The zinc responsive regulator Zur controls expression of coelibactin gene cluster in Streptomyces coelicolor

Dimitris Kallifidas, Ben Pascoe, Gillian A. Owen, Claire M. Strain-Damerell, Hee-Jeon Hong, and Mark S.B. Paget

Supplementary Data

Microarray methods

Detailed descriptions of the microarray experiment can be found in the submission at ArrayExpress (Accession number E-MAXD-56) and are briefly described here. Triplicate cultures of S121 (Dzur::apr) and M145 (wt) were treated with RNAprotect (Qiagen) to prevent further RNA synthesis and degradation, then RNA isolated from mycelium using the total RNA isolation method developed by the University of Surrey Microarray Group (http://www.surrey.ac.uk/SBMS/Fgenomics/Microarrays/docs/RNeasy_0803.pdf ). RNA was converted to cDNA in the presence of random oligonucleotides (Invitrogen Cat.#48190-011) and SuperScript II and either Cy3-dCTP (S121) or Cy5-dCTP (M145). PCR product spotted microarrays (scp48) were obtained from the University of Surrey Microarray Group and processed as recommended by their on-line protocols (http://www2.surrey.ac.uk/fhms/microarrays/). Briefly, slides were pre-hybridised and hybridised with Cy3 and Cy5 samples using the Pronto! Universal Microarray

Hybridisation kit (Corning, Cat.# 40026) according to the supplier's instructions. Following washing steps, slides were scanned using an Axon Genepix 4000B scanner. Following the manual marking of bad spots to prevent their further analysis, data was imported into Genespring GX 7.3.1. To account for dye bias data was transformed using the Lowess method prior to per spot and per chip normalisation. A Lowess curve was fit to the log intensity verses log-ratio plot. 20% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. The Student’s t-test in Genespring was used to identify genes that were significantly up-regulated in S121 compared to M145 (P= <0.05).

Table S1. Genes induced 2-fold in S121 (Dzur::apr) compared to the parental strain M145 (P = <0.05). This list includes genes that do not possess a likely upstream Zur binding site and might therefore not be direct targets of Zur.

Gene / Gene function / S121/M145 / P-value
SCO0027 / hypothetical protein / 2.1 / 0.000374
SCO0079 / putative integral membrane transport protein / 2.6 / 0.000843
SCO0111 / putative oxidoreductase / 2.0 / 5.42E-05
SCO0125 / possible oxidoreductase / 2.3 / 0.0453
SCO0354 / putative binding-protein-dependent transport protein. / 2.2 / 0.0374
SCO0437 / conserved hypothetical protein / 3.8 / 2.91E-05
SCO0471 / putative araC family transcriptional regulator / 2.0 / 0.0244
SCO0472 / putative secreted protein / 11.5 / 0.0195
SCO0473 / putative solute-binding lipoprotein / 4.6 / 0.019
SCO0475 / ABC transporter protein, integral membrane subunit / 8.7 / 3.66E-06
SCO0476 / ABC transport protein, ATP-binding subunit / 4.2 / 0.00286
SCO0553 / hypothetical protein SCF11.33c / 2.0 / 0.0181
SCO0744 / conserved hypothetical protein SCF81.03 / 2.2 / 0.0063
SCO0897 / conserved hypothetical protein / 2.3 / 0.000859
SCO1512 / hypothetical protein SCL2.02 / 3.4 / 5.89E-07
SCO1740 / putative membrane protein / 2.9 / 0.00289
SCO2210 / glutamine synthetase / 2.1 / 0.0423
SCO2505 / putative ABC-transporter metal-binding lipoprotein / 42.1 / 0.0152
SCO2506 / putative metal transport ABC transporter / 2.3 / 0.0107
SCO2899 / putative GroES-family molecular chaperone. / 2.2 / 0.00446
SCO3310 / hypothetical protein / 2.1 / 0.00339
SCO3426 / hypothetical protein / 3.7 / 0.0368
SCO3428 / putative 50S ribosomal protein L33 / 7.1 / 0.000397
SCO3429 / putative 50S ribosomal protein L28 / 7.4 / 3.04E-06
SCO3430 / putative 30S ribosomal protein S14 / 4.2 / 0.00985
SCO3431 / possible membrane protein / 2.5 / 0.0497
SCO3471 / extracellular agarase precursor / 2.1 / 0.0141
SCO3535 / hypothetical protein / 2.4 / 0.000174
SCO3559 / putative oxidoreductase / 2.2 / 0.0186
SCO3746 / putative membrane protein / 3.0 / 0.0109
SCO3765 / putative integral membrane protein / 2.4 / 0.000245
SCO3818 / putative two-component system response transcriptional regulator / 2.8 / 1.07E-05
SCO3833 / putative transcriptional regulator / 2.0 / 0.00846
SCO4213 / hypothetical protein / 2.1 / 0.0293
SCO4558 / putative acetyltransferase / 2.3 / 0.0282
SCO4938 / putative ECF-sigma factor / 2.4 / 0.032
SCO5142 / putative secreted protein / 2.1 / 0.00448
SCO5160 / hypothetical protein SCP8.23 / 2.2 / 0.0107
SCO5224 / putative araC-family transcriptional regulator / 2.0 / 8.10E-05
SCO5444 / putative glycogen phosphorylase / 2.6 / 0.0429
SCO5526 / urease alpha subunit / 2.2 / 0.0198
SCO5684 / putative two-component system response regulator / 2.2 / 0.00153
SCO5857 / FAD-dependent oxidoreductase / 2.6 / 0.0228
SCO5982 / putative regulator / 2.1 / 0.0205
SCO6518 / putative transcriptional regulator / 2.0 / 0.0324
SCO7026 / hypothetical protein SC1H10.15. / 3.0 / 0.0493
SCO7547 / putative sulfatase / 2.9 / 0.0148
SCO7676 / putative ferredoxin / 5.3 / 0.00644
SCO7677 / putative secreted solute-binding protein / 5.0 / 0.0172
SCO7686 / putative cytochrome P450 / 2.0 / 0.000597
SCO7687 / putative thioesterase / 2.2 / 0.00274
SCO7787 / hypothetical protein / 2.4 / 0.0202
SCO0027 / hypothetical protein / 2.1 / 0.000374


Table S2 Oligonucleotides used in this study.

Name / Sequence / Gene
Q-PCR primers
q0475 For / 5'-GTGACGTTTGTGCAAAGAGC / SCO0475
q0475 Rev / 5'-AGATGGCGGATACCAGGAT / SCO0475
q0476 For / 5'-GTACGTCTGACCGAGCTGTG / SCO0476
q0476 Rev / 5'-CAGGTGAGTTGCCTCAGGA / SCO0476
q0477 For / 5'-ATCAACCTCACCAGCGTCTT / SCO0477 / SCO0475
q0477 Rev / 5'-CAGGGTCTCGGTGTAGAAGC / SCO0477 / SCO0475
q7676 For / 5'-TTCGACCAGGACGAGGAG / SCO7676 / SCO0475
q7676 Rev / 5'-GAAGAGTGAGCGCTCCAGA / SCO7676 / SCO7676
q7677 For / 5'-GACCCTCGACTTCTTCAACG / SCO7677 / SCO7676
q7677 Rev / 5'-ATGTCGATTCCCTTGAGCAC / SCO7677 / SCO7676
q7681 For / 5'-ACACGTTGCGTGGCTACTAC / SCO7681 / SCO7676
q7681 Rev / 5'-GGTTGATCTGGTCCTTGAGC / SCO7681 / SCO7676
q7682 For / 5'-CCGTACTGCACCTCTCCTTC / SCO7682 / SCO7676
q7682 Rev / 5'-ACGTAGTCGCGGAAAGTGAT / SCO7682 / SCO7676
q2505 For / 5'-GCCACCAAGGTCTTCTTCAC / znuA / SCO7676
q2505 Rev / 5'-CTGCTGGAGTTCCTTGATCC / znuA / SCO7676
q5820 For / 5'-CTCTTCCTGGACCTCATCCA / hrdB / SCO7676
q5820 Rev / 5'-GGCGTACGTGGAGAACTTGT / hrdB / SCO7676
Electromobility shift assay primers / SCO7676 / SCO7676
0475/76_F1 / 5’-CGGTATCTCGCAGGTGAGTTGCCTCAGGACC / SCO0475/76 intergenic / SCO7676
0475/76_R1 / 5’- CCACAAGGCTCTTTGCACAAACGTGACTTCG / SCO0475/76 intergenic / SCO7676
7676_F1 / 5’-GCTGTTCCTGCTGCTCGT / SCO7676 / SCO7676
7676_R1 / 5’-CTCGTCCTGGTCGAAGACAC / SCO7676 / SCO7676
7681/82_F1 / 5’-AGCTCCGCGTAGGTCCAC / SCO7681/82 intergenic / SCO7676
7681/82_R1 / 5’-TACCGACCAGGGACATCAAC / SCO7681/82 intergenic / SCO7676