Identification of AMPs
A metagenomic library consisting of 8448 fosmids from the plant-attached rumen microbiome15 was screened for antimicrobial activity by a combination of agar-based functional screening, sequencing of positive clones and bioinformatic prediction of AMP sequences. To test the antimicrobial activity of fosmid clones, lawns of pathogens including methicillin-sensitive Staphylococcus aureus (MSSA) RN4220, Escherichia coli K12, Salmonella enterica serovar Typhimurium SL1344, Listeria monocytogenes NCTC 11994 (serovar 4b) and Enterococcus faecalis JH2-2 were inoculated on appropriate agar medium and the clones were gently transferred on top using multichannel pin replicators. After an incubation period of 24 h at the appropriate temperatures, 255 fosmids produced clearing zones in the pathogen lawn, indicating that the metagenome clones carried inserts with antimicrobial activity. Of these clones, twenty-four were selected randomly and sequenced using the GS FLX system. A summary of the assembly metrics of the twenty-four metagenomic inserts with antimicrobial activity is provided in Supplementary Table S1. The full fosmid sequences are available in the GenBank database under the BioProject PRJNA264884; BioSample IDs: SAMN063330279 to SAMN063330302. Prediction of AMPs from the identified open reading frames (ORFs) yielded a total of 181 AMPs. A peptide library consisting of 135 of these AMPs (≤25 amino acids (AAs)) was synthesized and screened for antimicrobial activity using the SPOT technique (synthesis of large numbers of peptides on marked spots on cellulose membrane sheets allowing for subsequent highthroughput screening).16 Twenty-five active AMPs were identified in this screen (Supplementary Table S2). Candidates, Lynronne-1 (19 AAs: LPRRNRWSKIWKKVVTVFS-NH2), Lynronne-2 (20 AAs: HLRRINKLLTRIGLYRHAFG-NH2) and Lynronne-3 (20 AAs: NRFTARFRRTPWRLCLQFRQ-NH2) encoded in fosmid clones SABPL29H11, SABPL5A1 and SABPL12(2)A3, respectively and were selected for further analysis due to their broad spectrum antibacterial activity and lower minimum inhibitory concentration (MIC) values. The nucleotide and protein sequences of the ORFs from which they were derived are available in the GenBank database under accession numbers KY628802, KY628803 and KY628804, respectively. Sequences upstream and downstream of these ORFs are shown in Supplementary Fig. S1. The likely producers of Lynronne-1, Lynronne-2 and Lynronne-3 were identified as Prevotella ruminicola 23 (CP002006.1), Uncultured bacterium Contig939 (KC246977.1) and Uncultured bacterium Contigcl_1559 genomic sequence (KC246861.1) respectively (Supplementary Table S3 and Supplementary Fig. S2). Structural modelling using PEP-FOLD17 indicate that these peptides adopt a α-helical conformation of amphipathic nature, an arrangement typical of many α-helical AMPs18 (Fig. 1). The AMPs have a net positive charge of +6, +5 and +6, respectively with a hydrophobicity ratio of ≥40%.
Fig. 1 Predicted structures for peptides: Lynronne-1, Lynronne-2 and Lynronne-3. a Lynronne-1, b Lynronne-2 and c Lynronne-3. Main-chain and side chains depicted in ribbon and stick representation respectively and colored according to atom type: carbon, oxygen and nitrogen in green, red and blue respective. Two orientations are shown rotated about the shown axis. Ct and Nt (C and N terminals) as well as selected residues are depicted in the figure. Figures were rendered using PyMol