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

Wang, Ying; Cen, Xufeng; Zhao, Guoping; Wang, Jin

doi:10.1128/jb.00989-12

Cited by 159 publications

(197 citation statements)

References 22 publications

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“…To determine the precise GlnR-binding sequences for regulation of actII-ORF4 expression, DNase I footprinting assays were conducted using a FAM-labeled actII-ORF4 probe, as reported previously (17). A 32-nt GlnR-protected area was present in the actII-ORF4 promoter region, spanning from nucleotide position Ϫ77 to Ϫ46 relative to the transcriptional start site (TSS) (Fig.…”

Section: Defining the Precise Glnr-binding Sites Upstream Of Actii-ormentioning

confidence: 99%

“…DNase I Footprinting Assays-DNase I footprinting assays were performed using purified His 6 -GlnR protein as described by Wang et al (17). The probes for actII-ORF4p and redZp containing the respective promoter regions were obtained by PCR amplification using the primer pairs, actII-ORF4p-FAM-fw/rv and redZp-FAM-fw/rv, respectively, which were then cloned into the T-vector pUC18B-T (TOLO BIOTECHNOLOGY, Shanghai, China) and verified by DNA sequencing.…”

Section: Tablementioning

confidence: 99%

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Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces

Zhu

Zheng

et al. 2016

Journal of Biological Chemistry

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Edited by Joel GottesfeldGlnR, an OmpR-like orphan two-component system response regulator, is a master regulator of nitrogen metabolism in the genus Streptomyces. In this work, evidence that GlnR is also directly involved in the regulation of antibiotic biosynthesis is provided. In the model strain Streptomyces coelicolor M145, an in-frame deletion of glnR resulted in markedly increased actinorhodin (ACT) production but reduced undecylprodigiosin (RED) biosynthesis when exposed to R2YE culture medium. Transcriptional analysis coupled with DNA binding studies revealed that GlnR represses ACT but activates RED production directly via the pathway-specific activator genes actII-ORF4 and redZ, respectively. The precise GlnR-binding sites upstream of these two target genes were defined. In addition, the direct involvement of GlnR in antibiotic biosynthesis was further identified in Streptomyces avermitilis, which produces the important anthelmintic agent avermectin. We found that S. avermitilis GlnR (GlnRsav) could stimulate avermectin but repress oligomycin production directly through the respective pathway-specific activator genes, aveR and olmRI/RII. To the best of our knowledge, this report describes the first experimental evidence demonstrating that GlnR regulates antibiotic biosynthesis directly through pathway-specific regulators in Streptomyces. Our results suggest that GlnR-mediated regulation of antibiotic biosynthesis is likely to be universal in streptomycetes. These findings also indicate that GlnR is not only a master nitrogen regulator but also an important controller of secondary metabolism, which may help to balance nitrogen metabolism and antibiotic biosynthesis in streptomycetes.

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Section: Defining the Precise Glnr-binding Sites Upstream Of Actii-ormentioning

confidence: 99%

Section: Tablementioning

confidence: 99%

Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces

Zhu

Zheng

et al. 2016

Journal of Biological Chemistry

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“…However, the structure-functional aspects of the consensus sequences are complicated, e.g., they may consist of either triple 22-bp GlnR binding boxes as in the amtB promoter (18) or double a sites as in the promoters of nasA (14) and SCO5163 (19) in S. coelicolor. Besides, in Streptomyces venezuelae, the double a sites (GTnACn6-GTnAC) were found to be the most common GlnR binding consensus sequences, while in several cases, there was either only one 22-bp GlnR binding box (GTnAC-n6-GAnAC) or no identifiable GlnR binding consensus sequences being characterized (20).…”

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confidence: 99%

Three of Four GlnR Binding Sites Are Essential for GlnR-Mediated Activation of Transcription of the Amycolatopsis mediterranei nas Operon

Wang

Shao

et al. 2013

J Bacteriol

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In Amycolatopsis mediterranei U32, genes responsible for nitrate assimilation formed one operon, nasACKBDEF, whose transcription is induced by the addition of nitrate. Here, we characterized GlnR as a direct transcriptional activator for the nas operon. The GlnR-protected DNA sequences in the promoter region of the nas operon were characterized by DNase I footprinting assay, the previously deduced Streptomyces coelicolor double 22-bp GlnR binding consensus sequences comprising a1, b1, a2, and b2 sites were identified, and the sites were then mutated individually to test their roles in both the binding of GlnR in vitro and the GlnR-mediated transcriptional activation in vivo. The results clearly showed that only three GlnR binding sites (a1, b1, and b2 sites) were required by GlnR for its specific binding to the nas promoter region and efficient activation of the transcription of the nas operon in U32, while the a2 site seemed unnecessary.A mycolatopsis mediterranei is widely studied for its importance in producing the antimycobacterial rifamycins. It was found that nitrate, a significant nitrogen source for bacteria in the biosphere, remarkably stimulated the yield of rifamycin SV by as much as 171% in A. mediterranei U32 fermentation, which simultaneously altered the expression of a series of genes related to both primary metabolism and secondary metabolism and thus was illustrated as the nitrate-stimulating effect (1). The assimilation of nitrate in bacteria requires nitrate reductase and nitrite reductase, which sequentially catalyze the reduction of nitrate to nitrite and then to ammonium (2). Although the assimilatory nitrate reductase was characterized from A. mediterranei decades ago, a nas operon consisting of nasACKBDEF genes encoding 7 enzymes/ proteins responsible for nitrate assimilation and necessary for the nitrate-stimulating effect was just recently characterized (3). The transcription of the nas operon was activated by the addition of extracellular nitrate sources (3), while the activator for nas operon transcription remains unknown.

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“…DNase I footprinting assays were performed essentially as described by Wang et al (2012). For preparation of the sense-strand probe for the espR promoter, the region upstream of espR was amplified by PCR with primers TemERForward and TemER-Reverse, and the 250 bp amplicon was cloned into the pMD-18T vector.…”

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confidence: 99%

“…Samples were extracted with phenol/chloroform and precipitated with ethanol, and then DNA pellets were dissolved in 30 ml MiniQ water. Preparation of the DNA ladder, electrophoresis and data analysis were essentially as described by Wang et al (2012), except that the Genescan-LIZ500 size standard (Applied Biosystems) was used. The MprA-protected regions upstream of espR were mapped in a similar way.…”

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EspR, a regulator of the ESX-1 secretion system in Mycobacterium tuberculosis, is directly regulated by the two-component systems MprAB and PhoPR

et al. 2015

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EspR, a regulator of the ESX-1 secretion system in Mycobacterium tuberculosis, is directly regulated by the two-component systems MprAB and PhoPR INTRODUCTIONESX-1 (ESAT-6 secretion system-1) is a major virulence determinant of Mycobacterium tuberculosis, the causative agent of human tuberculosis. ESX-1 exports pathogenic and immunogenic proteins such as ESAT-6 (EsxA), EspA and other substrate proteins into host cells (Simeone et al., 2009). The secretion of EspA and ESAT-6 is mutually dependent, with deletion of espA resulting in loss of ESAT-6 secretion, and vice versa (Fortune et al., 2005). EspA is encoded within the espA operon, while esxA is located in RD-1 (Region of difference 1), which contains most ESX-1 genes (Mahairas et al., 1996). The intergenic region upstream of espA contains known or predicted binding sites for several different regulatory factors (Hunt et al., 2012;Pang et al., 2013;Rickman et al., 2005; Rosenberg et al., 2011), suggesting that ESX-1 activity could be modulated via altering espA expression.EspR, a key transcriptional regulator of ESX-1, directly regulates espA (Raghavan et al., 2008; Rosenberg et al., 2011). An espR mutant exhibited decreased transcription of the espA operon, loss of ESAT-6 secretion and reduced virulence (Raghavan et al., 2008). Higher-order oligomerization of EspR appears to be involved in cooperatively linking distant DNA sites, such as the three EspR binding sites in the espA promoter (Blasco et al., 2011; Rosenberg et al., 2011). Three EspR sites were also identified in the espR promoter (Blasco et al., 2012). However, these EspR sites are much closer than in the espA promoter, suggesting that EspR may have aAbbreviations: CRP, cAMP receptor protein; EMSA, electrophoresis mobility shift assay; ESX-1, ESAT-6 secretion system-1; L6, lineage 6; RD-1, Region of difference 1; TCS, two-component system.

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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

Cited by 159 publications

References 22 publications

Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces

Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces

Three of Four GlnR Binding Sites Are Essential for GlnR-Mediated Activation of Transcription of the Amycolatopsis mediterranei nas Operon

EspR, a regulator of the ESX-1 secretion system in Mycobacterium tuberculosis, is directly regulated by the two-component systems MprAB and PhoPR

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