# Pigment which can be observed in the culture condition of LB medium and 37°C. 2.2 PCR and sequencing Four genes of VC1344, VC1345, VC1345, and VC1347 (corresponding to the N16961 genome) were amplified using the primer pairs listed

in Table 2 (S-1344, S-1345, S-1346 and S-1347 respectively). The PCR products were purified and sequenced. Sequence alignments and comparisons were performed using Ixazomib concentration the CLUSTAL X program (version 2.0). Table 2 Primers used in this study Primer pairs Primer sequences S-1344 U 5′ AAG GCA AGG GTT TTT GTG 3′   L 5′ TGT CGG TGC ATG TTG ATG 3′ S-1345 U 5′ GCG CAA AGG TAA TCA AGG 3′   L 5′ GTT ATC CAA CGC CTG CTG 3′ S-1346 U 5′ GCA GCA GGT GGA AAA TCG 3′   L 5′ ATT GAG GGC AAT ACG CAC 3′ S-1347 U 5′ TTT TTG GTG CGA TTG AGC 3′   L 5′ TGC CGA TGA AGA ATC TGC 3′ RT-1344 U 5′ TTT GTG GAT CGT TAT GGC 3′   L 5′ AAT GCC ATC TTT CAT CGG 3′ RT-1344-45 U 5′ MK0683 supplier TGC ACC GAT GAA AGA TGG 3′   L 5′ CAC CCG CAC TTT CAC TTC 3′ RT-1345 U 5′ GAA GTG AAA GTG CGG GTG 3   L 5′ TTG GAA CGC TTT CGG ATG 3′ RT-1345-46 U 5′ CAT CCG AAA GCG TTC CAA 3′   L 5′ AAA TCT CGG CTC ACC ACC 3′ RT-1346 U 5′ GGT GGT GAG CCG AGA TTT 3′   L 5′ GCG ACA

GGT GAA AAA GCC 3′ RT-1346-47 U 5′ ACA CGA GCA CTG TGT GCG 3   L 5′ GGC GCG TGA CTC GTA AAC 3′ RT-1347 U 5′ AGC ATC ATG CCG AGT TTC 3′   L 5′ ATA TTC CCC TGC CGT ATG 3′ 1345:1U U 5′ CAT GCC ATG GAT GCA TAA ATG GAT C 3′ 1345:525L L 5′ GAT CGA AGG CAC GTC CAA CAC GGC AGG ATC AAA CAC CGC GTG ATT G 3′ 1345:555U U 5′ GGA CGT GCC TTC GAT C 3′ 1345:1122L L 5′ CAT GCC ATG GCT ACT CCT TTT TAC TC 3′ 16S U 5′ AGA GTT TGA TCA TGG CTC AG 3′   L 5′ AAG GAG GTG ATC CAA CCG CA 3′ Reverse transcription PCR was used to detect if these four genes were transcribed together. Total RNA of strains N16961 and 95-4 was extracted P-type ATPase using an RNeasy Mini Kit (Qiagen), transcribed

to cDNA and used as templates. Four pairs of primers designed within of the ORF of each gene, RT-1344, RT-1345, RT-1346 and RT-1347 (Table 2), and three pairs of primers spanning the intervals between these four genes, RT-1344-45, RT-1345-46, and RT-1346-47 (Table 2), were used in the amplification. The total mRNA without reverse transcription were used as negative control, 2.3 Filling in of the 15-bp gap in the VC1345 gene Two pairs of primers were used to amplify the upstream and downstream fragment of the 15-bp gap in the VC1345 gene of pigment-producing strain 95-4. The primers were as follows: 1345:1U, 1345:525L, 1345:555U and 1345:1122L (Figure 1 and Table 2).