Current Microbiology

The SARP family regulator Txn9 and two-component response regulator Txn11 are key activators for trioxacarcin biosynthesis in Streptomycesbottropensis

Kui Yang1,2,*, Li-Hua Qi2,*, Mei Zhang2, Xian-Feng Hou2, Hai-Xue Pan2, Gong-Li Tang2, Wei Wang1,#, Hua Yuan2,#

1School of Perfume and Aroma Technology, Shanghai Institute of Technology, 100 Haiquan Road, Shanghai 201418, China; 2State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China

*These two authors contributed equally to this work.

#Corresponding author

Y.H.: ; W.W.:

Table S1 Primers used in this study

Primer / DNA sequence (5’ to 3’) / Relevant comments
Tg09F / GTCACCGGCCAGTACCGACTGATGCAGGCGCTGAAGCCGATTCCGGGGATCCGTCGACC / Used for txn9 inactivation
Tg09R / CCGAACAACTCCGAGCGAGGAGCCAAGGACGTCCTGGTCTGTAGGCTGGAGCTGCTTC
Tg10F / CATTCGCTCATCGCCACGGTCGTGTTGGCGTATGGCGTGATTCCGGGGATCCGTCGACC / Used for txn10 inactivation
Tg10R / GTCGCGGTAGGTCCTCGCCACCAGCCTCATGTCCGACAGTGTAGGCTGGAGCTGCTTC
Tg11F / GTCCGAGTGCTCATCGCCGAAGACGTCGCCATGCTCCGTATTCCGGGGATCCGTCGACC / Used for txn11 inactivation
Tg11R / AGGACTCTCCTGGACGTCCCAGGCCGACAGCGCCAATTC TGTAGGCTGGAGCTGCTTC
Tg22F / GCTTCCGGCACGACGCGTTGTCGTCGTTGCTGCGAAGCCATTCCGGGGATCCGTCGACC / Used for txn22 inactivation
Tg22R / GAGGCCGGAGTTCGTCGACCACCTCACGCAGTCTGCTGATGTAGGCTGGAGCTGCTTC
Tg34F / CGTGGACCTCGAACGGACAGCCGATGCTCACGAGGCGAAATTCCGGGGATCCGTCGACC / Used for txn34 inactivation
Tg34R / AGCTGGAGCAGGTCCTGCCCTCGCGTCTGCGGCAGAGAGTGTAGGCTGGAGCTGCTTC
Tg48F / GGCTGCGGACAAGGGTTCGAGGGGCTCAACCGTCACCTGATTCCGGGGATCCGTCGACC / Used for txn48 inactivation
Tg48R / ACCTACACCAATCCGCTGCGCTACCTGAACGGCGTGCGCTGTAGGCTGGAGCTGCTTC
Re09F / ATAGAATTC GCGACGAGATGGCCTTCCG / Used for txn9mutant complementation
Re09R / ATACATATG CGTTCACGGTCCTCGCAC
Re11F / ATACATATG GAGTGGCCGAGGACGGATG / Used for txn11mutant complementation
Re11R / ATAGAATTC CGCGTGAACCTCCCGTACC
txn0F / GAAGCGGAACTGCGGGTCATT / Used for differential expression analysis
txn0R / TGCCGACCTTAGACGGTGCAG
txn1F / CATTTCGCCGCTGAATCGC / Used for differential expression analysis
txn1R / CGTGACGAAGAGCGGGATGA
txn2F / CTCCGTGGATGCGTGTTGTT / Used for differential expression analysis
txn2R / GCCATGACGTACTTGCCGTAG
txn4F / CAACTCCGAGAACGCCACAGC / Used for differential expression analysis
txn4R / CGACGACTTCCACGCTGATGC
txn5F / CACGGTACTGGACCTCATGGACG / Used for differential expression analysis
txn5R / GCGAACGATGTCAGCGTCTGG
txn5-6F / AGCAGCAGAGCCGTGTCCTTG / Used for co-transcriptional analysis
txn5-6R / CAGGTCATCGGGCAGGTTCA
txn6-7F / CTTCAGCCGGTCTGCGGTGCCT / Used for co-transcriptional and differential expression analysis
txn6-7R / GTGATCCCTCCGACCGTCA
txn9F / aCATATGATGGAAATCAAGGTCCTGGGA / Used for differential expression analysis
txn9R / aGGATCCGAATTCAAGCTTACTCGAGGAGAGCGGCGACCTCCAGGTC
txn10F / GCCGCTAATCGGTTTCCAGG / Used for differential expression analysis
txn10R / TGGCGGGAGATGTCCAGAA
txn12F / TTCTCCTGAGTCCGAGCGTGTTC / Used for differential expression analysis
txn12R / CCACGTTGTAGTGGCCCTTTCC
txn12-13F / CGGCGTACATACAGGCGAGCAT / Used for co-transcriptional analysis
txn12-13R / CGGTGGGTCGTGGACTTCGT
txn13-14F / GTGGACCGTGGGACCGAAGA / Used for co-transcriptional and differential expression analysis
txn13-14R / ACCGTGGCGACACTCCCTTCTC
txn15F / TCCTCGACGCGGAACACTTCT / Used for differential expression analysis
txn15R / CCAGCTCCTTGACCCTTGTGGC
txn16F / TGACCGCCCTTTCCGTGAGTG / Used for differential expression analysis
txn16R / CGCAGGGGTACACGCAGTAGAA
txn15-16F / TGCCGGGGATGGCTCTTGG / Used for co-transcriptional and differential expression analysis
txn15-16R / CGGATGGAGCGATGGGGAG
txn17F / ATGCCGAACGGAACCTTTGC / Used for differential expression analysis
txn17R / ACCAACGACGACGCCGGATC
txn17-18F / AGGTGGCGGGGCTGTTCGA / Used for co-transcriptional analysis
txn17-18R / CGATGCCGACGAGGAACACC
txn18-19F / GCTCGGCAACACGACCTACCA / Used for co-transcriptional and differential expression analysis
txn18-19R / GCCTGAACGATGCCGAAGTG
txn21F / TCCTGGACGGACGTGACTGC / Used for differential expression analysis
txn21R / CAGGAACGCCATACCGAGGA
txn22F / ACTTCCTGGTGCCGATGGTCC / Used for differential expression analysis
txn22R / CGGAACGGGAAGAGGAACAGC
txn23F / CGGCAGGATGAGCTGTGGCT / Used for differential expression analysis
txn23R / CCATCACCTGTCCGCAGTTCC
txn25F / GGTGTACGACCTGATCGGCTTCG / Used for differential expression analysis
txn25R / GCTCGGACAGCAGTTCCTCACA
txn27F / ACGCCTTCCACGTCGTCTGTC / Used for differential expression analysis
txn27R / GGGTGCTCTGGAGGACCGAAT
txn27-28F / ACGGACAGACGACGTGGAAGGC / Used for co-transcriptional and differential expression analysis
txn27-28R / AGGCTCTGCATATCCGCGAACT
txn30F / GGGATATGGGTGACCGAGCAG / Used for differential expression analysis
txn30R / ACGGGAAGCGGGAAGACCA
txn31F / GGAACGTCATCGTCGAGACCC / Used for differential expression analysis
txn31R / CCGATGTCCACGACGAGATGA
txn33-34F / GTGGCGAAGGCGACGGTGTA / Used for co-transcriptional and differential expression analysis
txn33-34R / CCTCGGTTGGCTGGCGTTT
txn34-35F / GCGAAACGCAGTTGCTCCC / Used for co-transcriptional and differential expression analysis
txn34-35R / CAACCCGCATCTGTCCTTCG
txn35F / GTGCCGTCGTCCCAAGAACC / Used for differential expression analysis
txn35R / TGAGGGTGAAGCCGAATGTGA
txn36F / GAGCGGCGTCTGGAGGAGTT / Used for differential expression analysis
txn36R / CACGACGGGTTGTGGATCTTGT
txn35-36F / TGATGAGCCAGGCGTCCAC / Used for co-transcriptional analysis
txn35-36R / GTCTGGAGGAGTTCCTGGAGACG
txn36-37F / TCTCCAGGAACTCCTCCAGACGC / Used for co-transcriptional and differential expression analysis
txn36-37R / GCCAACGAATCGGCTCTGGT
txn37F / GGACAACGGGCTCTGTGGGA / Used for differential expression analysis
txn37R / CGCACCAGAGCCGATTCGTT
txn41F / TCGTCGTCAACACCCATTTCCA / Used for differential expression analysis
txn41R / GCCTTCCTGTGCCACTCCTTGA
txn42F / CGCTGACCGATTCGTGGCA / Used for differential expression analysis
txn42R / TCCACCGCCAGCAGCAACTC
txn44F / TACCTGGACCGAGGACCTCACG / Used for differential expression analysis
txn44R / GGCGTCGTAGACGTGGGAGAT
txn45F / AACCCGCATCTTCCATTCCC / Used for differential expression analysis
txn45R / TCGACGGACTGCTTCTTGTGATC
txn45-46F / CCACAGGACGAGATCGGGTTT / Used for co-transcriptional analysis
txn45-46R / CGCCAATGAGCTGCGAGACA
txn46-47F / CCTCGAAGCCGATTCCTTTGTT / Used for co-transcriptional analysis
txn46-47R / ACCGACTCCGAGGTACTGAGCAT
txn47-48F / CGGGTGGCTGCTCCGTGAA / Used for co-transcriptional analysis
txn47-48R / CCAACCACGCAACTGCCTCTAC
txn48F / GCTCCCTTTCCGCACCTTCA / Used for differential expression analysis
txn48R / GGGTAGAGGCAGTTGCGTGGTT
txn48-49F / GCCGTGAAGGTGCGGAAAG / Used for co-transcriptional and differential expression analysis
txn48-49R / CACCCTGAGAACTGGGCGAAA
txn50-51F / CGCATAGGTGTAGGTGCTGAAAG / Used for co-transcriptional and differential expression analysis
txn50-51R / CCGTGGTGGATGGCAAGGA
txn51-52F / TCGGCGATGCCCGCTTTGGT / Used for co-transcriptional analysis
txn51-52R / GTCCTCATCGGCAACACTCCC
txn54F / GAAACTCCTCAGCCCTGTCCC / Used for differential expression analysis
txn54R / ACCTTCCAGGTCCGGCAGTC
txn55F / TGTCCGTGGCAGGAGACCG / Used for differential expression analysis
txn55R / CAGGCGGATTTGAGGACACTCG
YZ-9-F / CACGGGATTTCGGTGGTGC / Used for txn9 mutant verification
YZ-9-R / AGAGCGGCGACCTCCAGGT
YZ-10-F / GTGAACGCCTTATCGAGTGGTG / Used for txn10 mutant verification
YZ-10-R / CGCCCATGAGACGAAACCAT
YZ-11-F / GCCCGTCCGAGTGCTCATC / Used for txn11 mutant verification
YZ-11-R / CACGATGGAGGTCAGGTAGTTGC
YZ-22-F / ACGCACCGTACGTGGGTCT / Used for txn22 mutant verification
YZ-22-R / CCGAGAAGGAGGTTGTCGAAGG
YZ-34-F / CGTCAGTCCCCGCACCATC / Used for txn34 mutant verification
YZ-34-R / CCCAGATCACGCAGGTAGGACAC
YZ-48-F / GCTCCCTTTCCGCACCTTCA / Used for txn48 mutant verification
YZ-48-R / CACGGTGCCGATGGTGAGTT

Table S2 The intergenic distance between twoadjacent txn genes with the same transcriptional direction

Gene pair / Distance (bp)
txn0-1 / 810
txn3-4 / 33
txn5-6 / 157
txn6-7 / 64
txn7-8 / -1
txn8-9 / 347
txn10-11 / 21
txn12-13 / 137
txn13-14 / 76
txn14-15 / 28
txn15-16 / 80
txn17-18 / 130
txn18-19 / 61
txn20-21 / 10
txn23-24 / -4
txn26-27 / 27
txn27-28 / 117
txn28-29 / -1
txn29-30 / 5
txn31-32 / -1
txn33-34 / 76
txn34-35 / 82
txn35-36 / 77
txn36-37 / 157
txn37-38 / 28
txn39-40 / 26
txn40-41 / 20
txn42-43 / 20
txn45-46 / 93
txn46-47 / 80
txn47-48 / 120
txn48-49 / 181
txn49-50 / 28
txn50-51 / 50
txn51-52 / 50
txn52-53 / 33

Table S3 The txn operons and regulation profiles by Txn9 and Txn11

Operon / Gene
(original name) / Proposed function / Activated by Txn9 / Activated by Txn11
1 / txn0(Rg1) / hypothetical protein / — / —
2 / txn1(B1) / dTDP-glucose 4,6-dehydratase / — / —
3 / txn2(B2) / dTDP-glucose synthase / — / —
4 / txn3(B3) / dehydrogenase E1 component / — / √
txn4(B4) / dehydrogenase E1 component
5 / txn5(A4) / acyl transferase / — / √
6 / txn6(A3) / acyl carrier protein / — / √
txn7(A2) / keto-acyl synthase beta
txn8(A1) / keto-acyl synthase alpha
7 / txn9(Rg2) / SARP family pathway specific regulatory protein / √ / √
8 / txn10(Rg3) / two-component sensory kinase / — / —
txn11(Rg4) / LuxR type two-component response regulator
9 / txn12(P1) / AMP-binding domain-containing protein / — / —
10 / txn13(C1) / cyclase/dehydrase / — / —
txn14(A5) / 3-oxoacyl-ACP synthase III
txn15(P2) / 2-isopropylmalate synthase/homocitrate synthase family protein
11 / txn16(P3) / CoA-transferase family III / — / —
12 / txn17(P4) / ketoreductase / — / —
txn18(Rr1) / transmembrane efflux protein
txn19(O1) / alcohol dehydrogenase
13 / txn20(U1) / hypothetical protein / √ / √
txn21(O2) / cytochrome P450 monooxygenase
14 / txn22(U2) / winged helix DNA-binding domain containing protein / — / —
15 / txn23(O3) / pyridine nucleotide-disulfide oxidoreductase / √ / √
txn24(O4) / ferredoxin
16 / txn25(H1) / secreted tripeptidylaminopeptidase / √ / √
17 / txn26(O5) / cytochrome P450 / √ / √
txn27(H2) / hydrolase
txn28(B5) / NDP-hexose-4 ketoreductase
txn29(B6) / dTDP-4-deoxyglucose 3,5-epimerase
txn30(M1) / NDP-hexose 3-C-methyltransferase
18 / txn31(B7) / dTDP-4-keto-6-deoxy-L-hexose2,3-dehydratase / √ / √
txn32(B8) / oxidoreductase
19 / txn33(Rr2) / major facilitator superfamily permease / — / —
txn34(Rg5) / DeoR family transcriptional regulator
20 / txn35(O6) / cytochrome P450 / √ / √
txn36(O7) / hypothetical protein
21 / txn37(O8) / acyl-CoA dehydrogenase / √ / √
txn38(O9) / ligand-binding SRPBCC domain
22 / txn39(C2) / short-chain dehydrogenase/reductase SDR / √ / √
txn40(O10) / flavin reductase-like, FMN-binding protein
txn41(C3) / beta-lactamase domain-containing protein
23 / txn42(B9) / glycosyltransferase / √ / √
txn43(O11) / cyclase
24 / txn44(U3) / hypothetical protein / √ / √
25 / txn45(B10) / glycosyltransferase / √ / √
txn46(C4) / oxidoreductase
txn47(M2) / O-methyltransferase
26 / txn48(U4) / winged helix DNA-binding domain containing protein / — / —
txn49(B11) / acyltransferase 3
txn50(B12) / glycosyltransferase
txn51(M3) / O-demethylpuromycin-O-methyltransferase
txn52(O12) / cytochrome P450 monooxygenase
txn53(M4) / O-demethylpuromycin-O-methyltransferase
27 / txn54(U5) / mycothiol maleylpyruvate isomerase N-terminal domain / — / —
28 / txn55(Rg6) / conserved hypothetical protein / — / —

Note: “—”, no obvious impact; “√”, absolute activation; “√”, obvious activation.