Dataset Information

Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145.

ABSTRACT: otrA resembles elongation factor G (EF-G) and is considered to be an oxytetracycline (OTC)-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE* promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coelicolor M145 by intergeneric conjugation, yielding the recombinant strain S. coelicolor M145-OA. As expected S. coelicolor M145-OA exhibited higher resistance levels specifically to OTC and aminoglycosides gentamycin, hygromycin, streptomycin, and spectinomycin. However, unexpectedly, S. coelicolor M145-OA on solid medium showed an accelerated aerial mycelia formation, a precocious sporulation, and an enhanced actinorhodin (Act) production. Upon growth in 5-L fermentor, the amount of intra- and extracellular Act production was 6-fold and 2-fold higher, respectively, than that of the original strain. Consistently, reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the transcriptional level of pathway-specific regulatory gene actII-orf4 was significantly enhanced in S. coelicolor M145-OA compared with in S. coelicolor M145.

SUBMITTER: Zhao YF

PROVIDER: S-EPMC6137418 | biostudies-literature | 2018 Sept.

REPOSITORIES: biostudies-literature

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Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145.

Zhao Yan-Fang YF Lu Dan-Dan DD Bechthold Andreas A Ma Zheng Z Yu Xiao-Ping XP

Journal of Zhejiang University. Science. B 20180901 9

otrA resembles elongation factor G (EF-G) and is considered to be an oxytetracycline (OTC)-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE<sup>*</sup> promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coelicolor M145 by intergeneric conjugation, yielding the recombinant strai ...[more]

PMID: 30178637

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Project description:The transcription factor AdpA is a key regulator controlling both secondary metabolism and morphological differentiation in Streptomyces. Due to its critical functions, its expression undergoes multilevel regulations at transcriptional, posttranscriptional, and translational levels, yet no posttranslational regulation has been reported. Sulfane sulfur, such as hydro polysulfide (HSnH, n ≥ 2) and organic polysulfide (RSnH, n ≥ 2), is common inside microorganisms, but its physiological functions are largely unclear. Here, we discovered that sulfane sulfur posttranslationally modifies AdpA in Streptomyces coelicolor via specifically reacting with Cys62 of AdpA to form a persulfide (Cys62-SSH). This modification decreases the affinity of AdpA to its self-promoter PadpA, allowing increased expression of adpA, further promoting the expression of its target genes actII-4 and wblA. ActII-4 activates actinorhodin biosynthesis, and WblA regulates morphological development. Bioinformatics analyses indicated that AdpA-Cys62 is highly conserved in Streptomyces, suggesting the prevalence of such modification in this genus. Thus, our study unveils a new type of regulation on the AdpA activity and sheds a light on how sulfane sulfur stimulates the production of antibiotics in Streptomyces. IMPORTANCE Streptomyces species produce a myriad of natural products with (potential) clinical applications. While the database of biosynthetic gene clusters is quickly expanding, their regulation mechanisms are rarely known. Sulfane sulfur species are commonly present in microorganisms with unclear functions. Here, we discovered that sulfane sulfur increases actinorhodin (ACT) production in S. coelicolor. The underlying mechanism is that sulfane sulfur specifically reacts with AdpA, a global transcription factor controlling both ACT gene cluster and morphological differentiation-related genes, to form sulfhydrated AdpA. This modification changes the dynamics of AdpA-controlled gene networks and leads to high expression of ACT biosynthetic genes. Given the wide prevalence of AdpA and sulfane sulfur in Streptomyces, this mechanism may represent a common regulating pattern of all AdpA-controlled biosynthetic pathways. Thus, this finding provides a new strategy for mining and activating valuable biosynthetic gene clusters.

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 biosynthesis - 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 (ACT) production; (ii) decreases calcium-dependent antibiotic (CDA) 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 ACT 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.

Dataset Information

Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145.

Publications

Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145.

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