S1 Graphical 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.</div

Comparison of gene cassettes associated with NRPS in bin 43, 137 and <i>R</i>. <i>koreense</i> JCM 31441, <i>P</i>. <i>ginsengisoli</i> JCM 30745, and <i>N</i>. <i>denitrificans</i> 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.</p

Detailed annotation of the gene cassettes associated with NRPS clusters in <i>Rhodoferax</i> bin 43, <i>Rhodocyclaceae</i> bin 137, and three strains’ genomes with antifungal activity.

Detailed annotation of the gene cassettes associated with NRPS clusters in Rhodoferax bin 43, Rhodocyclaceae bin 137, and three strains’ genomes with antifungal activity.</p

Taxonomic classification at the phylum level for the bins showing NRPS and NRPS/PKS hybrid clusters.

Taxonomic classification at the phylum level for the bins showing NRPS and NRPS/PKS hybrid clusters.</p

Antifungal activities of <i>R</i>. <i>koreense</i> JCM 31441, <i>P</i>. <i>ginsengisoli</i> JCM 30745, <i>N</i>. <i>denitrificans</i> JCM 17722, <i>Bacillus subtilis</i> NBRC 109107, and <i>R</i>. <i>sediminis</i> JCM 32677 against <i>C</i>. <i>plurivorum and V</i>. <i>dahliae</i>.

An overnight culture of each bacterial strain was inoculated in a straight line on an R2A agar plate with an inoculating loop. (TIF)</p

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.</p

Secondary metabolite genes in <i>R</i>. <i>koreense</i> JCM 31441, <i>P</i>. <i>ginsengisoli</i> JCM 30745, and <i>N</i>. <i>denitrificans</i> JCM 17722 obtained with Anti-SMASH.

The red box shows NRPS genes with the C domain of syringomycin synthetase. (TIF)</p

NRPS-related gene (C domain) identified in the bins using NaPDoS.

NRPS-related gene (C domain) identified in the bins using NaPDoS.</p

General information of metagenomic bins.

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.</div

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.</p