The Streptomyces coelicolor Small ORF trpM Stimulates Growth and Morphological Development and Exerts Opposite Effects on Actinorhodin and Calcium-Dependent Antibiotic Production.

Alberto Vassallo,Teresa Faddetta,Emilia Palazzotto,Giovanni Renzone,Giuseppe Gallo,Andrea Scaloni,Luigi Botta,Anna Maria Puglia

doi:10.3389/fmicb.2020.00224

Abstract

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.

Highlights

Bacteria belonging to the phylum Actinomycetales are widely recognized as a very prolific source of biologically active natural compounds, such as antibiotics, immunosuppressants, and herbicides
Since qRT-PCR showed that the expression of trpM in Sco-trpMKI was already twofold greater than that in Sco-EV (Supplementary Figure S1), thiostrepton was not added to the growth medium as inducer to avoid the introduction of any possible negative effects on antibiotic production and on protein expression pattern as described by Wang et al (2017) and Chiu et al (1999), respectively
The results described in this work revealed that trpM boosts the morpho-physiological differentiation of S. coelicolor

Summary

Introduction

Bacteria belonging to the phylum Actinomycetales are widely recognized as a very prolific source of biologically active natural compounds, such as antibiotics, immunosuppressants, and herbicides. Supplementation of specific nutriments exerts a control on S. coelicolor morphological and physiological differentiation: as an example, Palazzotto et al (2015) demonstrated that the supplementation of L-tryptophan (L-Trp) promotes sporulation and stimulates the production of CDA – that contains proteinogenic and non-proteinogenic amino acids, including L-Trp and D-Trp – and the production of ACT – that does not contain any Trp in its structure. In this regard, it is noteworthy that in S. coelicolor the expression of genes involved in L-Trp biosynthesis (i.e., trp genes) is not repressed by Trp supplementation (Hu et al, 1999; Palazzotto et al, 2016). Trp genes are organized either as gene clusters (i.e., trpC1MBA and trpC2D2GE2) or single genes (i.e., trpE3, trpE1 and priA/trpF) spread in the genome with trpC2D2GE2 localized within the CDA biosynthetic gene cluster

Methods

Results

Conclusion