The molecular bases for the symbiosis of the amphibian skin microbiome with its host are poorly understood. Here, we used the odor-producer Pseudomonas sp. MPFS and the treefrog Boana prasina as a model to explore bacterial genome determinants and the resulting mechanisms facilitating symbiosis. Pseudomonas sp. MPFS and its closest relatives, within a new clade of the P. fluoresens Group, have large genomes and were isolated from fishes and plants, suggesting environmental plasticity. We annotated 16 biosynthetic gene clusters from the complete genome sequence of this strain, including those encoding the synthesis of compounds with known antifungal activity and of odorous methoxypyrazines that likely mediate sexual interactions in Boana prasina. Comparative genomics of Pseudomonas also revealed that Pseudomonas sp. MPFS and its closest relatives have acquired specific resistance mechanisms against host antimicrobial peptides (AMPs), specifically two extra copies of a multidrug efflux pump and the same two-component regulatory systems known to trigger adaptive resistance to AMPs in P. aeruginosa. Subsequent molecular modeling indicated that these regulatory systems interact with an AMP identified in Boana prasina through the highly acidic surfaces of the proteins comprising their sensory domains. In agreement with a symbiotic relationship and a highly selective antibacterial function, this AMP did not inhibit the growth of Pseudomonas sp. MPFS but inhibited the growth of another Pseudomonas species and Escherichia coli in laboratory tests. This study provides deeper insights into the molecular interaction of the bacteria-amphibian symbiosis and highlights the role of specific adaptive resistance toward AMPs of the hosts.
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We are grateful to members of the Vences lab and the DSMZ for many helpful suggestions while conducting this work, especially Miguel Vences, Joana Sabino Pinto, Heike Freese, and Juliane Hartlich. We thank the Pupo Lab for their assistance handling the Pseudomonas sp. MPFS strain and Simone Severitt for excellent technical assistance regarding PacBio genome sequencing. We also thank Santiago Di Lella, Sasha Greenspan, Julian Ferreras, and Livia Zaramela for productive comments and discussions. This research was supported by São Paulo Research Foundation FAPESP grants (#2013/50741-7, #2013/50954-0, #2020/02207-5; postdoctoral fellowships #2014/20915-6 and #2017/23725-1 to AEB, #2017/26162-8 to MLL). The research was also supported by a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; 88881.062205/2014-01, to CFBH, and MLL; and postdoctoral fellowship 88887.464731/2019-00 to CAF). CNPq for research fellowships (306623/2018-8). AEB and MMM are researchers of CONICET. Collection permits and genetic heritage access permits for both treefrogs and bacteria were issued by SISBIO (Permits 41508-8, 50071-1, 50071-2, 57098-1) and SISGEN (A1FC113, A9EC80A, A299B7D), respectively.
The authors declare no competing interests.
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Brunetti, A.E., Bunk, B., Lyra, M.L. et al. Molecular basis of a bacterial-amphibian symbiosis revealed by comparative genomics, modeling, and functional testing. ISME J (2021). https://doi.org/10.1038/s41396-021-01121-7