Biosynthetic gene cluster identification in plasmids and characterization of plasmids from animal-associated microbiota

Abstract

Individual bacteria in complex microbial communities can acquire and accumulate new traits. These traits are reflective of their environment, being niche-specific. A major player in trait sharing is horizontal gene transfer (HGT). Plasmids, extrachromosomal DNA molecules, have a role in HGT and can change the host’s phenotype. Considering the transformative role of plasmids in bacterial lifestyle, we investigated the prevalence, distribution and products of biosynthetic gene clusters (BGCs) present in plasmids. Sequences available on the National Center for Biotechnology Information (NCBI) database (n=101 416) were run through two bioinformatic pipelines for BGC detection that apply different approaches, deepBGC and antiSMASH (antibiotics and secondary metabolites analysis shell). The highest percentage of plasmids with BGCs was detected in Actinobacteria but, apart from Chlamidiae and Tenericutes, all phyla had BGCs in their plasmids, with predictions varying according to the software used. The BGCs identified comprised a range of classes, indicating that plasmid encoded BGCs could be leveraged for the discovery of new molecules. In order to apply that concept to real-life examples, plasmids were isolated from animal-associated microbial communities and characterized. Plasmids from Escherichia coli isolated from wild birds (n=36) were screened for phenotypes of interest in human and animal health. Seven isolates displayed plasmid-encoded antibiotic resistance. Taxonomic identification of the hosts of plasmids isolated from bovid-associated microbiomes (n=38) was determined via 16S rRNA gene, and placed the majority of the isolated in the phylum Firmicutes, apart from a single Klebsiella pneumoniae isolate. Twelve plasmids were sequenced. Three plasmids from different hosts (pRAM-12, pRAM-19-2 and pRAM-30-2) shared 100% nucleotide sequence and a gene cluster for the bacteriocin cloacin. Two of those hosts shared not one, but two plasmids, pRAM-19-1 and pRAM-30-1, despite being in different phyla. This highlights the intimacy of gene sharing and the importance of HGT. pRAM-28 and pRAM-21 shared a plasmid that harbors the BGC for the bacteriocin aureocin A70, the only four peptide bacteriocin known to date. Additional analysis revealed two putative novel lanthipeptide gene clusters in pRAM-2. These results suggest that the plasmidome is a neglected source of secondary metabolites with the potential for molecule discovery. Furthermore, it can be leveraged to study genetic exchange in a community and how plasmid-encoded featured can mediate interactions in a microbiome.