Supplementary information A tight tunable range for Ni(II ...€¦ · +RT-RT S LE rps1 rps1 rps1...

1

Supplementary information

A tight tunable range for Ni(II) sensing and buffering in cells

Andrew W. Foster1,2 †, Rafael Pernil1,2 †, Carl J. Patterson1,2, Andrew J. P. Scott1, Lars-Olof

Pålsson2, Robert Pal2, Ian Cummins1, Peter T. Chivers1.2, Ehmke Pohl1,2 and Nigel J.

Robinson1,2*

1Department of Biosciences, Durham University, DH1 3LE, UK; 2Department of Chemistry,

Durham University, DH1 3LE, UK

* email: [email protected]

† these authors contributed equally

Nature Chemical Biology: doi:10.1038/nchembio.2310

mailto:[email protected]

2

Supplementary results

Supplementary Table 1│ Data collection and refinement statistics

* Data set was collected from a single crystal. † Highest resolution shell is shown in parenthesis.

Apo-InrS*

Data collection

Space group I222

Cell dimensions

a, b, c (Å) 64.29, 77.79, 85.60

() 90, 90, 90

Resolution (Å) 2.4 (2.4-2.5) †

Rsym 0.042 (0.324)

I / I 27.7 (3.2)

Completeness (%) 95.5 (78.2)

Redundancy 6.1 (3.4)

Refinement

Resolution (Å) 2.4

No. reflections 7855

Rwork / Rfree 0.208/0.282

No. atoms

Protein (chain A/B) 692/706

Water 33

B-factors

Protein (chain A/B) 54.9/59.3

Water 50.5

R.m.s. deviations

Bond lengths (Å) 0.011

Bond angles () 1.40


3

Supplementary Table 2│ Metal- and DNA-affinities plus associated statistics for InrS

variants*.

Metal KMetal (M) Mutant

KMetal fold

weakening

KDNA (M)† ΔGC

(kcal mol-1)†

Set point

(M)‡‡

Mutant set

point fold

change

InrS apo - - nrsD 9.4(±2.0)10-9‡ - - -

- - nik 3.3(±0.7)10-7 - - -

Ni(II) 2.8(±0.4)10-12 - nrsD 2.3(±0.0)10-6‡ +3.3(±0.1) ‡ 3.010-10 -

- - nik 5.5(±0.9)10-6 +1.7(±0.1) 4.010-12 -

Cu(II) nd - nrsD 3.6(±0.8)10-6‡ +3.5(±0.1) ‡ - -

H21L apo - - nrsD 2.1(±0.7)10-8 - - -

- - nik 3.5(±0.1)10-7 - - -

Ni(II) 5.5(±1.3)10-11 20 nrsD 2.7(±0.2)10-6 +2.9(±0.2) 3.010-9 10

- - nik 7.5(±0.9)10-6 +1.8(±0.1) 6.010-11 15

Cu(II) nd - nrsD 1.1(±0.1)10-6 +2.4(±0.2) - -

H21E apo - - nrsD 5.6(±0.2)10-8 - - -

- - nik 6.2(±0.5)10-7 - - -

Ni(II) 8.6(±1.7)10-11 31 nrsD 2.7(±0.4)10-6 +2.3(±0.1) 2.010-9 7

- - nik 7.7(±0.2)10-6 +1.5(±0.0) 1.510-11 4

Cu(II) nd - nrsD 1.7(±0.2)10-6 +2.0(±0.1) - -

H78L apo - - nrsD 2.1(±0.7)10-8 - - -

Ni(II) <2.010-5 7.1106 nd - 7.010-4 2.3106

Cu(II) nd - nrsD 2.1(±0.3)10-6 +2.8(±0.2) - -

* All constants are means of at least triplicate determinations (‘n’ specified in each figure legend) with ± one standard deviation shown in parenthesis. nd = not determined † Promoter DNA used to obtain value shown to left.

‡ These values were determined previously

1.

‡‡ See supplementary figure 3.


4

Supplementary Table 3│Concentrations of abundant amino acids and glutathione in

Synechocystis.

Molecule Intracellular concentration (μM)*

Alanine 1,649(±224)

Arginine 126(±11)

Aspartic acid 283(±61)

Glutamine 634(±100)

Glutamic acid 2,390(±850)

Histidine 45(±21)

Isoleucine 73(±11)

Leucine 77(±4)

Serine 607(±98)

Threonine 205(±29)

Tyrosine 43(±10)

Valine 163(±35)

Total glutathione 299(±85)

* Means of triplicate biological determinations with ± one standard deviation shown in parenthesis.


5

Supplementary Table 4│Oligonucleotides used in this work.

Oligonucleotide Sequence (5´→3´)

1 Quikchange

H21E_F

GCCCATCCCCATGTCGAGAGCCAAGAATCCTTAC

2 Quikchange

H21E_R

GTAAGGATTCTTGGCTCTCGACATGGGGATGGGC

3 nikProFA_F [HEX]-AAATCCATATCCCCCTTCCCCCCAGAGGGTATT

4 nikProFA_R AATACCCTCTGGGGGGAAGGGGGATATGGATTT

5 nrsRS-

nrsBACDSeg_F

CTAAACTGTCATGCAACGTCC

6 nrsRS-

nrsBACDSeg_R

CTTCTTCCGGTGCCAGAC

7 inrS region SR_F3 CCTCGAGACTAACTCTGATTTCTGGTTTTGTTATCGGC

8 inrS region SR_R3 CAATACGCCTCGAGACAGGATCCAAAAACCCTATAAAAACCGGTC

9 slr0168-SR_F2 GACTATTCAATACACCCCCCTAGCC

10 slr0168-SR_R2 CCAGAAGTCGATAAAAACCCATGG

11 slr0168-SR-QC-

XhoI_F

CCAATCAAAATAACAGTCTCGAGGATTTATATGGAGACC

12 slr0168-SR-QC-

XhoI_R

GGTCTCCATATAAATCCTCGAGACTGTTATTTTGATTGG

13 Quikchange H21L_F GCCCATCCCCATGTCCTGAGCCAAGAATCCTTAC

14 Quikchange

H21L_R

GTAAGGATTCTTGGCTCAGGACATGGGGATGGGC

15 Quikchange H78L_F GATTAATTTTGGATGACCTGATGAATGAGTGCATCACCAG

16 Quikchange

H78L_R

CTGGTGATGCACTCATTCATCAGGTCATCCAAAATTAATC

17 Slr0168-inrS-Seq_F GTTAGACCGCTTCAGTGACCTG

18 T7 universal primer TAATACGACTCACTATAGGG

19 SP6 universal

primer

ATTTAGGTGACACTATAG

20 slr0168-SR_F4 GTTGTGACAGAATATCAAGATGCC

21 slr0168-SR_R4 GCTCTGGTTTCTAGGTCTTCTGC

22 inrS-RT_R GTCTAGGGCTTCCTTCAACTC

23 nrsD-RT_F CCCAAGCCATTAGTTTGCTGG

24 nrsD-RT_R TAAAAAAGGCGTTAAACACATTCAGC

25 nikM-RT_F TGTCTGCGGCAAATTAATCG

26 nikM-RT_R GGCGCAGTTGAAAAATATGATAGG

27 rps1-RT_F CTCTGATTGACATTGGGGCG

28 rps1-RT_R GAGCGCTGATGTGGGAGCCG

29 inrS amplification_F GGGCATATGACTTCCCAACCCGTACCCC

30 inrS amplification_R GGGGGATCCCTATAAAAACCGGTCTAGGGC


6

αB1

αA1

NA

αA2

CA

αA3

CBαB

3

NB

αB2

a

InrS H21/

RcnR H3 InrS C53 InrS H78 C82b

InrS 1 MTSQPVPHPSARHSHAHPHVHSQESLQKLVNRLSRIEGHIRGVKTMVQENRPCPEVLIQVAAVRGALDRVARLILDDHMNECITRAAAEGNIEQELAELKEALDRFL---- 107

EcRcnR 1 ------------------MSHTIRDKQKLKARASKIQGQVVALKKMLDEPHECAAVLQQIAAIRGAVNGLMREVIKGHLTEHIVHQGDELKREEDLDVVLKVLDSYIK--- 90

B1WW97 25 TQAKIEPTHNHNHGASHKHVHSEASLKQIINRLSRIEGHIRGVKTMVSESRPCPEVLIQLAAVRGAIDRVSRLILDEHLNECVARAAQEGNIEEELQELKAALDRFLP--- 132

B0JIQ2 10 PLHNHDETGHDHESKGTPHVHSEASLKQIINRLSRIEGHIRGIKTMVSENRPCPEVLMQIAAIRGAIDRVARMILDEHLSECIARAAQEGSIETEIEELKAALDRFLP--- 117

B7KLC9 20 SHQGTHSHPHDSTGTPHAHVHTEASLRQIINRLSRIEGHIRGIKTMVQESRPCPEVLVQIAAVRGAIDRVARIILDEHLSECIARAAEEGNIDEEIEQLKAALDRFLP--- 127

B2J3G0 26 TEHEHTDHTHGTGEPAHPHVHSEESLRRIANRLSRIEGHVRGIKTMVQQNSPCPDVLLQIAAVRGALDKVARIVLDEHLTECIARAAHEGNIDVEIKELKAALDRFLP--- 133

Q10YR8 15 TLEKDIISEDHHHSSAHPHVHSEESLKRLINRLSRIEGHVRGIKTMIKDHRACPDVLIQIAAVRGALDRVARIVLDNHLSDCIVRANQDGDIDVEIAALKSALDRFLP--- 122

Q8YTG1 26 AEHDHKHHTHGQAESVHAHVHSEESLRRIVNRLSRIEGHIRGIKTMVQQNSPCPDVLLQIAAVRGALDKVARIVLDEHLTECIARASKEGNIEEEIKQLKAALDRFLP--- 133

Q3M516 26 AEHDHKHHTHGQAESVHAHVHSEESLRRIVNRLSRIEGHIRGIKTMVQQNSPCPDVLLQIAAVRGALDKVARIVLDEHLTECIARASKEGNIAEEIEQLKAALDRFLP--- 133

B7JUF2 19 SLEDTHIENHHHHHGGHPHIHSEESLKKITNRLSRIEGHIRGVKTMVTENRACPEVLVQLAAVRGAIDRVSRLILDEHLSECIARAAKEGNIDAEIEELKAALDRFLP--- 126

Q8DIK8 1 -MSTESHSHFSPSVHSHPHHHSEESLRAIVNRLSRIEGHVRGIKTMVQDSRPCPEVLIQIAAVRGALDRVARLILDEHLDECVTRAAQEGRIDQELAELKAALDHFLG--- 107

D5A592 15 SDRHHGDASNHSHSHPHPHVHSDESLRRIINRLSRIEGHIRGVKNMVQESRPCPDVLVQIAAVRGALDRVARIILDEHLTECIARAAKEGNIDVEIEELKAALDRFLP--- 122

B8HSQ0 16 PLGAPSSLAAHSHPHAHPHVHSEESLRAIVNRLSRIEGHIRGIKTMVQESRACPDVLVQIAAVRGALDRVARMILDAHLTECIARAAEAGNIEAEIEELKTALDRFLN--- 123

Q2JP51 24 EEWELALEKPPLSGRAQPHHHDPQSRRKLTNRLARIEGHVRGIRSMIEQDQPCPDVLLQIAAVKGALDRVARLILDDHIRHCIRHAIETGNIEVELEELQRALDRFIS--- 131

Q2JSG2 58 EEWELALERPPLSGRAQPHHHDPQSRRKLIHRLARIEGHVRGIRSMIEQDQPCPDVLLQIAAVKGALDRVARLILDDHIRHCIRHAIESGNIEVELEELQRALDRYIS--- 165

Q7NPK7 1 -MASEVRPSLPTSPTGTTHTHDPVHTKVLLDRLARIEGHVRGIGNMVREDRPCPDVLVQISAVRAALNQVAKLVLKEHLSDCVVHAVENGGAHEEIQALNQAIDRYID--- 107

α1 α2 α3

Supplementary Figure 1│ Structure and sequence analysis of InrS. a. Ribbon

representation of the InrS tetramer, with analogy to Cu(I)-sensing CsoR2-5. Superscript

letters reflect chain and subscript numbers reflect sequential α-helices starting from the

amino-terminus (N), C = C-terminus. An expanded view of the region in the red box is shown

in figure 1. The structure reveals a tetramer assembled from sub-units composed of three

helix bundles. b. InrS and E. coli RcnR aligned with cyanobacterial InrS homologues

deduced to detect Ni(II). Residues previously deduced to bind Ni(II) comprise C53 (yellow),

H78 and C82 (gray)1. InrS H21 aligns with His residues in the homologues (red). There are

also multiple histidine residues (green) N-terminal to H21. Secondary structure elements

shown below the alignment were taken from the structure of InrS reported here (5FMN), with

the dashed line of α1 indicating a longer helix in chain A relative to B in the determined

structure.


7

0 10 20 30 40 50[Ni(II)] (μM)

0

0.05

0.1

0.15

0.2

A3

33

nm

c

0

5

10

15

20

0 5 10 15 20[Ni(II)] (μM)

ε(

10

3M

-1 c

m-1

)

d

a

300 400 500 600 700Wavelength (nm)

0

5

10

20

15

ε(

10

3M

-1 c

m-1

)

01 2

[Ni(II)]/[H21L]0

5

10

15

20

ε(

10

3M

-1 c

m-1

) b

300 400 500 600 700Wavelength (nm)

0

5

10

20

15

ε(

10

3M

-1 c

m-1

)

1 2[Ni(II)]/[H21E]

0

5

10

15

20

ε(

10

3M

-1 c

m-1

)

0

Supplementary Figure 2│ Ni(II)-binding properties of purified InrS variants. a. Apo-

subtracted spectra of H21L (10 μM protomer) titrated with NiCl2. Inset, binding isotherm at

333 nm. b. As ‘a’ with H21E. c. Representative (n = 3) Ni(II)-InrS absorbance upon titration

of EGTA (1 mM) and InrS (10 μM protomer) with NiCl2. Solid black line is a fit to a model

describing competition from InrS for one molar equivalent of Ni(II), KNi(II) = 2.8(±0.4) 10–12

M. Dashed lines represent simulated curves with KNi(II) 10-fold tighter and weaker than the

fitted value. d. InrS (10.4 μM protomer) Ni(II)-binding isotherm at 333 nm 6, to compare a

simulated fit using KNi(II) determined in this work (solid line) and a simulated fit using weaker

KNi(II) of 7 10–8 M and 4.5 10–6 M for the first and second pairs of Ni(II)-binding sites per

tetramer respectively (dashed line), suggested in other studies7. The value determined in

this work fits the data.


8

0

0.2

0.4

0.6

0.8

-14 -12[Ni(II)]buffered (log10M)

DN

A o

ccupancy

-8-10 -6 -4

H78L

InrS

H21L

H21E

0

0.02

0.04

0.06

0.08

-14 -12

[Ni(II)]buffered (log10M)

DN

A o

ccupancy

-8-10 -6 -4

InrS

H21L

H21E

Response

to [Ni(II)]

observed

in vivo

nrsDPro

nikPro

Supplementary Figure 3│ InrS DNA-binding as a function of Ni(II) concentration.

Deduced fractional occupancy of the nrsD (upper) and nik (lower) promoter in the indicated

strains, as a function of buffered [Ni(II)]. From these plots it is possible to define set points on

the nrsD promoter of 3 10–10, 2 10–9, 3 10–9 and 7 10–4 M for InrS, H21E, H21L and

H78L, respectively, using 95% clearance of InrS from nrsD as a standard. The equivalent set

points for InrS, H21E and H21L on the nik promoter are 4 10–12, 1.5 10–11 and 6 10–11

M, respectively. The set points for which regulation is observed in vivo (indicated by colored

arrows), for InrS on the nrsD promoter (Fig. 3c), H21E and H21L on the nik promoter (Fig.

4d) range from 1.5 10–11 to 3 10–10 M.


9

32.52

a

+RT

nrsD

rps1

rps1-RT

c

InrS

H2

1L

H2

1E

H7

8L

ΔΔwt

b InrS H21L H21E H78La b

Co

ntr

ol

a b a b a b a b a b a b a b

Contr

ol

Co

ntr

ol

Co

ntr

ol

kb

f

rps1

rps1

+RT

-RT

InrS

H2

1L

H2

1E

H7

8L

rps1

rps1

rps1

rps1

+RT

-RT

+RT

-RT

1 μM 3 μM 5 μM 10 μM 15 μM

wtΔPnrsΔinrS

ΔΔ

d

InrS

H2

1L

H2

1E

H7

8L

i

rps1

rps1

j

wt

Δin

rS

+RT

-RT

e [Ni(II)] (μM)

rps1

rps1

+RT

-RT

0.1

7

0.0

8

0 0.1

7

0.0

8

0 0.1

7

0.0

8

0 0.1

7

0.0

8

0

H78LInrS H21L H21E

g

h

Supplementary Figure 4│ Generation of strains expressing InrS variants and RT-PCR

controls. a. Deletion of the nrs promoter1,8 in the ΔinrS strain6 by insertion of the Km

cassette with segregation to all chromosomal copies confirmed by PCR (integration gives a

2.8 kb product, unaltered gives 1.9 kb) (full gel shown in supplementary figure 19). b.

Insertion of the inrS variant plasmids in the slr0168 region of strain ΔinrS ΔPnrs, with

segregation to all chromosomal copies confirmed by PCR. Oligonucleotide pair a

(oligonucleotides 20 and 21, Supplementary Table 4) only amplifies from Synechocystis

wild type genomic DNA (control), generating a diagnostic 1.8 kb fragment, while

oligonucleotide pair b (oligonucleotides 20 and 22) only amplifies in mutants to generate a

diagnostic 1.55 kb fragment (full gels shown in supplementary figure 20). c. nrsD transcript

abundance (by RT-PCR) in wild type Synechocystis (wt), Δinrs ΔPnrs (ΔΔ), InrS, H21L,

H21E and H78L strains cultured in 0.17 µM Ni(II) plus rps1 loading controls (RT = reverse

transcriptase). Integration of inrS genes, in all cases, results in partial or full repression of

nrsD relative to the double mutant host (ΔΔ) (full gel shown in supplementary figure 21). d.

Ni(II)-sensitivity of strains on standard BG11 media plus the indicated [NiSO4]. e. rps1

loading controls for nrsD expression data in figure 3c and variant nikM expression in figure

4d (full gels shown in supplementary figure 22). f. rps1 loading controls for nrsD expression

data in figure 3d, [Ni] = (0.42 μM) (full gel shown in supplementary figure 6). g. as ‘f’ [Ni] =

(0.67 μM) (full gel shown in supplementary figure 7). h. as ‘ f’ [Ni] = (1.17 μM) (full gels

shown in supplementary figure 23). i. rps1 loading controls for nikM expression data in figure

4a (upper) (full gel shown in supplementary figure 24). j. rps1 loading controls for nikM

expression data in figure 4a (lower) (full gels shown in supplementary figure 11 and 25).


10

3c.

3d. 0.42 μM Ni(II)S4f. +RT

S4f. -RT

Supplementary Figure 5│ Full gel image for data shown in figure 3c.

Supplementary Figure 6│ Full gel image for data shown in figure 3d (0.42 μM Ni(II)) and

supplementary figure 4f.


11

3d. 1.17 μM Ni(II)

3d. 0.67 μM Ni(II)S4g. +RT

S4g. -RT

Supplementary Figure 7│ Full gel image for data shown in figure 3d (0.67 μM Ni(II)) and

supplementary figure 4g.

Supplementary Figure 8│ Full gel image for data shown in figure 3d (1.17 μM Ni(II)).


12

H21L

H78L 223 TTGGATGACCTGATG----------AATGAGTGCATCACCAGGGCGGCGGCGGAAGGCAA 272

H78L* TTGGATGACCTGATGGTCGATATTTAATGAGTGCATCACCAGGGCGGCGGCGGAAGGCAA

*************** ***********************************

Leu Asp Asp Leu Met Asn Glu Cys Ile Thr Arg Ala Ala Ala Glu Gly

Leu Asp Asp Leu Met Val Asp Ile StopStop

H78L

H78L*

nrsD

rps1

rps1

+RT

-RT

a

InrS

H21E

ΔΔwt

b0.42 μM Ni(II)

H78L*

Supplementary Figure 9│ After prolonged culture a strain expressing the H78L variant

acquired a mutation predicted to alleviate nickel toxicity. a. nrsD transcript abundance

along with rps1 loading controls in the indicated strains (wild type Synechocystis (wt), Δinrs

ΔPnrs (ΔΔ)) following continuous culture for ~6 months in standard medium (which contains

0.17 μM NiSO4) (full gel shown in supplementary figure 26). For the experiment, cells were

cultured in standard medium further supplemented with 0.25 μM NiSO4 (48 h). The inrS gene

was amplified from H78L* using oligonucleotide pair 29 and 30 (Supplementary Table 4),

and cloned into pGEM-T easy. Plasmid DNA was prepared from independent colonies and

sequenced. b. Alignment of H78L coding sequence with sequence retrieved from three

sequenced clones of H78L* showing two inserted stop codons.


13

4a. Upper

4a. Lower S4j. -RT

Supplementary Figure 10│ Full gel image for data shown in figure 4a (upper panel).

Supplementary Figure 11│ Full gel image for data shown in figure 4a (lower panel) and

supplementary figure 4j (-RT).


14

[H21E] (nM)10 100 1,000 10,000

0

10

20

30

apo

Ni(II)

nikPro

Δr o

bs (10

-3)

Supplementary Figure 12│ H21E binding to the nik-promoter region. Anisotropy change

on titration of nikProFA (the identified InrS binding site plus 7 flanking base pairs) (10 nM)

with H21E in 5 mM EDTA (open symbols) or Ni(II)-bound forms (closed symbols). Symbol

shapes represent individual experiments (Ni(II) n = 3, apo n=4). Simulated curves generated

as in Fig. 2d–f.


15

4d. H21L

4d. H21E

.

Supplementary Figure 13│ Full gel image for data shown in figure 4d.


16

0 1 2 3 4 5 60.01

0.1

1

t (ns)

Inte

nsity

(a.u

.)

0 1 2 3 4 5 60.01

0.1

1

t (ns)In

tensity

(a.u

.)

a b

InrS H78L

c

H78LInrS

0.67 μM Ni(II)

140d

0

20

40

60

80

100

120

Flu

ore

scence

0 0.17 0.67[Ni(II)] (μM)

Supplementary Figure 14│ Detection of cellular Ni(II) by Newport Green fluorescence.

a. Fluorescence decay of lnrS cells cultured in standard (black) or standard plus 0.5 μM

Ni(II) (red) media, after addition of Newport Green. With 0.5 μM Ni(II) supplementation the fit

(red line) gives a single exponential decay with a lifetime of 2.4 ± 0.1 ns. For lnrS cells

cultured in standard medium, the fit (black line) reveals a bi-exponential decay with lifetimes

0.2 ± 0.1 ns (30% yield) and 2.2 ± 0.1 ns (70% yield) (error in lifetime yields < ±5% from

replicates). The initial fast decay is generated by apo-fluorophore. b. As ‘a’ for H78L cells.

The fit with Ni(II) gives a single exponential decay with a lifetime of 2.4 ± 0.1 ns while in the

absence of extra Ni(II) there is a bi-exponential decay with lifetimes 0.2 ± 0.1 ns (20% yield)

and 2.5 ± 0.1 ns (80% yield). The reduced yield of the fast fluorescence decay phase of

Newport Green in H78L cells grown in standard medium relative to InrS cells (initial fast-

phases for black lines in ‘b’ versus ‘a’) implies a (slightly) larger exchangeable Ni(II)-content

in H78L cells. The presence of residual fast decay for both strains cultured in standard

medium implies that Newport Green is not metal saturated, while the absence of fast decay

with further Ni(II) supplementation suggests that the assay may approach its upper limit in

elevated Ni(II). c. Fluorescence of cells, previously cultured in standard medium with 0.5 μM

NiSO4, upon addition of Newport Green (5 μm scale bar). d. Mean (of 10-15 technical

replicates with error bars representing one standard deviation) InrS (black bars) and H78L

(gray bars) relative cell fluorescence 1 h after addition of Newport Green.


17

hox- / -

0 2 4 6 80

60

120

180

H78L / -

InrS / -

InrS / +

H78L / +

hox- / +

t (min)

Curr

ent

(nA

)

a Hydrogenase activity

(nmol H2 mg-1 min-1)

b Strain/

Ni(II) supplement

InrS / -

InrS / +

H78L / -

H78L / +

hox- / -

hox- / +

10.0 ( 0.8)

9.3 ( 0.8)

11.3 ( 1.5)

10.2 ( 1.5)

0.0 ( 0.0)

0.0 ( 0.1)

Supplementary Figure 15│ Hydrogenase activity in InrS, H78L and hox– strains. a.

Representative (n = 3 biological replicates) hydrogenase assays showing current generated

using InrS, H78L and hox– strains (deficient in hydrogenase). Cells had previously been

cultured in standard (+) or minimal (–) media containing 0.17 M Ni(II) or no added Ni(II),

respectively. b. Mean (of triplicate biological replicates with ± one standard deviation)

hydrogenase activities of strains InrS, H78L, and hox– control.


18

0 2 4 6 8 10 12 14[Ni] (μM)

a

0

5

10

15

20

ε 333

nm

(1

03

M-1

cm

-1)

0 60t (min)

equilibrium

0

20

ε 333

nm

(x1

03

M-1

cm

-1) [Ni] = 7.92 μM

10

Cyto

so

lic -

His

Cyto

so

lic +

1 m

M A

TP

Cyto

so

lic -

GS

H

0

5

10

15

20

ε 333

nm

(1

03

M-1

cm

-1)

[Ni] = 7.92 μM

equilibrium

b

Supplementary Figure 16│ Control assays for Ni(II)-binding to InrS and H21L in

competition with metabolites at cellular concentrations. a. As figure 5b at equilibrium.

Inset, the kinetics of Ni(II) binding to H21L upon addition of H21L (10 μM protomer) to

cytosol-matched buffer containing 7.92 μM NiCl2 (all values are means of triplicate analyses

with error bars showing ± one standard deviation). b. As figure 5c at equilibrium for

conditions not shown in figure 5d (all values are means of triplicate analyses with error bars

showing one standard deviation).


19

Km

eta

l(log

10M

)

[M]buffered (log10M)

Observed [Ni(II)]bufferedCueR

Zn30

[Ar]3d104s2

Mg12

[Ne]2s2

Mn25

[Ar]3d54s2

26

Fe[Ar]3d64s2

28

Fur

MntR

[Ar]3d84s2

Ni

Cu29

[Ar]3d104s1

-15 -10 -5

-20

-15

-10

-5

CsoR

CsoR

ZurZntR

Zur

ZurZiaR

CzrAInrS

NikR

Fur

Mg

Riboswitch

Zur

Observ

ed t

uneable

range

of

InrS

KN

i(II

) on n

rsD

Supplementary Figure 17│ Relationship between buffered [metal] and the affinities of

metal-sensors, showing the tuneable ranges for InrS. Estimates of available metal

concentrations for magnesium, manganese, iron, nickel, copper, and zinc in bacterial cells9-

15 (plotted as log10) against KMetal of cytoplasmic sensors for their cognate metal from E.

coli (black)16-19, Bacillus subtilis (blue)20-23, Synechocystis PCC 6803 (green)1 and

Staphylococcus aureus (red)24,25 (also plotted as log10). Line represents linear regression fit

of the data. Cobalt is not included26,27. Zur in B. subtilis has known allosterically effective

sites of different affinities giving two set points20. Observed tuneable ranges for InrS KNi(II)

and [Ni(II)]buffered in culture (green bars) are estimated from the promoters-sensors that

respond to Ni(II). The estimated InrS KNi(II) and [Ni(II)]buffered ranges are not identical because

other parameters such as KDNA also contribute to the set point for sensing as illustrated in

supplementary figure 3. The green dash on the x-axis represents the calculated set point of

InrS on the nikM promoter (Supplementary Fig. 3).


20

inrS ---ATGACTTCCCAACCCGTACCCCATCCTTCTGCCCGCCATAGTCACGCCCATCCCCAT

InrSplasmid CATATGACTTCCCAACCCGTACCCCATCCTTCTGCCCGCCATAGTCACGCCCATCCCCAT

H21Lplasmid CATATGACTTCCCAACCCGTACCCCATCCTTCTGCCCGCCATAGTCACGCCCATCCCCAT

H21Eplasmid CATATGACTTCCCAACCCGTACCCCATCCTTCTGCCCGCCATAGTCACGCCCATCCCCAT

H78Lplasmid CATATGACTTCCCAACCCGTACCCCATCCTTCTGCCCGCCATAGTCACGCCCATCCCCAT

inrS GTCCACAGCCAAGAATCCTTACAAAAGTTAGTTAATCGCCTCTCCCGCATTGAAGGCCAT

InrSplasmid GTCCACAGCCAAGAATCCTTACAAAAGTTAGTTAATCGCCTCTCCCGCATTGAAGGCCAT

H21Lplasmid GTCCTGAGCCAAGAATCCTTACAAAAGTTAGTTAATCGCCTCTCCCGCATTGAAGGCCAT

H21Eplasmid GTCGAGAGCCAAGAATCCTTACAAAAGTTAGTTAATCGCCTCTCCCGCATTGAAGGCCAT

H78Lplasmid GTCCACAGCCAAGAATCCTTACAAAAGTTAGTTAATCGCCTCTCCCGCATTGAAGGCCAT

inrS ATTCGGGGAGTGAAAACCATGGTGCAGGAAAATCGTCCCTGCCCAGAGGTGTTAATTCAG

InrSplasmid ATTCGGGGAGTGAAAACCATGGTGCAGGAAAATCGTCCCTGCCCAGAGGTGTTAATTCAG

H21Lplasmid ATTCGGGGAGTGAAAACCATGGTGCAGGAAAATCGTCCCTGCCCAGAGGTGTTAATTCAG

H21Eplasmid ATTCGGGGAGTGAAAACCATGGTGCAGGAAAATCGTCCCTGCCCAGAGGTGTTAATTCAG

H78Lplasmid ATTCGGGGAGTGAAAACCATGGTGCAGGAAAATCGTCCCTGCCCAGAGGTGTTAATTCAG

inrS GTGGCGGCAGTCCGGGGGGCATTAGACCGGGTGGCTAGATTAATTTTGGATGACCACATG

InrSplasmid GTGGCGGCAGTCCGGGGGGCATTAGACCGGGTGGCTAGATTAATTTTGGATGACCACATG

H21Lplasmid GTGGCGGCAGTCCGGGGGGCATTAGACCGGGTGGCTAGATTAATTTTGGATGACCACATG

H21Eplasmid GTGGCGGCAGTCCGGGGGGCATTAGACCGGGTGGCTAGATTAATTTTGGATGACCACATG

H78Lplasmid GTGGCGGCAGTCCGGGGGGCATTAGACCGGGTGGCTAGATTAATTTTGGATGACCTGATG

inrS AATGAGTGCATCACCAGGGCGGCGGCGGAAGGCAATATTGAGCAGGAGTTGGCGGAGTTG

InrSplasmid AATGAGTGCATCACCAGGGCGGCGGCGGAAGGCAATATTGAGCAGGAGTTGGCGGAGTTG

H21Lplasmid AATGAGTGCATCACCAGGGCGGCGGCGGAAGGCAATATTGAGCAGGAGTTGGCGGAGTTG

H21Eplasmid AATGAGTGCATCACCAGGGCGGCGGCGGAAGGCAATATTGAGCAGGAGTTGGCGGAGTTG

H78Lplasmid AATGAGTGCATCACCAGGGCGGCGGCGGAAGGCAATATTGAGCAGGAGTTGGCGGAGTTG

inrS AAGGAAGCCCTAGACCGGTTTTTATAG---

InrSplasmid AAGGAAGCCCTAGACCGGTTTTTATAGGGA

H21Lplasmid AAGGAAGCCCTAGACCGGTTTTTATAGGGA

H21Eplasmid AAGGAAGCCCTAGACCGGTTTTTATAGGGA

H78Lplasmid AAGGAAGCCCTAGACCGGTTTTTATAGGGA

a

b

10

15

2025

37

50

75100

150

250

kDa

InrS

H21L

H21E

H78L

Supplementary Figure 18│ Sequencing of InrS variant overexpression plasmids and

purity of proteins. a. Alignment of sequenced InrS overexpression plasmids with inrS

nucleotide sequence. The start and stop codons are highlighted in yellow and blue

respectively with 3 flanking nucleotides from the plasmid vector shown. Introduced mutations

green. b. Analysis of purity of representative samples of InrS variants. Purified proteins were

resolved on a 4–20 % Mini-PROTEAN TGX gradient SDS-PAGE gel (BioRad), molecular

weights of markers are noted to the left. H21L, H21E and H78L co-migrate with InrS, the

identity of which has been confirmed by mass spectrometry analysis6.


21

S4a.

32.5

2

kb

S4b. InrS

Control

S4b. InrS

Mutant

2.52

1.5

kb

2.52

1.5

kb

S4b. H21L

Control

S4b. H21L

Mutant

2.52

1.5

kb

S4b. H21E

Control

S4b. H21E

Mutant

2.52

1.5

kb

S4b. H78L

Control

S4b. H78L

Mutant

a b

c d

Supplementary Figure 19│ Full gel image for data shown in supplementary figure 4a.

Supplementary Figure 20│ Full gel image for data shown in supplementary figure 4b for a.

InrS, b. H21L, c. H21E and d. H78L.


22

S4c. nrsD +RTS4c. rps1 +RT

S4c. rps1 -RT

S4e. InrS

rps1 +RT

S4e. H21L

rps1 +RT

S4e. H21E

rps1 +RT

S4e. H78L

rps1 +RT

S4e. InrS

rps1 -RT

S4e. H21L

rps1 -RT

S4e. H21E

rps1 -RT

S4e. H78L

rps1 -RT

a b

Supplementary Figure 21│ Full gel image for data shown in supplementary figure 4c.

Supplementary Figure 22│ Full gel image for data shown in supplementary figure 4e for a.

rps1 +RT and b. rps1 –RT.


23

S4h. +RT S4h. -RT

a b

S4i. +RT S4i. -RT

Supplementary Figure 23│ Full gel image for data shown in supplementary figure 4h for a.

rps1 +RT and b. rps1 –RT.

Supplementary Figure 24│ Full gel image for data shown in supplementary figure 4i.


24

S4j. +RT

S9a. nrsD +RTS9a. rps1 +RT

S9a. rps1 -RT

Supplementary Figure 25│ Full gel image for data shown in supplementary figure 4j (rps1

+RT).

Supplementary Figure 26│ Full gel image for data shown in supplementary figure 9a.


25

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