Dataset Information

WblC- and tetracycline-dependent shifts in the composition of Streptomyces coelicolor ribosomal proteome

ABSTRACT: WblC, also known as WhiB7, is a widely conserved WhiB-like transcription factor in actinomycetes that activates transcription of many targets upon antibiotic challenge to bring about intrinsic resistance to a wide range of translation-targeting antibiotics. As we found that WblC controls many genes involved in translation and that WblC promotes translation rate upon antibiotic stress in the model actinomycetes Streptomyces coelicolor, we speculated that WblC might alter the protein composition of ribosome during antibiotic stress. To test this, we prepared 70S ribosome fraction from wild-type S. coelicolor cells untreated or treated with tetracycline and ΔwblC mutant treated with tetracycline, and then compared the protein compositions of each 70S samples by mass spectrometric quantification.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Streptomyces Coelicolor A3(2)

SUBMITTER: Ju-Hyung Lee

LAB HEAD: Jung-Hye Roe

PROVIDER: PXD015538 | Pride | 2020-05-27

REPOSITORIES: Pride

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Publications

The WblC/WhiB7 Transcription Factor Controls Intrinsic Resistance to Translation-Targeting Antibiotics by Altering Ribosome Composition.

Lee Ju-Hyung JH Yoo Ji-Sun JS Kim Yeonbum Y Kim Jong-Seo JS Lee Eun-Jin EJ Roe Jung-Hye JH

mBio 20200414 2

Bacteria that encounter antibiotics can efficiently change their physiology to develop resistance. This intrinsic antibiotic resistance is mediated by multiple pathways, including a regulatory system(s) that activates specific genes. In some Streptomyces and Mycobacterium spp., the WblC/WhiB7 transcription factor is required for intrinsic resistance to translation-targeting antibiotics. Wide conservation of WblC/WhiB7 within Actinobacteria indicates a critical role of WblC/W ...[more]

PMID: 32291305

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Project description:In actinomycetes, antibiotic production is often associated with a morpho-physiological differentiation program that is regulated by complex molecular and metabolic networks. Many aspects of these regulatory circuits have been already elucidated and many others still deserve further investigations. In this regard, the possible role of many small open-reading frames(smORFs) in actinomycete morpho-physiological differentiation is still elusive. In Streptomyces coelicolor, inactivation of the smORF trpM (SCO2038) - whose product modulates L-tryptophan metabolism - impairs production of antibiotics and morphological differentiation. Indeed, it was demonstrated that TrpM is able to interact with PepA (SCO2179), a putative cytosol aminopeptidase playing a key role in antibiotic production and sporulation. In this work, a S. coelicolor trpM knock-in (Sco-trpMKI) mutant strain was generated by cloning trpM into overexpressing vector to further investigate the role of trpM in actinomycete growth and morpho-physiological differentiation. Results highlighted that trpM: i) stimulates growth and actinorhodin production; ii) decreases calcium-dependent antibiotic production; iii) has no effect on undecylprodigiosin production. Metabolic pathways influenced by trpM knock-in were investigated by combining two-difference in gel electrophoresis/nanoliquid chromatography coupled to electrospray linear ion trap tandem mass spectrometry (2D-DIGE/nanoLC-ESI-LIT- MS/MS) and by LC-ESI-MS/MS procedures, respectively. These analyses demonstrated that over-expression of trpM causes an over-representation of factors involved in protein synthesis and nucleotide metabolism as well as a down-representation of proteins involved in central carbon and amino acid metabolism. At the metabolic level, this corresponded to a differential accumulation pattern of different amino acids - including aromatic ones but tryptophan – and central carbon intermediates. PepA was also down-represented in Sco-trpMKI. The latter was produced as recombinant His- tagged protein and was originally proven having the predicted aminopeptidase activity. Altogether, these results highlight the stimulatory effect of trpM in S. coelicolor growth and actinorhodin biosynthesis, which are elicited through the modulation of various metabolic pathways and PepA representation, further confirming the complexity of regulatory networks that control antibiotic production in actinomycetes.

Project description:Background The genomes of Streptomyces coelicolor and Streptomyces lividans bear a considerable degree of orthology. While S. coelicolor is the model streptomycete for studying antibiotic synthesis and differentiation, S. lividans is almost exclusively considered as the preferred host, among actinomycetes, for cloning and expression of exogenous DNA. We used whole genome microarrays as a comparative genomics tool for identifying the subtle yet crucial differences between these two chromosomes. Results We identified five large S. coelicolor genomic islands (≥25 kb) and 18 smaller islets absent in S. lividans chromosome. Many of these regions show anomalous GC bias and codon usage patterns. Six of them are in close vicinity of tRNA genes while nine are flanked with near perfect repeat sequences indicating that these are probable recent evolutionary acquisitions into S. coelicolor. Embedded within these segments are at least four DNA methylases and two probable methyl-sensing restriction endonucleases. Comparison with S. coelicolor transcriptome and proteome data revealed that some of the missing genes are active during the course of growth and differentiation in S. coelicolor. In particular, a pair of methylmalonyl CoA mutase (mcm) genes involved in polyketide precursor biosynthesis, an acyl-CoA dehydrogenase implicated in timing of actinorhodin synthesis and bldB, a developmentally significant regulator whose mutation causes complete abrogation of antibiotic synthesis belong to this category. Conclusion Our findings provide tangible hints for elucidating the genetic basis of important phenotypic differences between these two streptomycetes. Importantly, absence of certain genes in S. lividans identified here could potentially explain the relative ease of DNA transformations or the conditional lack of actinorhodin synthesis in S. lividans. Further genetic studies based on these results will enable one to target specific sequences in the genetically well-characterized S. coelicolor to adapt it for industrial processes. Keywords: Comparative Genomic Hybridization

Dataset Information

WblC- and tetracycline-dependent shifts in the composition of Streptomyces coelicolor ribosomal proteome

Publications

The WblC/WhiB7 Transcription Factor Controls Intrinsic Resistance to Translation-Targeting Antibiotics by Altering Ribosome Composition.

Similar Datasets

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