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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

Thanapeptin genetic and chemical analysis. (A) The upper panel shows the interaction of SH-C52 in interaction with R. solani, as imaged by MALDI imaging mass spectrometry (IMS). Thanamycin and thanapeptin detection by MALDI IMS are shown. (B) The middle panel presents the thanapeptin gene cluster with the position of the transposon insertions, and the modules encoded by the gene clusters and the prediction for the amino acids assembled. On the middle left, the partial spectrometry profile of the MALDI IMS of the SH-C52 and R. solani interaction is shown. (C) At the bottom the tentative thanapeptin structure obtained from tandem mass spectrometry data is presented. Dashed arrows indicate amino acids that are difficult to predict from genome information, and that needed to be resolved by mass spectrometry.
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Figure 5: Thanapeptin genetic and chemical analysis. (A) The upper panel shows the interaction of SH-C52 in interaction with R. solani, as imaged by MALDI imaging mass spectrometry (IMS). Thanamycin and thanapeptin detection by MALDI IMS are shown. (B) The middle panel presents the thanapeptin gene cluster with the position of the transposon insertions, and the modules encoded by the gene clusters and the prediction for the amino acids assembled. On the middle left, the partial spectrometry profile of the MALDI IMS of the SH-C52 and R. solani interaction is shown. (C) At the bottom the tentative thanapeptin structure obtained from tandem mass spectrometry data is presented. Dashed arrows indicate amino acids that are difficult to predict from genome information, and that needed to be resolved by mass spectrometry.

Mentions: The thanapeptin gene cluster consists of three NRPS genes. BLAST analysis of the protein sequences showed similarity with the NRPSs for the production of the LP syringopeptin of P. syringae. Indeed, the first gene of the thanapeptin cluster starts with a specific condensation (C)-domain, a so-called C1 starter domain (data not shown), suggesting N-acylation of the first amino acid in the peptide moiety. The three NRPS genes of the thanapeptin gene cluster encode nine, four and nine adenylation (A)-domains, respectively. The prediction of the 22 amino acids activated by these A-domains is shown in Figure 5. The C-terminus of the last gene of the cluster encodes two thioesterase (TE)-domains, indicating termination of the thanapeptin synthesis. Despite the similarity between syringopeptin and thanapeptin (Figure 2), there also is a clear difference in modular organization as syringopeptin is produced from three NRPS modules, with five, five and twelve adenylation domains, respectively. Comparison with the P. corrugata CFBP5454 draft genome sequence shows that the corpeptin gene cluster sequence is incomplete and scattered over contigs. Nevertheless, for the annotated parts, protein identities with the SH-C52 NRPSs ThpA and ThpC ranged between 75 and 95%, whereas for ThpB identities ranged between 60 and 80%.


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)

Thanapeptin genetic and chemical analysis. (A) The upper panel shows the interaction of SH-C52 in interaction with R. solani, as imaged by MALDI imaging mass spectrometry (IMS). Thanamycin and thanapeptin detection by MALDI IMS are shown. (B) The middle panel presents the thanapeptin gene cluster with the position of the transposon insertions, and the modules encoded by the gene clusters and the prediction for the amino acids assembled. On the middle left, the partial spectrometry profile of the MALDI IMS of the SH-C52 and R. solani interaction is shown. (C) At the bottom the tentative thanapeptin structure obtained from tandem mass spectrometry data is presented. Dashed arrows indicate amino acids that are difficult to predict from genome information, and that needed to be resolved by mass spectrometry.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Thanapeptin genetic and chemical analysis. (A) The upper panel shows the interaction of SH-C52 in interaction with R. solani, as imaged by MALDI imaging mass spectrometry (IMS). Thanamycin and thanapeptin detection by MALDI IMS are shown. (B) The middle panel presents the thanapeptin gene cluster with the position of the transposon insertions, and the modules encoded by the gene clusters and the prediction for the amino acids assembled. On the middle left, the partial spectrometry profile of the MALDI IMS of the SH-C52 and R. solani interaction is shown. (C) At the bottom the tentative thanapeptin structure obtained from tandem mass spectrometry data is presented. Dashed arrows indicate amino acids that are difficult to predict from genome information, and that needed to be resolved by mass spectrometry.
Mentions: The thanapeptin gene cluster consists of three NRPS genes. BLAST analysis of the protein sequences showed similarity with the NRPSs for the production of the LP syringopeptin of P. syringae. Indeed, the first gene of the thanapeptin cluster starts with a specific condensation (C)-domain, a so-called C1 starter domain (data not shown), suggesting N-acylation of the first amino acid in the peptide moiety. The three NRPS genes of the thanapeptin gene cluster encode nine, four and nine adenylation (A)-domains, respectively. The prediction of the 22 amino acids activated by these A-domains is shown in Figure 5. The C-terminus of the last gene of the cluster encodes two thioesterase (TE)-domains, indicating termination of the thanapeptin synthesis. Despite the similarity between syringopeptin and thanapeptin (Figure 2), there also is a clear difference in modular organization as syringopeptin is produced from three NRPS modules, with five, five and twelve adenylation domains, respectively. Comparison with the P. corrugata CFBP5454 draft genome sequence shows that the corpeptin gene cluster sequence is incomplete and scattered over contigs. Nevertheless, for the annotated parts, protein identities with the SH-C52 NRPSs ThpA and ThpC ranged between 75 and 95%, whereas for ThpB identities ranged between 60 and 80%.

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