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Genome mining and metabolic profiling of the rhizosphere bacterium Pseudomonas sp. SH-C52 for antimicrobial compounds.

Van Der Voort M, Meijer HJ, Schmidt Y, Watrous J, Dekkers E, Mendes R, Dorrestein PC, Gross H, Raaijmakers JM - Front Microbiol (2015)

Bottom Line: Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Phytopathology, Wageningen University Wageningen, Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus

Activity of thanapeptin derivatives against Phytophthora infestans. (A) Activity of thanapeptin tested on plate. Numbers indicated in the pictures are the parent mass of the derivate tested in Da. (B) For the derivate of 2096 Da the growth-inhibitory activity was tested in liquid broth, at different concentrations, indicated in the picture in μg/ml.
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Figure 7: Activity of thanapeptin derivatives against Phytophthora infestans. (A) Activity of thanapeptin tested on plate. Numbers indicated in the pictures are the parent mass of the derivate tested in Da. (B) For the derivate of 2096 Da the growth-inhibitory activity was tested in liquid broth, at different concentrations, indicated in the picture in μg/ml.

Mentions: The role of the thanapeptin gene cluster in the activity of SH-C52 against fungi, oomycetes and bacteria was studied by comparing wild-type strain SH-C52 and two independent mutants, each with a single transposon insertion in the thanapeptin NRPS gene cluster. The two mutants lost their antagonistic activity against oomycete pathogens, whereas they still had activity against fungi (Figure 6) and bacteria (data not shown). From the wild-type strain, thanapeptin derivatives were purified and seven derivatives were tested for activity against the oomycete P. infestans. Substantial differences in anti-oomycete activity were observed between the compound derivatives, with the strongest activity for those with the lowest mass, i.e., compounds with the masses 2082, 2096, 2108, and 2122 Da (Figure 7A). In addition, two derivatives with strong activity, 2096 and 2122 Da, were also tested against the oomycete pathogens Saprolegnia parasitica and P. ultimum. Similar results were obtained as in the assays with P. infestans, with the strongest activity for the derivative with a mass of 2096 Da, and slightly lower activity for the derivative with a mass of 2122 Da (data not shown). Subsequently, the activity of the derivative with a mass of 2096 Da was tested at different concentrations against the three oomycete pathogens in a liquid broth. For P. infestans (Figure 7), a clear reduction in mycelial growth was observed at a concentration of 0.25 μg/ml. For syringopeptin or corpeptin, no anti-oomycete activity has been reported to date (Vassilev et al., 1996; Emanuele et al., 1998). No apparent antifungal activity or activity against Gram-negative bacteria was observed for any of the thanapeptin derivatives, whereas activity was observed against the Gram-positive bacterium B. megaterium (data not shown), which is in line with results observed previously for syringopeptin and corpeptin (Vassilev et al., 1996; Emanuele et al., 1998).


Genome mining and metabolic profiling of the rhizosphere bacterium Pseudomonas sp. SH-C52 for antimicrobial compounds.

Van Der Voort M, Meijer HJ, Schmidt Y, Watrous J, Dekkers E, Mendes R, Dorrestein PC, Gross H, Raaijmakers JM - Front Microbiol (2015)

Activity of thanapeptin derivatives against Phytophthora infestans. (A) Activity of thanapeptin tested on plate. Numbers indicated in the pictures are the parent mass of the derivate tested in Da. (B) For the derivate of 2096 Da the growth-inhibitory activity was tested in liquid broth, at different concentrations, indicated in the picture in μg/ml.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493835&req=5

Figure 7: Activity of thanapeptin derivatives against Phytophthora infestans. (A) Activity of thanapeptin tested on plate. Numbers indicated in the pictures are the parent mass of the derivate tested in Da. (B) For the derivate of 2096 Da the growth-inhibitory activity was tested in liquid broth, at different concentrations, indicated in the picture in μg/ml.
Mentions: The role of the thanapeptin gene cluster in the activity of SH-C52 against fungi, oomycetes and bacteria was studied by comparing wild-type strain SH-C52 and two independent mutants, each with a single transposon insertion in the thanapeptin NRPS gene cluster. The two mutants lost their antagonistic activity against oomycete pathogens, whereas they still had activity against fungi (Figure 6) and bacteria (data not shown). From the wild-type strain, thanapeptin derivatives were purified and seven derivatives were tested for activity against the oomycete P. infestans. Substantial differences in anti-oomycete activity were observed between the compound derivatives, with the strongest activity for those with the lowest mass, i.e., compounds with the masses 2082, 2096, 2108, and 2122 Da (Figure 7A). In addition, two derivatives with strong activity, 2096 and 2122 Da, were also tested against the oomycete pathogens Saprolegnia parasitica and P. ultimum. Similar results were obtained as in the assays with P. infestans, with the strongest activity for the derivative with a mass of 2096 Da, and slightly lower activity for the derivative with a mass of 2122 Da (data not shown). Subsequently, the activity of the derivative with a mass of 2096 Da was tested at different concentrations against the three oomycete pathogens in a liquid broth. For P. infestans (Figure 7), a clear reduction in mycelial growth was observed at a concentration of 0.25 μg/ml. For syringopeptin or corpeptin, no anti-oomycete activity has been reported to date (Vassilev et al., 1996; Emanuele et al., 1998). No apparent antifungal activity or activity against Gram-negative bacteria was observed for any of the thanapeptin derivatives, whereas activity was observed against the Gram-positive bacterium B. megaterium (data not shown), which is in line with results observed previously for syringopeptin and corpeptin (Vassilev et al., 1996; Emanuele et al., 1998).

Bottom Line: Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Phytopathology, Wageningen University Wageningen, Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus