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Molecular basis of a bacterial-amphibian symbiosis revealed by comparative genomics, modeling, and functional testing


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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Fig. 1: Circular plot of the 7.1 Mbp Pseudomonas sp. MPFS chromosome and COG functional categories.
Fig. 2: Phylogenetic relationships of Pseudomonas obtained with maximum likelihood based on protein sequence alignments of orthologous genes from 141 type strains (type), 17 non-type strains, and the yet undescribed Pseudomonas sp. MPFS from this study (arrowhead, red, bold).
Fig. 3: Phylogenomic tree showing the position of Pseudomonas sp. MPFS in the most closely related clades and overview of ecological niches used by different clades of Pseudomonas.
Fig. 4: Biosynthetic gene clusters (BGCs) from Pseudomonas sp. MPFS and from strains within the P. piscis, P. protegens, and P. chlororaphis subgroups with reliable secondary metabolite assignment (sequence similarity ≥75% with clusters of known function).
Fig. 5: Bacterial growth curves of three Pseudomonas strains and Escherichia coli exposed to different concentrations of the cationic antimicrobial peptide raniseptin-Prs from Boana prasina.
Fig. 6: Tertiary structure model of the sensory periplasmatic domain of the sensory histidine kinases PhoQ and CprS from Pseudomonas aeruginosa strain PAO1 (left panel) and Pseudomonas sp. MPFS (right panel), and modeled docking with the peptide raniseptin-Prs.


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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.

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Conceptualization: AEB, BB, MLL, NPL, JO; methodology: AEB, BB, MLL, MMM, CS; data curation: AEB, BB, MLL, MMM, CS, JO; modeling, CAF; investigation: AEB, BB, MLL, CAF, MMM, JO; writing the original draft: AEB with substantial contributions from MLL, CAF, MMM, JO; reviewing and editing: AEB, BB, MLL, CAF, MMM, CS, CFBH, NPL, JO; funding acquisition: CFBH, NPL, JO.

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Correspondence to Andrés E. Brunetti, Norberto P. Lopes or Jörg Overmann.

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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).

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