Supplementary Table S1 Bacterial Strains and Plasmids Used in This Study

Supplementary material

A regulatory gene SCO2140 is involved in antibiotic production and morphological differentiation of Streptomyces coelicolor A3(2)

Table S1 Bacterial strains and plasmids used in this study

Bacterial strains/plasmids / Relevant characteristics / Source/references
Streptomyces
S. coelicolor M145 / A derivative of the wild-type strain S. coelicolor A3(2) lacking plasmids SCP1 and SCP2 / [5]
SCO2140DM / The SCO2140 disruption mutant of S. coelicolor / This study
SCO2140CM / The complemented strain of SCO2140DM / This study
SCO2140OE / The SCO2140 overexpressed strain / This study
Staphylococcus aureus / Indicator strain for CDA bioassays / [1]
E. coli
JM109 / recA1endA1gyrA96thi-1hsdR17supE44relA1Δ(lac-proAB)/F’[traD36 proAB+ lacIq lacZΔM15] / Invitrogen
DH5α / F- φ80lacZ M15 endArecAhsdR (rk-mk-) supEthigyrArelAΔ(lacZYA-argF) U169 / Invitrogen
ET12567/pUZ8002 / damdcmhsdScattet/pUZ8002 / [5]
C43(DE3) / F-ompT gal hsdSB (rB-mB-) dcm lon λDE3 and two uncharacterised mutations described in the reference / [8]
Plasmids
pKC1132 / suicide vector for Streptomyces containing oriT RK2 / [5]
pIJ8600 / The expression vector containing the promoter of tipAp / [5]
pUC119::Kanr / pUC119 containing kanamycin resistance gene (Kanr) / [17]
pBluescript KS+ / Routine cloning and subcloning vector / Stratagene
pIMEP / pSET152containing an ermE* promoter and ribosomal binding site sequence / [16]
pD2140 / plasmid used for the construction of SCO2140DM / This study
pH2140 / pIJ8600 containing the intact SCO2140coding region / This study
pIMEP::SCO2140 / pIMEP containing the intact SCO2140coding region / This study
pET23b / Expression vector used in E. coli / Novagen
pET23b::SCO2140 / SCO2140 gene cloned in pET23b / This study

Table S2 Primers used in this study

Primers / Sequence (5’-3’)
2140UF / AGCAAGCTTCGCTGTGCTGTCTGCTGTT
2140UR / AGGGATCCGGTCCACGCTGGTCTTGATG
2140DF / GAGAATTCGCACCTACTCCCAGCACGAC
2140DR / CCTCTAGATGGAAGGCGACCACGAA
2140YF1 / CCGTTCTGGGTTTCCCTTCA
2140YR1 / CGGCGGTTCCGATGAGTT
2140YF2 / GAGAGCGTCAGCGAGGTC
2140YR2 / GGGAGTAGGTGCGGAAGG
2140HF / CATATGATCAGCGCGATCGTCCTCA
2140HR / GGATCCTCAGTTGTCCAGGCCGAT
2140HF1 / GTGATCAGCGCGATCGTCCTCA
2140HR1 / TCAGTTGTCCAGGCCGATGGC
2140OF / CATATGATCAGCGCGATCGTCCT
2140OR / CTCGAGGTTGTCCAGGCCGATG
RTactF / GCTCCTCAGGCGGCACGA
RTactR / GCCGGCGGGTGTGGTACA
RTcdaF / GGAAAGCGACGCCTACTT
RTcdaR / AGGCTCGTCTTTCCGATT
act-PF / CCAGCCGTATCAGGAATGC
act-PR / ACATGGACACGTCCCAATAAG
cda-PF / AATAGCAAACGGCGCACTGA
cda-PR / CGCATTTCCGATTCTCTGGT

FigureS1 Amino acid aliment of SCO2140 with its orthologs from other Streptomyces(a). SAV, Streptomyces avermitilis; SBI, Streptomyces bingchenggensis; SCAB, Streptomyces scabiei; SCLAV, Streptomyces clavuligerus; SFUL, Streptomyces fulvissimus; SGR, Streptomyces griseus; SLIV, Streptomyces lividans; Strvi, Streptomyces violaceusniger; SVEN, Streptomyces venezuelae. bSequence alignment of SCO2140 with Lrp from E. coli and LrpA from Mycobacterium tuberculosis

Figure S2Determination of the undecylprodigiosin (RED)production in Streptomyces coelicolor. RED production in thewild-type strain (WT), the SCO2140 disruption strain (SCO2140DM) and the complemented strain (SCO2140CM) after grown for 24, 48, 72, 96 and 120 h in R5 medium. Error bars represent standard deviations from three independent experiments

Figure S3 EMSAs for detecting the binding of SCO2140 to the promoter regions of actII-ORF4 andcdaR.a Binding of SCO2140 to the promoter region of actII-ORF4. b Binding of SCO2140 to the promoter region of cdaR. 1-9 represent the varying quantities of SCO2140-His6 (0, 45, 180, 360, 450, 1800, 3600, 4500 and 6750 nM protein)

References

Anderson TB, Brian P, Champness WC (2001) Genetic and transcriptional analysis of absA, an antibiotic gene cluster-linked two-component system that regulates multiple antibiotics in Streptomyces coelicolor. Mol Microbiol39(3):553-566. doi:10.1046/j.1365-2958.2001.02240.x

5. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics.The John Innes Foundation, Norwich

8. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260(3):289-298. doi:10.1006/jmbi.1996.0399

16. Wang SL, Fan KQ, Yang X, Lin ZX, Xu XP, Yang KQ (2008) CabC, an EF-hand calcium-binding protein, is involved in Ca2+-mediated regulation of spore germination and aerial hypha formation in Streptomyces coelicolor. J Bacteriol 190(11): 4061-4068. doi:10.1128/JB.01954-07

17. Yang H, Wang L, Xie Z, Tian Y, Liu G, Tan H (2007) The tyrosine degradation gene hppD is transcriptionally activated by HpdA and repressed by HpdR in Streptomyces coelicolor, while hpdA is negatively autoregulated and repressed by HpdR. Mol Microbiol 65(4):1064-1077. doi:10.1111/j.1365-2958.2007.05848.x