Table_3_The Streptomyces coelicolor Small ORF trpM Stimulates Growth and Morphological Development and Exerts Opposite Effects on Actinorhodin and Calcium-Dependent Antibiotic Production.XLSX

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.

在放线菌（Actinomycetes）中，抗生素的合成往往与受复杂分子与代谢网络调控的形态生理分化程序密切相关。这些调控回路的诸多环节已被阐明，但仍有许多内容有待进一步研究。就此而言，众多小型开放阅读框（small open reading frames, smORFs）在放线菌形态生理分化中可能发挥的作用仍尚不明确。在天蓝色链霉菌（Streptomyces coelicolor）中，小型开放阅读框trpM（SCO2038，其编码产物可调控L-色氨酸生物合成）的失活会损害抗生素合成与形态分化。已有研究证实，TrpM可与PepA（SCO2179）相互作用，后者是一种假定的胞质氨肽酶，在抗生素合成与孢子形成中发挥关键作用。本研究通过将trpM克隆至过表达载体，构建了天蓝色链霉菌trpM敲入（Sco-trpMKI）突变菌株，以进一步探究trpM在放线菌生长及形态生理分化中的功能。研究结果显示，trpM可：(i) 促进菌体生长与放线菌素（actinorhodin, ACT）的合成；(ii) 抑制钙依赖性抗生素（calcium-dependent antibiotic, CDA）的合成；(iii) 对十一烷基灵菌红素的合成无显著影响。本研究分别采用双向差异凝胶电泳/纳升液相色谱-电喷雾线性离子阱串联质谱（2D-DIGE/nanoLC-ESI-LIT-MS/MS）与液相色谱-电喷雾质谱联用（LC-ESI-MS/MS）技术，对trpM敲入所影响的代谢通路进行了探究。分析结果表明，trpM过表达会使参与蛋白质合成与核苷酸代谢的因子表达上调，同时使参与中心碳代谢与氨基酸代谢的蛋白质表达下调。在代谢层面，这表现为多种氨基酸（包括芳香族氨基酸但不包括色氨酸）与中心碳代谢中间产物的积累模式发生改变。在Sco-trpMKI菌株中，PepA的表达同样被下调。本研究将PepA制备为重组His标签蛋白，并证实其具有预测的氨肽酶活性。综上，本研究结果揭示了trpM对天蓝色链霉菌生长与ACT合成的促进作用，该作用通过调控多种代谢通路及PepA的表达水平实现，进一步证实了放线菌中调控抗生素合成的网络具有高度复杂性。