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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

Antimicrobial and phospholipase D stimulating activity of brabantamide A in interaction with Phytophthora infestans. (A) Growth of P. infestans in liquid medium with different concentrations of brabantamide A. (B) Dry weight of P. infestans grown in liquid medium (n = 2). (C) Lipids extracted from P. infestans, separated by alkaline (left) or ethylactetate (EtAc) TLC (right) and analyzed by phosphoimaging. The origin of the chromatogram, phosphatidylinositolphosphate (PtdInsP), phosphatidic acid (PtdOH), phosphatidylinositol (PtdIns), phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylglycerol (PtdGro) and phosphatidylbutanol (PtdBut) are indicated. (D) PtdOH and PtdBut were quantified from the EtAc TLC and presented as the fold increase when compared to control conditions (n = 2). Error bars represent standard deviations.
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Figure 4: Antimicrobial and phospholipase D stimulating activity of brabantamide A in interaction with Phytophthora infestans. (A) Growth of P. infestans in liquid medium with different concentrations of brabantamide A. (B) Dry weight of P. infestans grown in liquid medium (n = 2). (C) Lipids extracted from P. infestans, separated by alkaline (left) or ethylactetate (EtAc) TLC (right) and analyzed by phosphoimaging. The origin of the chromatogram, phosphatidylinositolphosphate (PtdInsP), phosphatidic acid (PtdOH), phosphatidylinositol (PtdIns), phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylglycerol (PtdGro) and phosphatidylbutanol (PtdBut) are indicated. (D) PtdOH and PtdBut were quantified from the EtAc TLC and presented as the fold increase when compared to control conditions (n = 2). Error bars represent standard deviations.

Mentions: In previous studies, brabantamides were shown to have activity against specific Gram-positive bacteria (Reder-Christ et al., 2012; Schmidt et al., 2014). Here, we show that brabantamide A, at a concentration of 50 μM, has activity against the oomycete plant pathogens Phytophthora capsici, Pythium ultimum, and P. infestans (Figure 4). No antifungal activity was observed at this concentration (data not shown). For P. infestans, mycelial growth was already inhibited at concentrations above 5 μM resulting in a less dense mycelial mat (Figure 4A). Previous studies suggested that structural analogs of brabantamide A inhibit phospholipase A2 of rabbit (Thirkettle et al., 2000). When [32P]Pi-labeled hyphae of P. infestans were incubated for 15 min with brabantamide, no significant variation in the phospholipid levels was observed (data not shown). However, overnight exposure of P. infestans mycelium to brabantamide A led to an increase in the phosphatidylbutanol (PtdBut) levels. This already occurred at a concentration of 1 μM brabantamide A, the lowest concentration tested (Figure 4). Other phospholipid levels were not affected based on quantification. The enhanced PtdBut levels indicates a stimulatory effect of brabantamide A on phospholipase D activity. In contrast, no indication was found for the inhibition of phospholipase A2 in this assay.


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)

Antimicrobial and phospholipase D stimulating activity of brabantamide A in interaction with Phytophthora infestans. (A) Growth of P. infestans in liquid medium with different concentrations of brabantamide A. (B) Dry weight of P. infestans grown in liquid medium (n = 2). (C) Lipids extracted from P. infestans, separated by alkaline (left) or ethylactetate (EtAc) TLC (right) and analyzed by phosphoimaging. The origin of the chromatogram, phosphatidylinositolphosphate (PtdInsP), phosphatidic acid (PtdOH), phosphatidylinositol (PtdIns), phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylglycerol (PtdGro) and phosphatidylbutanol (PtdBut) are indicated. (D) PtdOH and PtdBut were quantified from the EtAc TLC and presented as the fold increase when compared to control conditions (n = 2). Error bars represent standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Antimicrobial and phospholipase D stimulating activity of brabantamide A in interaction with Phytophthora infestans. (A) Growth of P. infestans in liquid medium with different concentrations of brabantamide A. (B) Dry weight of P. infestans grown in liquid medium (n = 2). (C) Lipids extracted from P. infestans, separated by alkaline (left) or ethylactetate (EtAc) TLC (right) and analyzed by phosphoimaging. The origin of the chromatogram, phosphatidylinositolphosphate (PtdInsP), phosphatidic acid (PtdOH), phosphatidylinositol (PtdIns), phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylglycerol (PtdGro) and phosphatidylbutanol (PtdBut) are indicated. (D) PtdOH and PtdBut were quantified from the EtAc TLC and presented as the fold increase when compared to control conditions (n = 2). Error bars represent standard deviations.
Mentions: In previous studies, brabantamides were shown to have activity against specific Gram-positive bacteria (Reder-Christ et al., 2012; Schmidt et al., 2014). Here, we show that brabantamide A, at a concentration of 50 μM, has activity against the oomycete plant pathogens Phytophthora capsici, Pythium ultimum, and P. infestans (Figure 4). No antifungal activity was observed at this concentration (data not shown). For P. infestans, mycelial growth was already inhibited at concentrations above 5 μM resulting in a less dense mycelial mat (Figure 4A). Previous studies suggested that structural analogs of brabantamide A inhibit phospholipase A2 of rabbit (Thirkettle et al., 2000). When [32P]Pi-labeled hyphae of P. infestans were incubated for 15 min with brabantamide, no significant variation in the phospholipid levels was observed (data not shown). However, overnight exposure of P. infestans mycelium to brabantamide A led to an increase in the phosphatidylbutanol (PtdBut) levels. This already occurred at a concentration of 1 μM brabantamide A, the lowest concentration tested (Figure 4). Other phospholipid levels were not affected based on quantification. The enhanced PtdBut levels indicates a stimulatory effect of brabantamide A on phospholipase D activity. In contrast, no indication was found for the inhibition of phospholipase A2 in this assay.

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