Genome sequence of Streptomyces coelicolor JCM4020
The genome size of S. coelicolor JCM4020 was 8,634,640 bp, comprising a single linear chromosome. JCM4020 did not contain any plasmids. Its GC content was 72.2 mol% and the number of coding sequences (CDS) was 7799. The identified CDS were almost identical to those of the model actinomycetes strain S. coelicolor A3(2) M145, which has a genome size of 8,667,507 bp, 72.1 mol% GC content, and 7877 CDS.36 Complete comparison of CDSs using Multi BLAST between two strains is shown in Supplemental File 1. In this paper, the SCO numbers used for S. coelicolor A3(2) M145 gene and CDS designations are used for convenience. S. coelicolor JCM4020 was used in this experiment since the strain does not produce RED or ACT in monoculture conditions. Secondary metabolite gene clusters in the strain JCM4020 were predicted by antiSMASH37 (Supplemental Figure 1).
12 C 5+ ion irradiation and dose-dependent survival rates
Mutagenesis by carbon ion beams was performed at TIARA (Takasaki Ion Accelerators for Advanced Radiation Application), QST (National Institutes for Quantum Science and Technology). First, 100 Gy, 500 Gy, and 1,000 Gy of 12C5+ ions were irradiated. The survival rates were measured by counting the colony forming units (CFU), which were then compared with those upon no irradiation, giving values of 68.8 ± 25.1% for 100 Gy, 7.4 ± 0.68% for 500 Gy, and 0.52 ± 0.10% for 1,000 Gy irradiation (Table 1). We also tested the formation of aerial mycelium in this step using the colony morphology as an indication. Overall, 99.9 ± 0.065% of the formed colonies generated aerial mycelium at 0 Gy, 93.6 ± 1.2% at 100 Gy, 63.3 ± 4.0% at 500 Gy, and 40.4 ± 16.2% at 1,000 Gy. Second, in line with the procedure used in the first irradiation, 10 Gy, 50 Gy, 100 Gy, and 200 Gy of 12C5+ ions were irradiated. The survival rates were 55.0 ± 3.5% at 10 Gy, 36.1 ± 8.6% at 50 Gy, 36.0 ± 5.5% at 100 Gy, and 13.0 ± 4.1% at 200 Gy, which were relatively consistent with the rates after the first irradiation (Table 1). The formation of aerial mycelium was also tested, showing rates of 99.9 ± 0.065% for 0 Gy, 99.3 ± 0.35% for 10 Gy, 99.3 ± 0.43% for 50 Gy, 98.4 ± 1.1% for 100 Gy, and 90.6 ± 2.3% for 200 Gy. Overall, the survival rate and aerial mycelium formation rate showed a certain correlation with the irradiation dose.
Screening of RED production-deficient mutants
We chose mutant spore libraries from the first 100 Gy irradiation, second 100 Gy irradiation, and second 200 Gy irradiation for the screening of mutants, considering the survival rates. In total, we tested approximately 152,000 spores (based on estimation from the CFU) and selected 118 mutants (with a yield of 0.078%) that showed lower or lost production of RED upon incubation with T. pulmonis TP-B0596 (Figure 1, Supplemental Figure 1 and 2). The low yield (0.078%) of mutant may have been because we used the mutant spore library showing a relatively high survival rate (13.0–68.8%). Radiation doses giving a surviving fraction of 1–10% have been proposed for effective mutagenesis in microorganisms.33 The use of a sample with a lower survival rate may increase the number of mutants exhibiting abnormal RED production, but it may also increase multiple undesired mutations in the genome, leading to difficulty identifying the genes responsible for the phenotype. We then tested the growth on minimal medium and the formation of aerial mycelium for 118 mutants, 59 of which showed no deficiency (Figure 1). We excluded mutants that could not grow on the minimal medium from further analysis because they may exhibit specific auxotrophy by possessing a primary metabolism-related mutation that significantly affects growth, in turn affecting the secondary metabolism. We also excluded mutants that could not form aerial mycelium from further analysis at this point because the genes responsible for the bald phenotype have been extensively studied38,39 and to reduce the rediscovery of already-characterized genes. There was a concern that the mutations would occur in the biosynthetic gene cluster of RED, which directly affect the production. The production of RED by Streptomyces coelicolor A3(2) was reported to be activated by the addition of NaCl.40 We then tested the effect of salt stress by adding 1% NaCl to the medium because S. coelicolor JCM4020 did not produce RED on the tested normal medium in monoculture. Five mutants showed RED production comparable to that of the wild type in the salt-stressed condition (Figure 1 and 2). Although the mechanism of its induction by salt stress was unclear, it was confirmed that at least these mutants do not contain significant lesions in the biosynthetic gene cluster of RED.
Identified point mutations
Subsequently, we performed genome re-sequencing to identify the point mutations in the genome of screened mutants to investigate the signature of induced point mutations and further identify the gene(s) responsible for the production of secondary metabolites. Overall, 16 mutants were selected, namely, 3 mutants that produce RED in salt stress condition and 13 mutants that form aerial mycelium, which showed a consistent phenotype (Figure 1, Supplemental Figure 3). The genome was resequenced using a next-generation sequencer, MiSEQ (Illumina). Upon comparison with the sequence of the wild type, we identified 58 point mutations (Table 2 and 3). The identified point mutations were confirmed by further Sanger sequencing. Six mutants (Mt 202001, Mt 202004, Mt 202007, Mt 205011, Mt 208014, Mt 20980) contained an identical C-to-A transversion at genomic position 4,593,233 bp and the insertion of a G at genomic position 4,565,012 bp (Table 2). Moreover, two mutants (Mt 209010, Mt 203013) contained the insertion of a C at genomic position 4,564,124 bp (Table 2). It is unlikely for an identical point mutation to be induced at the same position, so these 14 (6+6+2) point mutations were considered to have arisen naturally during cell growth for spore preparation. Therefore, the other 44 point mutations were considered to have been induced by the carbon ions. Upon 100 Gy of irradiation, 14 point mutations from 7 mutants (average 2.0 point mutations/mutant) were found, while upon 200 Gy of irradiation, 30 point mutations from 9 mutants (average 3.3 point mutations/mutant) were found. The number of mutations showed a certain correlation with the irradiation dose. Among these 44 mutations, there were 31 base substitutions, 5 insertions, and 8 deletions (Table 3). The 44 point mutations were distributed relatively evenly across the whole genome. Meanwhile, we did not detect any large-scale genomic variations, such as large deletions, translocations, or inversions in the carbon ion-irradiated S. coelicolor JCM4020.
Identification of amino acid mutations
Amino acid mutations in the CDS caused by point mutations were identified. Among the identified 44 point mutations, at the amino acid level they caused 13 missense mutations, 2 nonsense mutations, 9 frameshifts, 1 amino acid insertion, 2 amino acid deletions, and 7 silent mutations, while the remaining 10 were in noncoding regions (Table 2). Overall, 27 amino acid mutations were considered to affect the function of the encoded protein. Two mutants (Mt 106003, Mt 201001) contained mutations in RED biosynthetic genes (redH and redP, respectively), indicating that these mutations directly cause deficiency of RED biosynthesis (Table 2, Supplemental Figure 3_5). As described previously, naturally arising point mutations involving the insertion of a G at 4,565,012 bp in six mutants and the insertion of a C at 4,564,124 bp in two mutants cause frameshift in the SarA (SCO4069) homolog originally found in Streptomyces coelicolor A3(2).41 As the deletion of sarA causes a defect in RED production41, it was considered that sarA mutants were accumulated by our screening method. Finally, six mutants containing a total of 14 amino acid mutations were considered for the identification of candidate genes involved in the production of RED induced by T. pulmonis stimulation.
Gene complementation for phenotypic recovery
Three of the 16 genome-resequenced mutants (Mt 108013, 203010, Mt 107004) formed aerial mycelium and produced RED in agar medium containing 1% NaCl in Bennett’s maltose at levels comparable to those in the wild type (Figures 2, Supplemental Figure 3_1). We performed gene complementation for Mt 108013 and Mt 203010. Mt 108013 possessed a mutation in glutamine synthase (GltB, SCO2026 homolog), and Mt 203010 possessed one in elongation factor G (EF-G) (FusA, SCO4661 homolog) (Table 2). In Mt 108013, C597 of the gltB gene was deleted, resulting in frameshift of the amino acid sequence (Supplemental Figure 4). The DNA fragment containing the gltB gene was amplified by PCR from the wild-type JCM4020 and cloned into the pTYM19t vector. The pTYM19t-gltB vector was introduced into Mt 108013 and the production of RED was compared with that of the wild type. Mt 108013 complemented by pTYM19-gltB recovered the capacity to produce RED in combined-culture with T. pulmonis, compared with Mt 108013 with an empty vector (Figure 3, Supplemental Figure 6). In Mt 203010, C147 of the fusA gene was deleted, which also resulted in frameshift of the amino acid sequence. (Supplemental Figure 5) The DNA fragment containing the fusA gene with 5′-UTR was amplified by PCR from the wild-type JCM4020 and cloned into the pTYM19t vector. The pTYM19t-fusA vector was introduced into Mt 203010 and its production of RED was compared with that of the wild type. Mt 203010 complemented by pTYM19-fusA recovered the production of RED in combined-culture with T. pulmonis, compared with Mt 203010 with an empty vector (Figure 3, Supplemental Figure 7).