title: Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Molecular Microbiology and Biotechnology, STEBICEF Department, University of Palermo, 90128, Palermo, Italy. giuseppe.gallo@unipa.it.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Gallo
authors.affiliation: Proteomic and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147, Naples, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Renzone
authors.affiliation: Laboratory of Molecular Microbiology and Biotechnology, STEBICEF Department, University of Palermo, 90128, Palermo, Italy.
authors.forename: Emilia
authors.initials: E
authors.lastname: Palazzotto
authors.affiliation: Naicons srl, 20139, Milan, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Monciardini
authors.affiliation: Proteomic and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147, Naples, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Arena
authors.affiliation: Laboratory of Molecular Microbiology and Biotechnology, STEBICEF Department, University of Palermo, 90128, Palermo, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Faddetta
authors.affiliation: Laboratory of Molecular Microbiology and Biotechnology, STEBICEF Department, University of Palermo, 90128, Palermo, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Giardina
authors.affiliation: Laboratory of Molecular Microbiology and Biotechnology, STEBICEF Department, University of Palermo, 90128, Palermo, Italy.
authors.forename: Rosa
authors.initials: R
authors.lastname: Alduina
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2970, Hørsholm, Denmark.|German Center for Infection Research (DZIF) partner site Tübingen, 72074, Tübingen, Germany.
authors.forename: Tilmann
authors.initials: T
authors.lastname: Weber
authors.affiliation: Sistema Informativo di Ateneo (SIA), Area Servizi di Rete, University of Palermo, 90128, Palermo, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Sangiorgi
authors.affiliation: Laboratory of Molecular Microbiology and Biotechnology, STEBICEF Department, University of Palermo, 90128, Palermo, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Russo
authors.affiliation: Laboratory of Molecular Microbiology and Biotechnology, STEBICEF Department, University of Palermo, 90128, Palermo, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Spinelli
authors.affiliation: Naicons srl, 20139, Milan, Italy.
authors.forename: Margherita
authors.initials: M
authors.lastname: Sosio
authors.affiliation: Proteomic and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147, Naples, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.affiliation: Laboratory of Molecular Microbiology and Biotechnology, STEBICEF Department, University of Palermo, 90128, Palermo, Italy.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Puglia
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D039903:Actinobacteria / Q000737:chemistry / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D001430:Bacteriocins / Q000737:chemistry / Q000378:metabolism*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D006094:Gram-Positive Bacteria / Q000187:drug effects / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics
chemical_list: D018528:ATP-Binding Cassette Transporters; D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D010455:Peptides; C528150:microbisporicin