title: Identification of antibacterial peptides from endophytic microbiome.
abstract: Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.
authors.affiliation: Genetics and Physiology, University of Oulu, PO Box 3000, FIN-90014, Oulu, Finland. mysore.tejesvi@oulu.fi.
authors.forename: M V
authors.identifier: http://orcid.org/0000-0002-6176-0981
authors.initials: MV
authors.lastname: Tejesvi
authors.affiliation: Genetics and Physiology, University of Oulu, PO Box 3000, FIN-90014, Oulu, Finland.|Pharmaceutical Microbiology Section, Institute for Medical Microbiology, Immunology, and Parasitology, University of Bonn, D-53115, Bonn, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Picart
authors.affiliation: Admescope Ltd, Typpitie 1, 90620, Oulu, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Kajula
authors.affiliation: Admescope Ltd, Typpitie 1, 90620, Oulu, Finland.
authors.forename: H
authors.initials: H
authors.lastname: Hautajärvi
authors.affiliation: Biochemistry and Molecular Medicine, University of Oulu, PO Box 3000, FIN-90014, Oulu, Finland.
authors.forename: L
authors.initials: L
authors.lastname: Ruddock
authors.affiliation: Novozymes AS, DK-2880, Bagsvaerd, Denmark.
authors.forename: H H
authors.initials: HH
authors.lastname: Kristensen
authors.affiliation: Department of Biochemistry, University of Trieste, Trieste, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Tossi
authors.affiliation: Pharmaceutical Microbiology Section, Institute for Medical Microbiology, Immunology, and Parasitology, University of Bonn, D-53115, Bonn, Germany.
authors.forename: H G
authors.initials: HG
authors.lastname: Sahl
authors.affiliation: Molecular Materials, University of Oulu, PO Box 3000, FIN-90014, Oulu, Finland.
authors.forename: S
authors.initials: S
authors.lastname: Ek
authors.affiliation: Molecular Materials, University of Oulu, PO Box 3000, FIN-90014, Oulu, Finland.
authors.forename: S
authors.initials: S
authors.lastname: Mattila
authors.affiliation: Genetics and Physiology, University of Oulu, PO Box 3000, FIN-90014, Oulu, Finland.
authors.forename: A M
authors.initials: AM
authors.lastname: Pirttilä
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D060026:Endophytes / Q000235:genetics*; D029791:Ericaceae / Q000382:microbiology*; D004926:Escherichia coli / Q000187:drug effects*; D005658:Fungi / Q000235:genetics*; D005820:Genetic Testing; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000187:drug effects*
chemical_list: D000900:Anti-Bacterial Agents; D010455:Peptides
keywords: Antimicrobial peptide; Endophyte; Microbiome; Unculturable