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A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.

Two novel Gram-positive-staining, acidophilic strains were isolated from soil samples. Both show typical features of filamentous actinomycetes. On the basis of 16S rRNA gene sequence analysis, the strains are members of the family Micromonosporaceae. The two strains contain hydroxydiaminopimelic acid, glycine, alanine and glutamic acid in the peptidoglycan. Fatty acid profiles clearly differentiate the two strains: cyclohexyl C17 : 0, i-C16 : 0 and ai-C17 : 0 are predominant in Delta1T, while the major components for Delta3T are ai-C17 : 0 and i-C16 : 0. The two strains also differ in their major menaquinones, MK-9(H8, H4, H6) for Delta1T and MK-9(H8, H6) for Delta3T, and in phospholipid patterns; Delta1T displays phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, methyl phosphatidylethanolamine and an unknown aminophospholipid, while Delta3T also contains minor amounts of several unknown phospholipids in addition to these phospholipids. The whole-cell sugars of both strains are galactose, arabinose and xylose. The G+C content of the DNA is 72.7 mol% for Delta1T and 71.9 mol% for Delta3T. On the basis of chemotaxonomic, physiological and phylogenetic data, we propose Rugosimonospora gen. nov. to accommodate the two strains, with the description of Rugosimonospora acidiphila gen. nov., sp. nov. (the type species; type strain Delta1T =DSM 45227T =NBRC 104874T) and Rugosimonospora africana sp. nov. (type strain Delta3T =DSM 45228T =NBRC 104875T).

Bacteria belonging to the order of Actinomycetales are the source of many clinically important drugs, especially antibiotics. Due to the large back-catalogue of molecules isolated from these bacteria, it is difficult to find novel compounds by using traditional methods and sources. In this thesis, we use state-of-the-art metabolomics and genomics techniques to identify new molecules from the “rare actinomycete” genus Planomonospora. In chapter 2, we discuss methods for the differentiation of known and unknown molecules, called dereplication, in detail. Further, we describe the creation of a computationally predicted tandem mass fragmentation library from a proprietary metabolite database, which can speed up and automate the identification of known compounds. In chapter 3, we discuss our study into the “rare actinomycete” genus Planomonospora. We correlate phylogenetic diversity with metabolite diversity, showing that specific groups of strains produce specific molecules. Further, we sequence the genomes of selected strains to gain insight into their theoretical biosynthetic capacity. Eventually, we establish criteria to rationally select metabolites with a high probability of novelty, for consecutive isolation. In chapter 4, we describe the characterization of a family of novel metabolites, prioritized in the previous study. Investigation of the biosynthetic pathway suggests that the metabolite is a ribosomally synthesized and post-translationally modified peptide. Indeed, we prove that a minimal, two-gene biosynthetic gene cluster is responsible for the production of the compound. One of the two genes is, to our knowledge, the shortest peptide-encoding gene ever reported. This work can be the steppingstone for future investigations into the under-appreciated genus Planomonospora in particular and “rare actinomycetes” in general.

The glycopeptide A40926, produced by the actinomycete Nonomuraea gerenzanensis, is the precursor of dalbavancin, a second-generation glycopeptide antibiotic approved for clinical use in the USA and Europe in 2014 and 2015, respectively. The final product of the biosynthetic pathway is an O-acetylated form of A40926 (acA40926). Glycopeptide biosynthesis in N. gerenzanensis is dependent upon the dbv gene cluster that encodes, in addition to the two essential positive regulators Dbv3 and Dbv4, the putative members of a two-component signal transduction system, specifically the response regulator Dbv6 and the sensor kinase Dbv22. The aim of this work was to assign a role to these two genes. Our results demonstrate that deletion of dbv22 leads to an increased antibiotic production with a concomitant reduction in glycopeptide resistance. Deletion of dbv6 results in a similar phenotype, although the effects are not as strong as in the Δdbv22 mutant. Consistently, quantitative RT-PCR analysis showed that Dbv6 and Dbv22 negatively regulate the regulatory genes (dbv3 and dbv4), as well as some dbv biosynthetic genes (dbv23 and dbv24), whereas Dbv6 and Dbv22 positively regulate transcription of the single, cluster-associated resistance gene. Finally, we demonstrate that exogenously added acA40926 and its precursor A40926 can modulate transcription of dbv genes but with an opposite extent: A40926 strongly stimulates transcription of the Dbv6/Dbv22 target genes while acA40926 has a neutral or negative effect on transcription of those genes. We propose a model in which glycopeptide biosynthesis in N. gerenzanensis is modulated through a positive feedback by the biosynthetic precursor A40926 and a negative feedback by the final product acA40926. In addition to previously reported control systems, this sophisticated control loop might help the producing strain cope with the toxicity of its own product. This work, besides leading to improved glycopeptide producing strains, enlarges our knowledge on the regulation of glycopeptide biosynthesis in actinomycetes, setting N. gerenzanensis and its two-component system Dbv6-Dbv22 apart from other glycopeptide producers.

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.

Background The filamentous actinomycete Microbispora ATCC-PTA-5024 produces the lantibiotic NAI-107, which is an antibiotic peptide effective against multidrug-resistant Gram-positive bacteria. In actinomycetes, antibiotic production is often associated with a physiological differentiation program controlled by a complex regulatory and metabolic network that may be elucidated by the integration of genomic, proteomic and bioinformatic tools. Accordingly, an extensive evaluation of the proteomic changes associated with NAI-107 production was performed on Microbispora ATCC-PTA-5024 by combining two-dimensional difference in gel electrophoresis, mass spectrometry and gene ontology approaches. Results Microbispora ATCC-PTA-5024 cultivations in a complex medium were characterized by stages of biomass accumulation (A) followed by biomass yield decline (D). NAI-107 production started at 90 h (A stage), reached a maximum at 140 h (D stage) and decreased thereafter. To reveal patterns of differentially represented proteins associated with NAI-107 production onset and maintenance, differential proteomic analyses were carried-out on biomass samples collected: i) before (66 h) and during (90 h) NAI-107 production at A stage; ii) during three time-points (117, 140, and 162 h) at D stage characterized by different profiles of NAI-107 yield accumulation (117 and 140 h) and decrement (162 h). Regulatory, metabolic and unknown-function proteins, were identified and functionally clustered, revealing that nutritional signals, regulatory cascades and primary metabolism shift-down trigger the accumulation of protein components involved in nitrogen and phosphate metabolism, cell wall biosynthesis/maturation, lipid metabolism, osmotic stress response, multi-drug resistance, and NAI-107 transport. The stimulating role on physiological differentiation of a TetR-like regulator, originally identified in this study, was confirmed by the construction of an over-expressing strain. Finally, the possible role of cellular response to membrane stability alterations and of multi-drug resistance ABC transporters as additional self-resistance mechanisms toward the lantibiotic was confirmed by proteomic and confocal microscopy experiments on a Microbispora ATCC-PTA-5024 lantibiotic-null producer strain which was exposed to an externally-added amount of NAI-107 during growth. Conclusion This study provides a net contribution to the elucidation of the regulatory, metabolic and molecular patterns controlling physiological differentiation in Microbispora ATCC-PTA-5024, supporting the relevance of proteomics in revealing protein players of antibiotic biosynthesis in actinomycetes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2369-z) contains supplementary material, which is available to authorized users.

We report the genome sequence of Planobispora rosea ATCC 53733, a mycelium-forming soil-dweller belonging to one of the lesser studied genera of Actinobacteria and producing the thiopeptide GE2270. The P. rosea genome presents considerable convergence in gene organization and function with other members in the family Streptosporangiaceae, with a significant number (44%) of shared orthologs. Patterns of gene expression in P. rosea cultures during exponential and stationary phase have been analyzed using whole transcriptome shotgun sequencing and by proteome analysis. Among the differentially abundant proteins, those involved in protein metabolism are particularly represented, including the GE2270-insensitive EF-Tu. Two proteins from the pbt cluster, directing GE2270 biosynthesis, slightly increase their abundance values over time. While GE2270 production starts during the exponential phase, most pbt genes, as analyzed by qRT-PCR, are down-regulated. The exception is represented by pbtA, encoding the precursor peptide of the ribosomally synthesized GE2270, whose expression reached the highest level at the entry into stationary phase. Copyright

The filamentous actinobacterium Microbispora sp. ATCC-PTA-5024 produces the lantibiotic NAI-107 (Maffioli et al., 2014), which is effective against multidrug-resistant Gram-positive pathogens (Jabés et al., 2011). In actinomycetes, the biosynthesis of antibiotics is generally elicited as a physiological response controlled by a complex regulatory network involving global regulators, playing pleiotropic roles, and pathway-specific regulators, which activate the biosynthesis of biologically active molecules (Bibb, 2005)

Background NAI-107, produced by the actinomycete Microbispora sp. ATCC-PTA-5024, is a promising lantibiotic active against Gram-positive bacteria and currently in late preclinical-phase. Lantibiotics (lanthionine-containing antibiotics) are ribosomally synthesized and post-translationally modified peptides (RiPPs), encoded by structural genes as precursor peptides. The biosynthesis of biologically active compounds is developmentally controlled and it depends upon a variety of environmental stimuli and conditions. Inorganic phosphate (Pi) usually negatively regulates biologically-active molecule production in Actinomycetes, while it has been reported to have a positive control on lantibiotic production in Firmicutes strains. So far, no information is available concerning the Pi effect on lantibiotic biosynthesis in Actinomycetes. Results After having developed a suitable defined medium, Pi-limiting conditions were established and confirmed by quantitative analysis of polyphosphate accumulation and of expression of selected Pho regulon genes, involved in the Pi-limitation stress response. Then, the effect of Pi on Microbispora growth and NAI-107 biosynthesis was investigated in a defined medium containing increasing Pi amounts. Altogether, our analyses revealed that phosphate is necessary for growth and positively influences both growth and NAI-107 production up to a concentration of 5 mM. Higher Pi concentrations were not found to further stimulate Microbispora growth and NAI-107 production. Conclusion These results, on one hand, enlarge the knowledge on Microbispora physiology, and, on the other one, could be helpful to develop a robust and economically feasible production process of NAI-107 as a drug for human use. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0133-0) contains supplementary material, which is available to authorized users.