Abstract
The plant microbiome represents an enormous untapped resource for discovering novel genes and bioactive compounds. Previously, we isolated Pseudomonas sp. SH-C52 from the rhizosphere of sugar beet plants grown in a soil suppressive to the fungal pathogen Rhizoctonia solani and showed that its antifungal activity is, in part, attributed to the production of the chlorinated 9-amino-acid lipopeptide thanamycin (Mendes et al., 2011). To get more insight into its biosynthetic repertoire, the genome of Pseudomonas sp. SH-C52 was sequenced and subjected to in silico, mutational and functional analyses. The sequencing revealed a genome size of 6.3 Mb and 5579 predicted ORFs. Phylogenetic analysis placed strain SH-C52 within the Pseudomonas corrugata clade. In silico analysis for secondary metabolites revealed a total of six non-ribosomal peptide synthetase (NRPS) gene clusters, including the two previously described NRPS clusters for thanamycin and the 2-amino acid antibacterial lipopeptide brabantamide. Here we show that thanamycin also has activity against an array of other fungi and that brabantamide A exhibits anti-oomycete activity and affects phospholipases of the late blight pathogen Phytophthora infestans. Most notably, mass spectrometry led to the discovery of a third lipopeptide, designated thanapeptin, with a 22-amino-acid peptide moiety. Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans. Of the remaining four NRPS clusters, one was predicted to encode for yet another and unknown lipopeptide with a predicted peptide moiety of 8-amino acids. Collectively, these results show an enormous metabolic potential for Pseudomonas sp. SH-C52, with at least three structurally diverse lipopeptides, each with a different antimicrobial activity spectrum.
Highlights
Pseudomonas species are ubiquitous in aquatic and terrestrial habitats, and are intensively studied for their abilities to promote plant growth and to suppress plant pathogens (Weller, 2007; Berendsen et al, 2012)
All fungi and oomycetes used in this study were cultured on Potato Dextrose Agar (PDA, Difco, Becton, Dickinson and Company, USA), except for Phytophthora infestans strain 88069 and Phytophthora capsici LT3239 which were cultured on Rye Sucrose Medium and on V8 medium (Latijnhouwers et al, 2004), respectively
P. infestans was cultured at 18◦C; whereas all other fungi and oomycetes were cultured at 25◦C
Summary
Pseudomonas species are ubiquitous in aquatic and terrestrial habitats, and are intensively studied for their abilities to promote plant growth and to suppress plant pathogens (Weller, 2007; Berendsen et al, 2012). A wide variety of bioactive compounds involved in pathogen control have been identified for Pseudomonads (Gross and Loper, 2009; Raaijmakers et al, 2010; Raaijmakers and Mazzola, 2012) These include siderophores, hydrogen cyanide, 2,4diacetylphloroglucinol, pyrrolnitrin, pyoluteorin, phenazines, 2,5-dialkylresorcinol, quinolones, gluconic acid, rhamnolipids, and various structurally diverse lipopeptides (Gross and Loper, 2009; D’Aes et al, 2010). To exploit the hidden genetic and metabolic potential in genome sequences, a number of search tools and approaches have been developed, including regulatorbased discovery (Hassan et al, 2010), metabolic networking, peptidogenomics and advanced mass spectrometry methods (Kersten et al, 2011; Watrous et al, 2012) This led to the discovery of NRPS gene clusters involved in the production of structurally novel LPs (Liu et al, 2014)
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