Abstract
BackgroundThe 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.ResultsMicrobispora 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.ConclusionThis 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 materialThe online version of this article (doi:10.1186/s12864-016-2369-z) contains supplementary material, which is available to authorized users.
Highlights
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 Streptomyces coelicolor, the most studied actinomycete used as model strain, the biosynthesis of antibiotics and other secondary metabolites is elicited as a developmental program and a physiological response to a variety of environmental stimuli and conditions, which depend on cell density, nature and/or quantity of carbon, nitrogen and phosphate sources [10,11,12,13,14]
Growth parameters and NAI-107 production kinetics When incubated in the KV6 complex medium, Microbispora ATCC-PTA-5024 WT showed a growth kinetics characterized by a stage of active mycelial cell growth and biomass accumulation (A), ranging from A-66 h to A-96 h, which was followed by a stage of general cell growth arrest and cellular lysis occurrences, as inferred by biomass content decline (D) from D-117 h to D-191 h (Fig. 1a)
Summary
The filamentous actinomycete Microbispora ATCC-PTA-5024 produces the lantibiotic NAI-107, which is an antibiotic peptide effective against multidrug-resistant Gram-positive bacteria. The continuous integration of bioinformatic tools and holistic technologies has allowed the development of consolidated strategies to manage the huge amounts of molecular information on gene expression and biochemical capabilities deriving from “omic” investigations, leading to novel perspectives and approaches to explore microbial strain physiology [15]. In this context, proteomics has recently been used for shedding light on the relationships among overall metabolic pathways and biosynthesis of interesting bioactive molecules in several actinomycete strains [16,17,18,19,20,21,22,23]
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