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A novel two-component system involved in the transition to secondary metabolism in Streptomyces coelicolor.

ABSTRACT: BACKGROUND: Bacterial two-component signal transduction regulatory systems are the major set of signalling proteins frequently mediating responses to changes in the environment. They typically consist of a sensor, a membrane-associated histidine kinase and a cytoplasmic response regulator. The membrane-associated sensor detects the environmental signal or stress, whereas the cytoplasmic regulatory protein controls the cellular response usually by gene transcription modulation. METHODOLOGY/PRINCIPALFINDINGS: The Streptomyces coelicolor two genes operon SCO5784-SCO5785 encodes a two-component system, where SCO5784 encodes a histidine-kinase sensor and SCO5785 encodes a response regulator protein. When the expression level of the regulator gene decreases, the antibiotic synthesis and sporulation is delayed temporarily in addition to some ribosomal genes became up regulated, whereas the propagation of the regulatory gene in high copy number results in the earlier synthesis of antibiotics and sporulation, as well as the down regulation of some ribosomal genes and, moreover, in the overproduction of several extracellular proteins. Therefore, this two-component system in S. coelicolor seems to influence various processes characterised by the transition from primary to secondary metabolism, as determined by proteomic and transcriptomic analyses. CONCLUSIONS/SIGNIFICANCE: Propagation of SCO5785 in multicopy enhances the production of antibiotics as well as secretory proteins. In particular, the increase in the expression level of secretory protein encoding genes, either as an artefactual or real effect of the regulator, could be of potential usefulness when using Streptomyces strains as hosts for homologous or heterologous extracellular protein production.

SUBMITTER: Rozas D

PROVIDER: S-EPMC3276577 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A novel two-component system involved in the transition to secondary metabolism in Streptomyces coelicolor.

Rozas Daniel D Gullón Sonia S Mellado Rafael P RP

PloS one 20120209 2

<h4>Background</h4>Bacterial two-component signal transduction regulatory systems are the major set of signalling proteins frequently mediating responses to changes in the environment. They typically consist of a sensor, a membrane-associated histidine kinase and a cytoplasmic response regulator. The membrane-associated sensor detects the environmental signal or stress, whereas the cytoplasmic regulatory protein controls the cellular response usually by gene transcription modulation. METHODOLOG ...[more]

PMID: 22347508

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Project description:Two-component systems (TCSs), the predominant signal transduction pathways employed by bacteria, play important roles in physiological metabolism in Streptomyces Here, a novel TCS, GluR-GluK (encoded by SCO5778-SCO5779), which is located divergently from the gluABCD operon encoding a glutamate uptake system, was identified as being involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor Under the condition of minimal medium (MM) supplemented with different concentrations of glutamate, deletion of the gluR-gluK operon (gluR-K) resulted in enhanced actinorhodin (ACT) but reduced undecylprodigiosin (RED) and yellow type I polyketide (yCPK) production, suggesting that GluR-GluK plays a differential role in antibiotic biosynthesis. Furthermore, we found that the response regulator GluR directly promotes the expression of gluABCD under the culture condition of MM with a high concentration of glutamate (75 mM). Using the biolayer interferometry assay, we demonstrated that glutamate acts as the direct signal of the histidine kinase GluK. It was therefore suggested that upon sensing high concentrations of glutamate, GluR-GluK would be activated and thereby facilitate glutamate uptake by increasing gluABCD expression. Finally, we demonstrated that the role of GluR-GluK in antibiotic biosynthesis is independent of its function in glutamate uptake. Considering the wide distribution of the glutamate-sensing (GluR-GluK) and uptake (GluABCD) module in actinobacteria, it could be concluded that the GluR-GluK signal transduction pathway involved in secondary metabolism and glutamate uptake should be highly conserved in this bacterial phylum.IMPORTANCE In this study, a novel two-component system (TCS), GluR-GluK, was identified to be involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor A possible GluR-GluK working model was proposed. Upon sensing high glutamate concentrations (such as 75 mM), activated GluR-GluK could regulate both glutamate uptake and antibiotic biosynthesis. However, under a culture condition of MM supplemented with low concentrations of glutamate (such as 10 mM), although GluR-GluK is activated, its activity is sufficient only for the regulation of antibiotic biosynthesis. To the best of our knowledge, this is the first report describing a TCS signal transduction pathway for glutamate sensing and uptake in actinobacteria.

Project description:As with most annotated two-component systems (TCSs) of Streptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, for morphogenesis and actinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using an S. coelicolor strain expressing MacR-Flag fusion protein, identified in vivo targets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assays in vitro and by ChIP-quantitative PCR in vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes, sco6728, sco4924, and sco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS from S. avermitilis and S. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism in StreptomycesIMPORTANCE TCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in the Streptomyces model strain S. coelicolor are unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that other Streptomyces species have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems in Streptomyces.

Project description:Antibiotic resistance currently presents one of the biggest threats to humans. The development and implementation of strategies against the spread of superbugs is a priority for public health. In addition to raising social awareness, approaches such as the discovery of new antibiotic molecules and the elucidation of resistance mechanisms are common measures. Accordingly, the two-component system (TCS) of Streptomyces coelicolor AbrB1/B2, offer amenable ways to study both antibiotic production and resistance. Global transcriptomic comparisons between the wild-type strain S. coelicolor M145 and the mutant ?abrB, using RNA-Seq, showed that the AbrB1/B2 TCS is implicated in the regulation of different biological processes associated with stress responses, primary and secondary metabolism, and development and differentiation. The ?abrB mutant showed the up-regulation of antibiotic biosynthetic gene clusters and the down-regulation of the vancomycin resistance gene cluster, according to the phenotypic observations of increased antibiotic production of actinorhodin and undecylprodigiosin, and greater susceptibility to vancomycin. The role of AbrB1/B2 in vancomycin resistance has also been shown by an in silico analysis, which strongly indicates that AbrB1/B2 is a homolog of VraR/S from Staphylococcus aureus and LiaR/S from Enterococcus faecium/Enterococcus faecalis, both of which are implied in vancomycin resistance in these pathogenic organisms that present a serious threat to public health. The results obtained are interesting from a biotechnological perspective since, on one hand, this TCS is a negative regulator of antibiotic production and its high degree of conservation throughout Streptomyces spp. makes it a valuable tool for improving antibiotic production and the discovery of cryptic metabolites with antibiotic action. On the other hand, AbrB1/B2 contributes to vancomycin resistance and is a homolog of VraR/S and LiaR/S, important regulators in clinically relevant antibiotic-resistant bacteria. Therefore, the study of AbrB1/B2 could provide new insight into the mechanism of this type of resistance.

Dataset Information

A novel two-component system involved in the transition to secondary metabolism in Streptomyces coelicolor.

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A novel two-component system involved in the transition to secondary metabolism in Streptomyces coelicolor.

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