Complex Regulatory Networks Governing Production of the Glycopeptide A40926

Glycopeptides (GPAs) are an important class of antibiotics, with vancomycin and teicoplanin being used in the last 40 years as drugs of last resort to treat infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus. A few new GPAs have since reached the market. One of them is dalbavancin, a derivative of A40926 produced by the actinomycete Nonomuraea sp. ATCC 39727, recently classified as N. gerenzanensis. This review summarizes what we currently know on the multilevel regulatory processes governing production of the glycopeptide A40926 and the different approaches used to increase antibiotic yields. Some nutrients, e.g., valine, l-glutamine and mal…

Additional file 4: of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Supplementary Results section. (PDF 123 kb)

Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024.

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. R…

A Two-Component regulatory system with opposite effects on glycopeptide antibiotic biosynthesis and resistance

AbstractThe 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 ge…

Additional file 5: of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Figure S1-S6 with corresponding figure legends. (PDF 511 kb)

Additional file 5: of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Figure S1-S6 with corresponding figure legends. (PDF 511 kb)

Artificial chromosome libraries of Streptomyces coelicolor A3(2) and Planobispora rosea

Using an Escherichia coli-Streptomyces shuttle vector derived from a bacterial artificial chromosome (BAC), we developed methodologies for the construction of BAC libraries of filamentous actinomycetes. Libraries of Streptomyces coelicolor, the model actinomycete, and Planobispora rosea, a genetically intractable strain, were constructed. Both libraries have an average insert size of 60 kb, with maximal insert larger than 150 kb. The S. coelicolor library was evaluated by selected hybridisations to DraI fragments and by end sequencing of a few clones. Hybridisation of the P. rosea library to selected probes indicates a good representation of the P. rosea genome and that the library can be u…

Draft Genome Sequence of the Microbispora sp. Strain ATCC-PTA-5024, Producing the Lantibiotic NAI-107.

ABSTRACT We report the draft genome sequence of Microbispora sp. strain ATCC-PTA-5024, a soil isolate that produces NAI-107, a new lantibiotic with the potential to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. The draft genome of strain Microbispora sp. ATCC-PTA-5024 consists of 8,543,819 bp, with a 71.2% G+C content and 7,860 protein-coding genes.

Additional file 4: of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Supplementary Results section. (PDF 123 kb)

Two master switch regulators trigger A40926 biosynthesis in Nonomuraea sp. strain ATCC 39727

ABSTRACT 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 …

Omics approaches to elucidate the molecular physiology of lantibiotc NAI-107 production in Microbispora ATCC-PTA-5024

A Genomic, Transcriptomic and Proteomic Look at the GE2270 Producer Planobispora rosea, an Uncommon Actinomycete.

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 G…

Inorganic phosphate is a trigger factor for Microbispora sp. ATCC-PTA-5024 growth and NAI-107 production

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…

Draft genome sequence of the Microbispora sp. strain ATCC-PTA-5024, producing the lantibiotic NAI-107

We report the draft genome sequence of Microbispora sp. strain ATCC-PTA-5024, a soil isolate that produces NAI-107, a new lantibiotic with the potential to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. The draft genome of strain Microbispora sp. ATCC-PTA-5024 consists of 8,543,819 bp, with a 71.2% G+C content and 7,860 proteincoding genes.

Microbial technologies for the discovery of novel bioactive metabolites

Soil microbes represent an important source of biologically active compounds. These molecules present original and unexpected structure and are selective inhibitors of their molecular targets. At Biosearch Italia, discovery of new bioactive molecules is mostly carried out through the exploitation of a proprietary strain collection of over 50000 strains, mostly unusual genera of actinomycetes and uncommon filamentous fungi. A critical element in a drug discovery based on microbial extracts is the isolation of unexploited groups of microorganisms that are at the same time good producers of secondary metabolites. Molecular genetics can assist in these efforts. We will review the development an…

Artificial chromosomes for antibiotic-producing actinomycetes.

Bacteria belonging to the order Actinomycetales produce most microbial metabolites thus far described, several of which have found applications in medicine and agriculture. However, most strains were discovered by their ability to produce a given molecule and are, therefore, poorly characterized physiologically and genetically. Thus, methodologies for genetic manipulation of actinomycetes are not available and efficient tools have been developed for just a few strains. This constitutes a serious limitation to applying molecular genetics approaches to strain development and structural manipulation of microbial metabolites. To overcome this hurdle, we have developed bacterial artificial chrom…

Additional file 2: Table S2. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from global proteome analysis at D substages. (XLS 107 kb)

Additional file 6: Table S5. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, abundance profile and mass spectrometry identification parameters of differentially represented spots containing multiple protein components. (XLS 45 kb)

Additional file 3: Table S3. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from membrane proteome analysis at A substages. (XLSX 37 kb)

Additional file 2: Table S2. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from global proteome analysis at D substages. (XLS 107 kb)

Additional file 6: Table S5. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, abundance profile and mass spectrometry identification parameters of differentially represented spots containing multiple protein components. (XLS 45 kb)

Additional file 7: Table S4. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented proteins due to NAI-107 exposure in Microbispora ATCC-PTA-5024 RP0 strain. (XLSX 32 kb)

Additional file 1: Table S1. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from global proteome analysis at A substages. (XLSX 48 kb)

Additional file 8: Table S6. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Numbers of KEGG orthology groups participating in molecular and metabolic processes as inferred from genome and proteome analyses, respectively. (XLS 24 kb)

Additional file 3: Table S3. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from membrane proteome analysis at A substages. (XLSX 37 kb)

Additional file 7: Table S4. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented proteins due to NAI-107 exposure in Microbispora ATCC-PTA-5024 RP0 strain. (XLSX 32 kb)

Additional file 8: Table S6. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Numbers of KEGG orthology groups participating in molecular and metabolic processes as inferred from genome and proteome analyses, respectively. (XLS 24 kb)

Additional file 1: Table S1. of Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024

Description, functional classification, abundance profile and mass spectrometry identification parameters of differentially represented Microbispora ATCC-PTA-5024 proteins identified from global proteome analysis at A substages. (XLSX 48 kb)