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Actinomycetes are soil-dwelling Gram-positive bacteria, industrially relevant as producers of a wide range of bioactive secondary metabolites, including many antibiotics of clinical and commercial importance.The understanding of actinomycete biology has been based on extensive studies on the model organism Streptomyces coelicolor over many years and on the availability of its complete genome sequence. This bacterium has an unusual complex developmental cycle that includes programmed cell death phenomena that make this bacterium a multicellular prokaryotic model.Morphological differentiation in S. coelicolor is strictly related to physiological differentiation: indeed the onset of morphological differentiation generally coincides with the production of secondary metabolites. During cell death, degradative proteins are synthesized and involved in an extensive degradation of some cellular constituents (proteins and lipids) used for a second growth phase, that is accompanied by antibiotic production.If on one hand, many factors with pleiotropic activity have been identified as key players to control both morphological and physiological differentiation in S. coelicolor, on the other hand, for most actinomycetes, mechanisms and factors governing morphological and physiological processes have not been deeply investigated.This chapter reviews the regulatory mechanisms known to control antibiotic production in actinomycetes and both genetic and physiological methods adopted to improve antibiotic yields.

The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by the dbv gene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation of dbv6 had no effect. In addition, overexpression of dbv3 led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35 , while Dbv3 positively controls the expression of four monocistronic transcription units ( dbv4 , dbv29 , dbv36 , and dbv37 ) and of six operons ( dbv2-dbv1 , dbv14-dbv8 , dbv17-dbv15 , dbv21-dbv20 , dbv24-dbv28 , and dbv30-dbv35 ). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation. IMPORTANCE This report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.