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. 2023 Nov 27;18(11):e0294843.
doi: 10.1371/journal.pone.0294843. eCollection 2023.

A small step to discover candidate biological control agents from preexisting bioresources by using novel nonribosomal peptide synthetases hidden in activated sludge metagenomes

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A small step to discover candidate biological control agents from preexisting bioresources by using novel nonribosomal peptide synthetases hidden in activated sludge metagenomes

Shun Tomita et al. PLoS One. .

Abstract

Biological control agents (BCAs), beneficial organisms that reduce the incidence or severity of plant disease, have been expected to be alternatives to replace chemical pesticides worldwide. To date, BCAs have been screened by culture-dependent methods from various environments. However, previously unknown BCA candidates may be buried and overlooked because this approach preferentially selects only easy-to-culture microbial lineages. To overcome this limitation, as a small-scale test case, we attempted to explore novel BCA candidates by employing the shotgun metagenomic information of the activated sludge (AS) microbiome, which is thought to contain unutilized biological resources. We first performed genome-resolved metagenomics for AS taken from a municipal sewage treatment plant and obtained 97 nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS)-related gene sequences from 43 metagenomic assembled bins, most of which were assigned to the phyla Proteobacteria and Myxococcota. Furthermore, these NRPS/PKS-related genes are predicted to be novel because they were genetically dissimilar to known NRPS/PKS gene clusters. Of these, the condensation domain of the syringomycin-related NRPS gene cluster was detected in Rhodoferax- and Rhodocyclaceae-related bins, and its homolog was found in previously reported AS metagenomes as well as the genomes of three strains available from the microbial culture collections, implying their potential BCA ability. Then, we tested the antimicrobial activity of these strains against phytopathogenic fungi to investigate the potential ability of BCA by in vitro cultivation and successfully confirmed the actual antifungal activity of three strains harboring a possibly novel NRPS gene cluster. Our findings provide a possible strategy for discovering novel BCAs buried in the environment using genome-resolved metagenomics.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Secondary metabolite clusters obtained by Anti-SMASH.
(a) Abundance of secondary metabolite gene cluster types in the 180 bins recovered from activated sludge obtained with Anti-SMASH. The red bar indicates the NRPS. The blue bar indicates NRPS/PKS hybrid clusters (NRPS-PKS and PKS-NRPS). (b) Comparisons with known NRPS and NRPS/PKS hybrid clusters from the MIBiG dataset in Anti-SMASH.
Fig 2
Fig 2. Phylogenetic tree of C domains (bin 43 and 137, the top 10 BLAST results on RefSeq, the NaPDoS reference sequences and previously reported environmental sequences).
The tree was generated by the NaPDoS pipeline (using FASTTREE and the maximum likelihood algorithm). Dots, squares, and triangles on nodes indicate the confidence value. The sequences from the bins are in red. The sequences of strains used for the antifungal activity test are shown in blue.
Fig 3
Fig 3. Comparison of gene cassettes associated with NRPS in bin 43, 137 and R. koreense JCM 31441, P. ginsengisoli JCM 30745, and N. denitrificans JCM 17722.
Numbers in gene boxes indicate amino acid sequence identity (%) to the corresponding gene of bin43 (Rhodoferax). NRPS domains are labeled as follows: C condensation, A adenylation, and T thiolation. Each domain in N. denitrificans JCM 17722 is located in different NRPS genes; Ga308623_01012623 possesses a T domain, and Ga0308623_1011965 possesses C and A domains.
Fig 4
Fig 4. Antifungal activities of R. koreense JCM 31441, P. ginsengisoli JCM 30745, N. denitrificans JCM 17722, and R. sediminis JCM 32677 against B. cinerea.
An overnight culture of each bacterial strain was inoculated in a straight line on an R2A agar plate with an inoculating loop. Rhodoferax sediminis JCM 32677 was used as a negative control.

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