhoP/GlnR counter-regulatory function subjected to further experimental proof. The in vivo ratio of nitrogen versus phosphate affects bacterial growth and metabolism, including the biosynthesis of secondary metabolites (6), and the metabolism of nitrogen and phosphate is coordinated via complex regulatory networks (14). In Streptomyces coelicolor, nitrogen metabolism is globally regulated by an orphan response regulator, GlnR (22), while the expression of phosphate-regulated genes is controlled by the PhoR-PhoP two-component system (18). When phosphate is insufficient, the sensor kinase PhoR is self-phosphorylated and subsequently transfers the high-energy phosphate group to its cognate response regulator PhoP. The phosphorylated PhoP then binds to the PHO boxes, which are comprised of several 11-nucleotide (nt) direct repeat units (DRus) in the promoter regions of its target genes, and regulates their expression (20). Based on microarray data (13), a connection between the phosphate metabolism controlled by PhoP and the nitrogen metabolism regulated by GlnR was proposed. Rodriguez-Garcia et al. (14) subsequently proved that PhoP repressed the expression of both glnR and the GlnR target genes, including glnA, glnII, and the amount operon (amtB-glnKglnD), through directly binding to the DRus in their promoters.The proposed cis-element for GlnR binding is complex and is comprised of two GlnR binding boxes, each consisting of one a site and one b site separated by 6 nucleotides (i.e., a1-site-n6-b1-site-n6-a2-site-n6-b2-site-n6 [a1-b1 and a2-b2]) located upstream of most GlnR target genes (including glnA, glnII, an...

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“…PhoP was found to bind a PHO box overlapping with the sequence recognized by AfsR. 36,37 Both PhoP and AfsR are positive regulators and compete for this sequence. Indeed, the affinity of AfsR for this PHO box is higher than that of PhoP, and therefore, PhoP interferes with optimal AfsR-mediated afsS expression.…”

Section: Phop Control Of Antibiotic and Other Secondary Metabolites Bmentioning

confidence: 99%

The master regulator PhoP coordinates phosphate and nitrogen metabolism, respiration, cell differentiation and antibiotic biosynthesis: comparison in Streptomyces coelicolor and Streptomyces avermitilis

2017

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Phosphate limitation is important for production of antibiotics and other secondary metabolites in Streptomyces. Phosphate control is mediated by the two-component system PhoR-PhoP. Following phosphate depletion, PhoP stimulates expression of genes involved in scavenging, transport and mobilization of phosphate, and represses the utilization of nitrogen sources. PhoP reduces expression of genes for aerobic respiration and activates nitrate respiration genes. PhoP activates genes for teichuronic acid formation and reduces expression of genes for phosphate-rich teichoic acid biosynthesis. In Streptomyces coelicolor, PhoP repressed several differentiation and pleiotropic regulatory genes, which affects development and indirectly antibiotic biosynthesis. A new bioinformatics analysis of the putative PhoP-binding sequences in Streptomyces avermitilis was made. Many sequences in S. avermitilis genome showed high weight values and were classified according to the available genetic information. These genes encode phosphate scavenging proteins, phosphate transporters and nitrogen metabolism genes. Among of the genes highlighted in the new studies was aveR, located in the avermectin gene cluster, encoding a LAL-type regulator, and afsS, which is regulated by PhoP and AfsR. The sequence logo for S. avermitilis PHO boxes is similar to that of S. coelicolor, with differences in the weight value for specific nucleotides in the sequence. INTRODUCTIONPhosphate is one of the essential nutrients of all living beings. In early studies, Martín and Demain 1 reported that, following phosphate starvation, there is a change in the metabolism of the antibiotic producing strains, which causes the slowdown of primary metabolism and triggers production of secondary metabolites. This early hypothesis has been largely confirmed by the research developed in the last few decades and is discussed in more detail below taking into account recent developments in the coordination of secondary metabolism. The aim of this article is to present an up-to-date integrative view of the PhoP regulation of metabolism in Streptomyces.

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Complex Transcriptional Control of the Antibiotic Regulator afsS in Streptomyces: PhoP and AfsR Are Overlapping, Competitive Activators

Abstract: The afsS gene of several Streptomyces species encodes a small sigma factor-like protein that acts as an activator of several pathway-specific regulatory genes (e.g., actII-ORF4 and redD in Streptomyces coelicolor).

Cited by 34 publications

References 42 publications

The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB

The master regulator PhoP coordinates phosphate and nitrogen metabolism, respiration, cell differentiation and antibiotic biosynthesis: comparison in Streptomyces coelicolor and Streptomyces avermitilis

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