Fig. S1. HPLC analysis of H42 and its mutants. Detection was performed at 304 nm. H42, the parental strain; nystatin A1, the nystatin A1 standard substance which was produced by Dr. Ehrenstorfer GmbH; ΔttmRIV, the ttmRIV deleted strain; ΔttmRIV::ttmRIV, ttmRIV gene complementary strain with native promoter; and H42::ttmRIV, the overexpressed ttmRIV strain with ermEp* promoter.TB, tetramycin B; NA1, nystatin A1; and TA, tetramycin A. The TB and TA peaks disappeared in the strain ΔttmRIV, but the NA1 peak was obviously increased, and these peaks were recovered to H42 level by complementation in the mutant. The TA peak was clearly enhanced in the H42::ttmRIV strain

Fig. S2. High-resolution mass spectra (HR-ESI-MS)analysis of TA, TB, and NA1. The peak TB, NA1, and TA of H42 in Fig. S1 were collected, and their HR-ESI-MS analyses were obtained using a Bruker micrOTOF-Q 125 mass spectrometer (Bruker Daltonics, Bremen, Germany). TB, [M+H]+ (m/z 712), [M+Na]+ (m/z 734); NA1, [M+H]+(m/z 926), [M+Na]+ (m/z 948); and TA, [M+H]+(m/z 696), [M+Na]+ (m/z 718)

Table S1Primersfor RT-PCR

Name / Sequence / Description
P-lysA-S / CCGCTGCCGCACCTACCT / lysA gene for internal control
P-lysA-AS / CAGGGTGATGCCGTGTTGC / lysA gene for internal control
P-E-S / GATGTCGGTGTACGTGGGG / Forward primer for ttmE
P-E-AS / TTGGCGTCGCTCTGGTCC / Reverse primer for ttmE
P-K-S / TGAAGCGGATTTCGGTGGG / Forward primer for ttmK
P-K-AS / ACGACAGCCCGTGTTCCTT / Reverse primer for ttmK
P-C-S / GGTGACGTACACGGGTGCCA / Forward primer for ttmC
P-C-AS / CCGGGTCTCGATGGAGTGGT / Reverse primer for ttmC
P-G-S / GTGCCCACGCAGGAGCAGAT / Forward primer for ttmG
P-G-AS / CGAGTGGAAGCGCAGGGTCT / Reverse primer for ttmG
P-F-S / GCGTATCACCATCGATTCG / Forward primer for ttmF
P-F-AS / GGTGATCGCCCCGGAG / Reverse primer for ttmF
P-S0-S / GCCCCACTCGCCACTGAT / Forward primer for ttmS0
P-S0-AS / GCGTTCGGCTTGGGGTAG / Reverse primer for ttmS0
P-L-S / GGCCGGTGGGTCAAGATG / Forward primer for ttmL
P-L-AS / CCCCTGCCGAAGATAGGTGTC / Reverse primer for ttmL
P-S1-S / GCACCTTGATCCGCTTGACCC / Forward primer for ttmS1
P-S1-AS / TCGGCATCAGCGGCACCAA / Reverse primer for ttmS1
P-D-S / GGTAGCGGCGGTCGTGGA / Forward primer for ttmD
P-D-AS / CGAAGTTGGGGAGGCTGAAGT / Reverse primer for ttmD
P-S4-S / CTGCTGGATCACCCGCTGTT / Forward primer for ttmS4
P-S4-AS / GGCATCGACCCGGTCTCG / Reverse primer for ttmS4
P-S3-S / GTCGCCGGTCGACAGTCC / Forward primer for ttmS3
P-S3-AS / GACTACGCCCACCTGCTCATC / Reverse primer for ttmS3
P-S2-S / GGTGGTGGGGCTGGACAT / Forward primer for ttmS2
P-S2-AS / CCGAGGAGCTCTGGTCCCT / Reverse primer for ttmS2
P-RVI-S / GCTCTAGAGTGCTGGATCCCGCTCTGAC / Forward primer for ttmRIV
P-RVI-AS / CCCAAGCTTTTACTTGATGAAGTCGTCCA / Reverse primer for ttmRIV
P-RIII-S / CCCCGCTACGCCCAACTT / Forward primer for ttmRIII
P-RIII-AS / CGCCATCGTCCTCCACCAG / Reverse primer for ttmRIII
P-RII-S / CGCCCTTGGTGGCTTCCT / Forward primer for ttmRII
P-RII-AS / GGTCGCCTGGCACCCCTA / Reverse primer for ttmRII
P-RI-S / GCCTCGCACAGCACCTTCC / Forward primer for ttmRI
P-RI-AS / CCCAGCACTGCGACGAACTCT / Reverse primer for ttmRI
P-J-S / GGTAGTTGCGGGTGATGAAGT / Forward primer for ttmJ
P-J-AS / GTCCAAGCGTGCGCTGAT / Reverse primer for ttmJ
P-A-S / GAACCCTCCGAACCCGACTG / Forward primer for ttmA
P-A-AS / GCCGAGAATGAGGCCGAAA / Reverse primer for ttmA
P-B-S / TCCAACCTGGGCTATGTCGC / Forward primer for ttmB
P-B-AS / GGCAAGGTGCGGATGAAGC / Reverse primer for ttmB
P-P-S / TCCGGGGCTGGTGCACTC / Forward primer for ttmP
P-P-AS / CCCTGGTGGGCGTTGCTG / Reverse primer for ttmP
P-a-S / GTCGCGGGCTACCACAAC / Forward primer for fragmenta
P-a-AS / AAGACTGACGAAAAGGAT / Reverse primer forfragmenta
P-b-S / CATCGAGAGTTGGGTTCCC / Forward primer for fragmentb
P-b-AS / ACGATATCCACCCGTCCTC / Reverse primer forfragmentb
P-c-S / TCACCAAGGACCACAGCTT / Forward primer for fragmentc
P-c-AS / CCGAGTGCGTCATGAACGG / Reverse primer forfragmentc
P-d-S / CCAGTGCCTGGGGCAGAAC / Forward primer for fragmentd
P-d-AS / ACGCGAACACCTCGGGGAG / Reverse primer forfragmentd
P-e-S / CGACTACCCCAAGCCGAAC / Forward primer for fragmente
P-e-AS / GCCCCCCACCGCCGATACG / Reverse primer forfragmente
P-f-S / GGCAGGACATCCCGACGAT / Forward primer for fragmentf
P-f-AS / GTTCCACGACCTGGGCTTC / Reverse primer forfragmentf
P-g-S / CACGAGGTTTCCAGCAGCA / Forward primer for fragmentg
P-g-AS / GACGACGCCTACGACGCAT / Reverse primer forfragmentg
P-h-S / GCGGAGCTTCTGTTCCTG / Forward primer for fragmenth
P-h-AS / CCTATGCGATGGGCGTGC / Reverse primer forfragmenth
P-i-S / ATCGTCAGAGCGGGATCCA / Forward primer for fragmenti
P-i-AS / ATGTTCGCGGCACGGTGAG / Reverse primer forfragmenti
P-j-S / CCGAGGAGTTGGTGGACGA / Forward primer for fragmentj
P-j-AS / CGACTGCTGACCGAGCTGT / Reverse primer forfragmentj
P-k-S / GGTCGCGCTCGGTCAGGGA / Forward primer for fragmentk
P-k-AS / ACGCACGGTCGCCAGCCTC / Reverse primer forfragmentk
P-l-S / GTGCTGCCTATATCAGGAT / Forward primer for fragmentl
P-l-AS / CGACCTTCAACAGGTATCC / Reverse primer forfragmentl
P-m-S / CGACGGTGCTCACGGTGGC / Forward primer for fragmentm
P-m-AS / GGGCGGCGGTGACGAAGAG / Reverse primer forfragmentm
P-n-S / CACCGCCTGTCCACCATCC / Forward primer for fragmentn
P-n-AS / CTTCCAGACGTTCACCCCG / Reverse primer forfragmentn

Table S2Primersfor EMSA probes preparation

Putative promoter region / Name / Sequencea / Probe size (bp)
ttmK / ttmKp1 / GTCGCGGGCTACCACAAC / 399
ttmKp2 / AAGACTGACGAAAAGGAT
ttmS1 / ttmS1p1 / CGCTGCTTCTGCTCCGTC / 401
ttmS1p2 / GGGCGTTTCGAGGTCGAG
ttmS2 / ttmS2p1 / GCGGAGCTTCTGTTCCTG / 443
ttmS2p2 / CCTATGCGATGGGCGTGC
ttmJttmA / ttmJ-Ap1 / CCGCCAGGTAGGAGCCGT / 499
ttmJ-Ap2 / CAGTGCCAACAGGACGAC
lysAb / lysA-S / CCGCTGCCGCACCTACCT / 523
lysA-AS / CAGGGTGATGCCGTGTTGC

aThe primers labeled with 5’-biotin were used as EMSA probes preparation

b The DNA region lysA was used as the nonspecific probe

1