Supporting information Figures S1-S9

The response to copper stress in Streptomyces lividans extends beyond genes under the direct control of a Copper sensitive operon Repressor protein (CsoR) Srivatsa Dwarakanath1, Amanda K. Chaplin1, Michael A. Hough1, Sébastien Rigali2, Erik Vijgenboom3 and Jonathan A.R. Worrall1* 1School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK. 2Centre for Protein Engineering, University of Liège, Institut de chimie B6a, B-4000 Liège, Belgium. 3Molecular Biotechnology, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300RA Leiden, The Netherlands. *To whom correspondence should be addressed: Jonathan Worrall jworrall@essex.ac.uk; Tel: +44 1206 872095

Figure S1: A coomassie stained 15 % SDS-PAGE gel showing two fractions of CsoRSl eluted from the G75 sephadex column following cleavage of the His-tag with thrombin. A UV-visible spectrum of purified CsoRSl in 20 mM Tris/HCl pH 7.5, 150 mM NaCl.

A B

Figure S2: Analytical ultracentrifugation. A) Sedimentation equilibrium data at pH 6.5 and 20 oC for apo-CsoRSl. Data were fitted using a single ideal species model with the residuals of the fits shown. A single species average molecular weight of 59.3 kDa was determined. B) Sedimentation equilibrium data at pH 6.5 and 20 oC for CuI-CsoRSl. Data were fittedusing a single ideal species model with the residuals of the fits shown. A single species average molecular weight of 56.9 kDa was determined.

Figure S3: Determination of Cu(I) binding stoichiometry to apo-CsoRSl using [CuI(BCA)2]3- as a non-competitive probe.

The absorbance at 562 nm in the visible spectrum of [Cu I(BCA)2]3- decreases to zero upon increasing amounts

of apo-CsoRStrep. A plot of the absorbance change at 562 nm as a function of protein:Cu ratio reveals a stoichiometry of 1:1. Conditions used: 20 mM [apo-CsoRSl], 20 mM [CuI]total and 60 mM [BCA].

44 hours

1326

ΔcsoR

90 hours

1326

ΔcsoR

R5 + 0.1 μM Cu

R5 + 10 μM Cu

R5 + 50 μM BCDA

R5 + 50 μM BPDA

SFM MM glucose,mannitol

Figure S4A: S. lividans 1326 (wild type) and the ΔcsoR mutant were plated on the media indicated and incubated at 30 oC. Pictures were taken after 44 hours and 90 hours. No significant differences between the two strains were observed regarding vegetative - and aerial growth and sporulation.

0 μM 250 μM 500 μM 750 μM 1000 μM

1326

ΔcsoR

Figure S4B S. lividans 1326 (wild type) and the ΔcsoR mutant were plated on MM (0.5% glucose, 0.5% mannitol) with the indicated [Cu2+] and incubated at 30 oC. Pictures were taken after 90 hours. No significant differences between the two strains were observed regarding vegetative - and aerial growth and sporulation. On this solid medium growth stops completely at 1000 μM Cu2+. Blown ups of individual colonies are taken with a stereo microscope and show that colony size is strongly reduced at 750 μM. Furthermore the colonies turn bluish at this copper concentration with the strongest staining for the ΔcsoR mutant.

Growthcurves in NMMP

1

10

100

1000

0 10 20 30 40

Time (hour)

Bio

mas

s (g

/lit

) x1

0

1326

1326 100μM Cu

1326 400μM Cu

Δ4136

Δ4136 100μM Cu

Δ4136 400μM Cu

Figure S5A. Growth curves of Streptomyces lividans wild type (1326) and the CsoR deletion mutant (ΔcsoR).

Cultures were grown in 50 mL NMMP with 0.5% mannitol and 0.5% glucose in 250 mL baffled polycarbonate flasks with 0.2 μm vent caps (Corning), 300C and 160 rpm in a NBS Innova 44 (2” orbit).

The inoculum was ~107 spores/mL from a fresh spore stock. For the growth curves shown in Figure S6B the inoculum of the strains was varied somewhat to avoid the complete overlap of the curves. Cu(II) was added to a final concentration of 100, 400 750, 1000 μM to the medium at the time indicated.

Growth was recorded by determining the dry biomass. Samples of 1.5 mL were collected in pre-dried eppendorftubes of known weight and mycelium was pelleted by centrifugation in an eppendorf centrifuge at 13.000 rpm for 10 minutes. After removal of the spent medium a short second run was applied and any residual liquid was removed. After 6 hours at 1050C the dry weight was determined on a analytical balance.

Growth rates derived from these growth curves are listed in Table 2 (see manuscript). SD values for the growth rates are 0,01

Figure S5B

Figure S6

Figure S7

Figure S8: S200 gel-filtration of apo-CsoRSl in complex with the 1045 DNA target. A pre-formed 1:4 complex of DNA:apo-CsoRSl (monomer) resulted in the formation of a new peak, but with significant amounts of free DNA still present. At a 1:8 ratio the majority of the DNA was found bound to the protein, therefore indicating that two tetramers are required to bind the DNA target. Similar profiles were observed with the other DNA targets.

Figure S9: Changes in the UV region of the C75A apo-CsoRSl base-lined spectrum upon addition of CuCl at pH 7.5, 20 oC. The inset shows the increase in absorbance at 240 nm plotted as a function of [Cu(I)]/[apo-C75A] revealing a saturation at ~ 1:1 molar ratio.