Regulation of a nickel-cobalt efflux system and nickel homeostasis in a soil actinobacterium Streptomyces coelicolor.

View Article Online View Journal

Metallomics Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: H. M. Kim, B. Ahn, J. Lee and J. Roe, Metallomics, 2015, DOI: 10.1039/C4MT00318G.

This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.

www.rsc.org/metallomics

Page 1 of 36

Metallomics View Article Online

DOI: 10.1039/C4MT00318G

1 2 3 4

Regulation of a nickel/cobalt efflux system and nickel

5

homeostasis in a soil actinobacterium Streptomyces

6

coelicolor

7 8

Hae Mi Kim, Bo-Eun Ahn‡, Ju-Hyung Lee, Jung-Hye Roe*

9 10 11 12 13 14 15 16 17 18 19 20 21 1


Published on 11 February 2015. Downloaded by Mahidol University on 13/02/2015 23:14:45.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Metallomics

Page 2 of 36 View Article Online

DOI: 10.1039/C4MT00318G

22 23

Table of contents entry

24

25 26 27

In nickel-tolerant Streptomyces coelicolor, highly nickel-sensitive regulator (Nur) for

28

nickel uptake systems and extremely insensitive regulator (NmtR) for nickel efflux

29

pump constitute nickel homeostasis system.

30 31 32 33 34 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 3 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

35

Abstract

36

In Streptomyces coelicolor, a soil actinobacterium capable of morphological

37

differentiation and complex secondary metabolism, nickel deficiency is sensed by

38

Nur, a Ni-specific Fur family regulator that controls nickel uptake systems

39

(NikABCDE and NikMNOQ) and both Fe-containing and Ni-containing superoxide

40

dismutases (SodF and SodN). On the other hand, nickel efflux system and its

41

regulator has not been elucidated. In this study, we demonstrate that an ArsR/SmtB

42

family metalloregulator NmtR, a close homologue of NmtR from Mycobacterium

43

tuberculosis, controls a putative efflux pump of P1-type ATPase (NmtA) in S.

44

coelicolor. NmtR binds to the nmtA promoter region to repress its transcription, and

45

is dissociated in the presence of Ni(II) and Co(II). Disruption of the nmtA gene makes

46

cells more sensitive to nickel and cobalt, consistent with its predicted role to encode

47

a Ni/Co-efflux pump. Growth of S. coelicolor in complex YEME medium is only

48

marginally inhibited by up to 0.5 mM Ni(II), with significant growth retardation at 1

49

mM. Nur-regulated sodF and nikA genes are repressed at less than 0.1 µM added

50

NiSO4 whereas NmtR-regulated nmtA transcription is induced at 0.5 mM or more

51

Ni(II). This reveals the extreme sensitivity of S. coelicolor to nickel deficiency as well

52

as tolerance to surplus nickel. How this organism and possibly other actinomycetes

53

have evolved to develop such a highly Ni-tolerant physiology and how the highly

54

sensitive regulator Nur and obtuse regulator NmtR achieve their characteristic Ni-

55

sensitivity are interesting questions to solve in the future.

3


DOI: 10.1039/C4MT00318G

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

56

Introduction

57

Nickel is used as a cofactor for several microbial enzymes such as urease, [NiFe]

58

hydrogenase, superoxide dismutase, carbon monoxide dehydrogenase, acetyl-

59

coenzyme A decarbonylase/synthase, aci-reductone dioxygenase, methyl-coenzyme

60

M reductase, and glyoxalase

61

bacteria, primarily due to its high affinity to ligands, replacing the essential metal of

62

metalloproteins or binding to residues of non-metalloenzymes, in addition to its ability

63

to cause oxidative stress 5. Nickel uptake, intracellular binding/trafficking, and efflux,

64

are tightly controlled to achieve homeostasis.

1-4

. At high concentrations, however, it is toxic to

65

How nickel uptake and efflux are coordinately regulated in a single organism

66

has been best studied in Escherichia coli. It expresses an ABC-transporter system

67

(NikABCDE) for nickel uptake under anaerobic conditions for the synthesis of [NiFe]

68

hydrogenases

69

the absence of oxygen and negatively regulated by NikR in the presence of nickel 8, 9.

70

Excess nickel is pumped out of the cytoplasm by an efflux pump RcnA

71

gene expression is repressed by the Ni(II)/Co(II)-responsive repressor RcnR under

72

low nickel condition, and is de-repressed when Ni(II) or Co(II) binds RcnR

73

rcnRA expresssion is also regulated by Fur to prevent induction of rcnA by iron

74

E.coli, nickel uptake was shown to be interlinked with efflux system at micromolar

75

concentration range of added nickel

76

type (NixA) and ABC transporter type (AbcABCD) uptake systems are present,

77

regulation of the nixA expression by repressor NikR has been reported

78

However, regulation of a nickel efflux system, an RND-type transporter encoded by

6, 7

. The nikABCDE gene expression is positively regulated by FNR in

12

10

. The rcnA

11, 12

. The 13

. In

. In Helicobacter pylori, where both Ni-CoT

4

14-19

.


DOI: 10.1039/C4MT00318G

Page 5 of 36


DOI: 10.1039/C4MT00318G

20

79

the cznABC is not well characterized

80

Ni/Co efflux pumps of P1 type ATPase (NmtA) and of cation diffusion-facilitator

81

family (Cdf) are regulated by NmtR and KmtR, respectively

82

KmtR are Ni(II)/Co(II)-responsive repressor of ArsR-SmtB family 21. Similar to RcnR,

83

they bind to the promoter/operator region of their target genes, whose expression is

84

de-repressed when metal levels increase. The genome of M. tuberculosis contains a

85

NiCoT gene encoding a putative uptake permease, whose function needs be verified.

. For Mycobacterium tuberculosis, putative

21-25

. Both NmtR and

86

In actinobacteria, nickel-binding proteins and enzymes have been studied

87

mostly in mycobacteria and streptomycetes. Nickel-containing superoxide dismutase

88

(SodN) is widely distributed among streptomycetes

89

that degrades a flavol quertin (QueD) were reported in a Streptomyces sp.

90

hydrogenases that oxidize H2 were reported in soil-dwelling Streptomyces spp. and

91

M. smegmatis

92

central metabolism by providing a nitrogen source when urea is readily available

93

How nickel uptake is regulated and coordinated with a nickel-containing enzyme has

94

been best studied in S. coelicolor, a model organism for antibiotic production and

95

morphological differentiation.

31, 32

26-29

. Ni-containing dioxygenase 30

. [NiFe]

. In M. tuberculosis, urease activity was reported to play a role in 33

.

96

In S. coelicolor, genes for high affinity nickel-uptake systems (nikABCDE and

97

nikMNQO) as well as those for Fe-containing (sodF) and nickel-containing (sodN)

98

superoxide dismutases are regulated by a Fur-family repressor Nur

99

nickel specifically and then gains the activity to bind to operator sites of its target

100

genes; nikA, nikM, and sodF. The sodN gene is indirectly regulated by Nur via sodF,

101

whose transcript is processed to produce a small regulatory RNA (s-SodF) that 5

34-37

. Nur binds



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Metallomics


DOI: 10.1039/C4MT00318G

37

102

contains an inhibitory anti-sodN sequence

103

antagonistically regulated by Nur in opposite ways. In contrast to the wealth of

104

information on Nur-regulated nickel-uptake/utilization systems, no information on

105

nickel-efflux system and its regulation is available. In this study, we demonstrate a

106

nickel efflux system that confers nickel tolerance and its regulator in S. coelicolor.

107

This provides novel information on how nickel homeostasis is achieved in a soil-

108

dwelling bacterium that is very tolerant to nickel.

. In this way both sodN and sodF is

109

110

Results and discussion

111

S. coelicolor has a nickel efflux system homologous to NmtRA of M.

112

tuberculosis.

113

Inspection of S. coelicolor genome reveals the presence of 16 ArsR-type

114

transcriptional regulators (StrepDB; http://strepdb.streptomyces.org.uk). Among

115

these, SCO6459 shares the highest sequence homology with the nmtR (Rv3744)

116

from M. tuberculosis (Mtb), whereas SCO1309 shares high similarity to kmtR

117

(Rv0827c). SCO6459 also shares the gene synteny, having a divergent gene

118

SCO6460 that encodes a putative P1-type ATPase, homologous to nmtA (Rv3743)

119

of Mtb (Fig. 1). SCO6459 and SCO6460 proteins share 63% and 62% identical

120

amino acids with MtbNmtR and MtbNmtA, respectively. The amino acid residues

121

proposed to contribute to metal selectivity (N-terminal Gly2, His3, and Glu91, His93,

122

His104, and His107 in MtbNmtR) are all conserved in SCO6459

123

sequence similarity and the functions revealed as below, we named these genes as 6

38

. Based on



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 7 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

124

nmtR and nmtA in S. coelicolor. The nmtRA genes in S. coelicolor are flanked by

125

largely uncharacterized genes encoding a hypothetical protein (SCO6458), possible

126

Ada-like regulatory protein (6461), and a putative cysteine methyltransferase (6462).

127

The gene synteny flanking the nmtRA is not conserved in other actinomycetes where

128

close homologs of nmtRA are present.

129

Previously proposed binding consensus for SmtB/ArsR regulators with 39

130

imperfect 12-2-12 inverted repeats

131

region between the divergent nmtR and nmtA genes. The transcription start site of

132

nmtR was detected in a genome-wide RNA sequencing analysis to coincide with the

133

first A residue of the initiation codon (data not shown; Fig. 1A). The 5’ end of the

134

nmtA transcript was hard to estimate, most likely due to highly stable GC-rich

135

secondary structure. High resolution S1 mapping revealed that the 5’ ends of nmtA

136

mRNA most likely reside within the 5 nucleotides (CCGCG) that includes the first G

137

residue in the initiation codon (GTG; Fig. 1A, data not shown). Prediction of putative

138

promoter elements (-10 and -35 regions) for nmtR and nmtA genes revealed that the

139

palindromic putative NmtR-binding site overlaps entirely with the -35 box and the

140

spacer of the nmtA promoter, and to a lesser extent with the nmtR promoter. This

141

sequence topology suggests that binding of NmtR to the predicted motif will repress

142

nmtA expression, and possibly its own expression as well.

was found within the 69 nt-long intergenic

143

144

NmtR is a repressor for nmtA expression.

145

To investigate the function of NmtRA system, we created ∆nmtR and ∆nmtA mutants. 7


DOI: 10.1039/C4MT00318G

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

146

First, we examined the expression profile of the nmtA gene in the wild type and

147

∆nmtR mutant by S1 mapping analysis. Results in Fig. 2 demonstrated that the nmtA

148

expression is induced when Ni(II) is added to YEME media at 0.5 mM or more for 40

149

min. In ∆nmtR mutant, the nmtA gene is constitutively expressed regardless of Ni(II)

150

addition. This clearly indicates that NmtR works as a repressor for nmtA expression.

151

The level of nmtR expression is relatively low, and increases only slightly at high

152

Ni(II).

153

We examined the expression profile of other Ni-sensitive genes, the sodF and

154

sodN. The amounts of the sodF mRNA and the small processed anti-sodN RNA from

155

sodF (s-SodF) were low in the wild type in the absence of added nickel, whereas the

156

sodN transcript was more abundant (Fig. 2, lane 1). This expression profile is typical

157

of the early exponential phase cells (OD600 ~0.3), from which RNA samples were

158

prepared

159

sodF and s-SodF RNA was elevated higher than in the wild type, whereas the sodN

160

RNA level was lowered (Fig. 2, lane 9). Since the ∆nmtR mutant grows as well as

161

the wild type in YEME, and we harvested both cells at the same growth time, the

162

elevated sodF gene expression (and decreased sodN expression) may reflect the

163

lower level of intracellular available Ni(II) in ∆nmtR than in the wild type grown in

164

YEME without added nickel. This observation is as expected if NmtA indeed

165

functions as a Ni-exporter, since NmtA is constitutively expressed in ∆nmtR. Addition

166

of more Ni(II) in the media decreased and increased the expression of sodF and

167

sodN genes, respectively, as expected from the fact that Nur gains its DNA-binding

168

activity in the presence of sufficient nickel.

37

. In ∆nmtR mutant in the absence of added nickel, however, the level of

8


DOI: 10.1039/C4MT00318G

Page 9 of 36


DOI: 10.1039/C4MT00318G

169

170

NmtA confers tolerance to nickel and cobalt in S. coelicolor.

171

If NmtA functions as a Ni-exporter, it will confer tolerance to high nickel. We

172

compared the viability of ∆nmtA and ∆nmtR mutants with the wild type on NA plates

173

containing 1 mM NiSO4. We also examined whether NmtA could function as a Co-

174

exporter as well. As demonstrated in Fig. 3, cell growth was very much retarded on

175

plates with 1 mM nickel or 0.5 mM cobalt, where colonies were visible after

176

incubation for 2 days. We found that ∆nmtA mutant was less tolerant to 1 mM Ni or

177

0.5 mM Co than the wild type and ∆nmtR mutant. This indicates that NmtA protein

178

indeed functions as a Ni/Co-exporter to protect S. coelicolor cells from nickel and

179

cobalt toxicity. The wild type and ∆nmtR mutant showed similar tolerance as

180

expected, since the level of nmtA gene expression is similar between the wild type

181

and ∆nmtR in the presence of 1 mM Ni(II) (Fig. 2). On NA plates, both the wild type

182

and ∆nmtR cells produced red antibiotic undecylprodigiosin, whereas ∆nmtA

183

produced almost no red antibiotic, suggesting that nickel accumulation hinders

184

secondary metabolism (data not shown).

185

186

Binding of NmtR to nmtA promoter/operator probe is inhibited by Ni(II) and

187

Co(II).

188

We examined the binding activity of NmtR to the promoter/operator region of the

189

nmtA DNA that contains the palindromic binding consensus for ArsR/SmtB family

190

regulators. EMSA assay demonstrated that the wild type cell extracts formed a 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

191

specific complex band, which is dissociated upon adding NiSO4, whereas ∆nmtR

192

extracts did not form any specific complex (Supplementary Fig. S1). To monitor the

193

metal-specificity of this bound complex, we examined the effect of various transition

194

metals by adding 0.1 mM each of FeSO4, ZnCl2, CoCl2, MnSO4, CuSO4, and NiSO4

195

to the wild type cell extracts before incubating with the DNA probe. Results in Fig. 4A

196

indicates that NmtR binding is specifically inhibited by Ni(II) and Co(II). We then

197

purified

198

promoter/operator DNA by EMSA. In the absence of added metal, the purified NmtR

199

bound to the labeled probes, upshifting half of them at less than 5 nM protein,

200

reflecting relatively tight binding (Supplementary Fig. S2). Upon adding NiSO4 from

201

0.05 to 1 mM, the complex dissociated gradually (Fig. 4B). Approximately half of the

202

labeled probe was dissociated free from the slower-moving complexes at around 0.1

203

mM NiSO4. Addition of CoCl2 also dissociated the complex, even though less

204

effectively than NiSO4. Half dissociation occurred by CoCl2 between 1.0 and 2.0 mM.

205

Based on these results, we were able to propose that SCO6459 (NmtR) protein is a

206

repressor that binds to the upstream region of the nmtA gene which encodes a Ni/Co

207

efflux pump. Its binding is inhibited specifically by Ni(II) and Co(II), leading to

208

induction of the nmtA transcription.

His-tagged

NmtR

protein

and

monitored

its

binding

to

nmtA

209

210

Induction of nmtA expression by Ni(II) and Co(II) at growth-inhibitory

211

concentrations.

212

We monitored Ni(II)-dependent gene expression in liquid culture. S. coelicolor cells 10


DOI: 10.1039/C4MT00318G

Page 11 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

213

were grown in liquid YEME media containing different amounts of NiSO4, and

214

measured for growth as well as expression of nickel-responsive genes. Results in

215

Fig. 5A demonstrate that up to 0.5 mM Ni(II) affected cell growth only marginally, with

216

similar exponential growth rates and cell mass accumulation. Ni(II) at 1 mM inhibited

217

cell growth significantly in liquid YEME, similarly as observed for growth on solid NA

218

plates (Fig. 3). We analyzed the expression of the nmtA gene in exponentially grown

219

cells by monitoring its transcript by S1 mapping. Results in Fig. 5B showed that the

220

nmtA expression began to get induced slightly at 0.5 mM Ni(II) and dramatically at 1

221

mM Ni(II). Parallel analyses of Nur-regulated sodF transcripts demonstrated that the

222

sodF gene was fully repressed even at 50 uM added Ni(II), consistent with the

223

disappearance of anti-sodN small RNA (s-SodF) and the full induction of sodN gene

224

expression (Fig. 5B, lane 2). Therefore, in S. coelicolor, NmtR responds to very high,

225

growth-inhibitory, concentrations of Ni(II). Effect of CoCl2 on cell growth and Ni(II)-

226

related gene expression was monitored in parallel. Cobalt inhibited cell growth

227

significantly at and above 0.25 mM, consistent with the sensitivity on solid plates

228

(Supplementary Fig. S3A). The nmtA transcripts were induced highly in cells grown

229

in YEME with 0.25 mM CoCl2 (Fig. S3B). This supports the hypothesis that NmtA

230

Ni/Co exporter is induced by Ni or Co at concentrations which inhibit cell growth.

231

Tolerance toward Ni was previously reported for another actinobacterium M. 22

232

smegmatis grown in LB medium

233

driven LacZ reporter activity in M. smegmatis increased continuously in a dose-

234

dependent manner up to 0.5 mM NiCl2 which is the maximal permissive

235

concentration in LB medium

22

. Previous study reported that nmtA promoter-

. In order to compare the behavior of S. coelicolor with 11


DOI: 10.1039/C4MT00318G

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

236

M. smegmatis in response to nickel, we grew M. smegmatis in the same liquid YEME

237

medium as used for S. coelicolor, and monitored the level of nmtA and nmtR

238

transcripts by quantitative real-time PCR. Cells were grown for 15 hr in YEME with

239

increasing concentrations of NiSO4 up to 0.5 mM. As presented in Fig. 5C, M.

240

smegmatis was tolerant to Ni(II) up to 0.1 mM in YEME, and significant growth

241

retardation was observed at 0.5 mM Ni(II). The level of nmtA RNA increased even at

242

non-inhibitory concentrations of Ni(II), with maximum induction at 0.5 mM Ni(II) by

243

more than 600-fold. Continuous induction of nmtA by non-inhibitory concentrations of

244

nickel in M. smegmatis appears different from the induction pattern in S. coelicolor.

245

This may reflect differences in the metal-responsiveness of NmtR and/or intracellular

246

metal-trafficking or metal-chelating environment between the two organisms.

247

248

Wide difference between Nur and NmtR in nickel sensitivity in vivo.

249

Since the two regulators that control influx and efflux of nickel are now identified in S.

250

coelicolor, we compared the sensitivity of Nur and NmtR toward nickel in vivo. For

251

this purpose, S. coelicolor cells were grown in YEME to OD600 of 1.0, and treated

252

with 0 to 1 mM NiSO4 for 40 min before cell harvest. Transcripts of nmtA, sodF, and

253

nikA gene were monitored by S1 mapping. Fig. 6A is a representative profile of

254

transcripts across the nickel concentrations we examined. The results exhibit striking

255

difference between Nur and NmtR. The production of sodF and nikA transcripts

256

regulated by Nur was fully repressed at 0.1 µM added Ni(II) in YEME, widely different

257

from the behavior of NmtR-regulated nmtA transcript that was de-repressed at >0.1 12


DOI: 10.1039/C4MT00318G

Page 13 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

258

mM Ni(II). We quantified the changes in the amount of these transcripts over a wide

259

range of Ni concentrations from 10 nM to 1 mM. The data in Fig. 6B, presented as %

260

expression relative to the fully induced (de-repressed) level, exhibited that Nur is

261

highly sensitive to Ni, with 50% activation at about 10 nM added Ni(II) (appKd,Ni, Nur

262

~1.2 x 10-8 M). In contrast, the DNA-binding activity of NmtR was half-inactivated at

263

about 0.8 mM Ni(II) (appKd,Ni,NmtR ~7.5 x 10-4 M), when full inactivation was set at 1

264

mM Ni(II). It is striking to observe that the sensitivity of Nur and NmtR in vivo toward

265

added nickel is different by more than four orders of magnitude. This observation

266

revealed that S. coelicolor responds to nickel availability in a highly sensitive way to

267

modulate the synthesis of nickel uptake systems within 1 to 100 nM range of

268

environmental Ni(II) in complex medium. On the other hand, it tolerates nickel from

269

0.1 µM to 0.1 mM Ni(II) without turning on efflux system. Only above 0.1 mM Ni(II),

270

synthesis of efflux system is induced as a steep response. As a comparison, we

271

performed similar experiment with cobalt. The result indicates that Nur responds to

272

cobalt much less efficiently than to nickel, gaining its full repressor activity at above

273

10 µM added CoCl2, whereas NmtR responds to cobalt even at 0.1 µM, continuously

274

losing activity by added cobalt up to 250 and 500 µM (Supplementary Fig. S4). The

275

relatively sensitive response of NmtR to added cobalt in vivo appears contradictory

276

to the relative insensitive response of purified NmtR to cobalt in vitro (Fig. 4B). Since

277

the in vivo response, monitored by nmtA transcripts, reflects the intracellular amount

278

of freely available metal, it can be hypothesized that more free cobalt is present than

279

free nickel inside S. coelicolor cells at the same added metal concentrations. This

280

needs further investigation.

13


DOI: 10.1039/C4MT00318G

Metallomics


DOI: 10.1039/C4MT00318G

281

The apparent Ni-sensitivity of Nur in S. coelicolor is comparable to or higher

282

than that of NikR in E. coli, which represses nikA promoter-driven LacZ activity by 50%

283

at about 10 nM added Ni in M63 minimal medium

284

activity at around 1 µM Ni(II) to turn off the synthesis of NikA system. Nickel titration

285

experiments in complex LB medium produced similar profile, shifting the curve to

286

higher nickel concentrations (half-maximal nikAp-LacZ at ~ 10 µM 12, 40. On the other

287

hand, RcnR gets inactivated above 0.1 µM Ni(II) to de-repress the synthesis of RcnA

288

efflux protein

289

and efflux is linked and is regulated within a very narrow range of environmental

290

nickel at low (~µM) concentrations, S. coelicolor demonstrates a vastly different

291

profile of achieving nickel homeostasis.

12

40

. NikR gained its full repressor

. In contrast to nickel homeostasis in E. coli where the nickel uptake

292

Nickel binding affinity (Kd,Ni) of Ni-specific regulators in vitro does not match

293

closely with the repression/induction profiles in vivo. For example, Kd,Ni for high

294

affinity nickel-binding sites in NikR from E. coli was reported to be ≤ 1 pM by EGTA

295

competition assay

296

for two binding sites in MtbNmtR dimer were estimated to be around 0.1 nM

297

apparent contradiction that MtbNmtR with higher Ni-affinity responds to nickel at

298

higher concentrations than RcnR does in vivo suggests the abundant existence of

299

nickel-sequestering

300

actinomycetes as well.

41, 42

, whereas that for RcnR is about 10-20 nM

molecules

in

tuberculosis,

M.

and

Nickel tolerating mechanisms in soil-dwelling streptomycetes. 14

. Kd,Ni values

likewise

301

302

11, 43

in

38

. The

other



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 15 of 36


DOI: 10.1039/C4MT00318G

303

Soils contain relatively high-level of nickel (16-40 ppm) compared with other

304

environments, such as oceans (0.3-0.6 ppb) and freshwaters (0.5-20 ppb)

305

resistant streptomycetes were isolated from soil samples of a polluted site at the

306

former uranium mine

307

serpentine soil also consists of streptomycetes

308

that S. coelicolor is very tolerant to nickel (Fig. 5A), coinciding with the observation

309

that NmtRA efflux system is induced at > 0.1 mM added nickel in complex YEME

310

medium. It can be postulated that Ni-tolerating mechanisms protect S. coelicolor

311

cells over wide concentration ranges, from 100 nM to 100 µM added Ni(II) in YEME,

312

following shut-off of synthesis of nickel uptake systems and before turning on the

313

synthesis of efflux pump. What could serve as protective mechanisms against nickel

314

in S. coelicolor until it decides to induce the synthesis of NmtA efflux pump?

315

Abundant Ni-binding proteins may serve as scavengers for surplus toxic nickel.

316

Inspection of the level of RNA abundance suggests that NiSOD and urease may be

317

abundant proteins in S. coelicolor (data not shown). A small nickel-binding protein

318

encoded by SCO4226 has been suggested to act as a Ni-storage protein

319

apparent

320

dehydrogenase/acetyl-coA synthase/decarbonylase were found in the genome of S.

321

coelicolor. We examined whether NiSOD could have contributed for S. coelicolor to

322

delay turning on NmtRA efflux system until encountering high inhibitory level of Ni.

323

Inspection of nmtA induction in ∆sodN and ∆nur mutants showed that the Ni-

324

responsive induction profile of nmtA did not change (Supplementary Fig. S5).

325

Therefore, NiSOD may not the critical buffering protein that sequesters nickel from

genes

45

44

. Nickel-

. A major portion of nickel-resistant bacteria isolated from

encoding

putative

46

. Our results clearly demonstrated

NiFe-hydrogenase,

15

carbon

47

. No

monoxide



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

326

being sensed by NmtR. There still remains a possibility that another nickel efflux

327

system homologous to KmtR-Cfd of Mtb may exist as a second efflux pump. The

328

divergent SCO1309 and SCO1310 genes encode MtbKmtR-like and MtbCdf-like

329

proteins, respectively, in S. coelicolor. Whether these gene products play a role in

330

nickel efflux and homeostasis needs be investigated. Unlike in M. tuberculosis,

331

where the cdf gene is constitutively expressed in complex media

332

SCO1310 is very low in S. coelicolor in YEME as suggested by a genome-wide

333

transcriptome analysis (communication with BK Cho). Further studies are in need to

334

reveal the complete constituents of nickel homeostasis in S. coelicolor.

21

, expression of

335

336

Experimental

337

Bacterial strains and culture conditions.

338

The wild type (M145) and mutant strains of S. coelicolor A3(2) were grown and

339

maintained according to standard procedures

340

suspensions were inoculated to YEME medium (0.3% yeast extract, 0.3% malt

341

extract, 0.5% peptone, 1% glucose, 10% sucrose, 5 mM MgCl2). For solid culture,

342

NA plates (0.4% beef extract, 0.4% peptone, 2% agar) were used. To add nickel,

343

NiSO4 at varying concentrations was added to the culture either continuously from

344

the time of inoculation to cell harvest, or briefly (≤40 min) to exponentially grown cell

345

cultures at OD600 of 0.3 - 1.2. The ∆nmtR and ∆nmtA mutant strains were obtained

346

by using PCR-targeted REDIRECT system

347

cells were grown in liquid YEME medium (with 0.05% Tween 80) at 37°C, with 16

49, 50

48

. For liquid culture, spore

. M. smegmatis wild type (mc2155)


DOI: 10.1039/C4MT00318G

Page 17 of 36


DOI: 10.1039/C4MT00318G

348

shaking at 150 rpm.

349 350

Cloning and Purification of NmtR.

351

The coding region of nmtR (SCO6459) was PCR-amplified from M145 genomic DNA

352

with a mutagenic primer pair: nmtR-pET-F (5’-GGA TGA ACC ATA TGG GTC ACG

353

GAG CC-3’; NdeI site underlined) and nmtR-pET-R (5’-GCG GAT CCT CAC TCG

354

GCC GTG TC-3’; BamHI site underlined). The PCR product (366 bp) was cloned into

355

pET15b expression vector (Novagen) through the NdeI and BamHI sites, and

356

introduced into E. coli BL21(DE3). Transformants were grown in LB broth at 37°C

357

until OD600 of 0.5, followed by induction with 1 mM IPTG for 1 hr. His-tagged NmtR

358

was purified from cell extracts by Ni-NTA column (Novagen), and dialyzed against

359

the buffer containing 20 mM Tris-HCl (pH 7.4), 500 mM NaCl, and 10% glycerol (or

360

50% for storage at -80°C). The purity of prepared protein was estimated to be >95%

361

by Coomassie brilliant blue staining after SDS-PAGE. Protein concentration was

362

determined by Bradford assay.

363 364

Electrophoretic mobility shift assay (EMSA).

365

The DNA probe for binding NmtR was generated by PCR amplification of the nmtA

366

promoter region from -93 to +24 nt relative to the start codon. The DNA was labelled

367

at 5’ ends with [γ-32P] ATP using T4 polynucleotide kinase. Binding reactions were

368

performed with approximately 8.8 fmole probe DNA and 40 µg cell extracts or 250

369

nM (final concentration) of purified NmtR protein in 40 µl binding buffer; 4 mM Tris-

370

HCl (pH 6.8), 1 mM DTT, 5 mM MgCl2, 20 mM KCl, 0.3 mg/ml BSA, 5% (v/v) glycerol, 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

371

and 140 ng poly(dI-dC). To find the effect of metal to dissociate the complex, 0.05-2

372

mM divalent metal salts (NiSO4, FeSO4, ZnCl2, CoCl2, MnSO4, or CuSO4) were

373

added to cell extracts or purified protein for 10 min prior to incubation with labeled

374

DNA probes for another 30 min. The reaction mixture was loaded on a 5%

375

polyacrylamide gel, and run in 0.5×TB buffer at room temperature at less than 20 mA.

376

Radioactive signals were detected and quantified by phosphor screen and image

377

analyzer (FLA-2000; Fuji).

378

379

S1 mapping analysis.

380

RNAs were isolated from cells grown in YEME to OD600 of 0.6 to 1.2. Harvested cells

381

were disrupted in modified Kirby mixture

382

of the maximum amplitude (600 W, 20 kHz). Following extraction with

383

phenol/chloroform, the supernatant was precipitated with isopropanol. The RNA

384

pellet was dissolved in DEPC-treated distilled water and quantified by measuring its

385

absorbance at 260 nm. To visualize rRNAs and check for contamination by genomic

386

DNA, RNA samples (10 µg each) were electrophoresed in 1.3% agarose gel in

387

MOPS buffer. For S1 mapping, DNA probes for nmtA (SCO6460), nikA, sodN, and

388

sodF transcripts were generated by PCR using M145 chromosomal DNA as a

389

template. The gene-specific probes for sod genes (sodF, s-SodF, sodN) were used

390

as described previously

391

produced PCR-amplified DNAs spanning from -144 to +162 nt and from -184 to +172

392

nt relative to the start codon of the nmtA and nikA coding region, respectively. The

37

48

using ultrasonicator with a microtip at 20%

. The primers used for nmtA and nikA-specific probes

18


DOI: 10.1039/C4MT00318G

Page 19 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

393

probe DNAs were radio-labeled at their 5’ ends with [γ-32P]-ATP by T4 polynucleotide

394

kinase. To each RNA sample (25 µg for sod and 100 µg for nmtA), probe DNA was

395

added for hybridization, and digested with S1 nuclease according to standard

396

procedures. The protected fragments were analyzed on a 6% polyacrylamide gel

397

containing 7 M urea. Radioactive signals were detected and quantified by phosphor

398

screen and image analyzer (FLA-2000; Fuji).

399

400

Quantitative real-time PCR (qRT-PCR)

401

RNA samples isolated from M. smegmatis cells grown in YEME were treated with

402

RQ1 DNase at 37°C for 30min. The amount of RNA was quantified by using ND-

403

2000

404

synthesized from 1ug of RNA using RevertAid reverse transcriptase (Thermo

405

Scientific) using random hexamers, as recommend by the manufacturer. qRT-PCR

406

was carried out for nmtA and nmtR RNAs using gene specific primers and SYBR

407

Green/ROX qPCR master mix (Fermentas) on a quantitative real-time PCR machine

408

(Stratagene Mx3000P, Agilent Technologies). qRT-PCR primers used were;

409

5’GTGTTCGACGGTGCGCTGTTG’ and 5’CCGTGTGGTCGCCACATCTTC3’ for

410

nmtA

411

5’CCCAGCGTCCGTAACAGGCGC3’

412

5’GGGAGCGAACAGGATTAGATAC3’ and 5’CCTTTGAGTTTTAGCCTTGCG3’ for

413

16S rRNA F and R primers. As a control for DNA contamination, the same reaction

414

was carried out for each sample in the absence of reverse transcriptase. The

Nanodrop

F

and

spectrophotometer

R

primers,

(Nanodrop

Technologies).

cDNA

5’GAATTGTCGGCGGCGGTCGGG3’ for

19

nmtR

F

and

R

was

and

primers,


DOI: 10.1039/C4MT00318G

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

415

amplification conditions for all reactions were 1 cycle of 50°C for 2 min and 95°C for

416

10 min, followed by 40 cycles of 95°C for 15s and 60°C for 1min. Analysis of qRT-

417

PCR data was carried out using comparative CT method. For each qRT-PCR run, the

418

calculated threshold cycle (CT) was normalized to the CT of the internal control 16S

419

rRNA gene amplified from the same sample. Statistical analysis was carried out

420

using MxPro QPCR software (Agilent Technologies).

421

422

Conclusion

423

In this study we demonstrated that NmtRA is a Ni(II)/Co(II)-specific efflux system in S.

424

coelicolor. NmtR responds to high level of Ni(II), with 50% activity modulation at ~7.5

425

x 10-4 M added nickel in vivo, to de-repress NmtA synthesis. This contrasts with the

426

high sensitivity of regulator Nur to nickel, whose 50% activity is modulated at ~1.2 x

427

10-8 M added nickel, to repress the synthesis of nickel uptake systems and induce

428

SodN production. This study revealed that highly sensitive Ni-specific uptake

429

regulator (Nur) and extremely insensitive efflux regulator (NmtR) constitute

430

regulatory system to control nickel homeostasis in a nickel-tolerant soil bacterium S.

431

coelicolor. In comparison with E. coli system, where the regulations of nickel-specific

432

uptake (NikR/NikA) and efflux (RcnR/RcnA) systems are coordinated and occur

433

within a narrow range of low environmental nickel concentrations, S. coelicolor

434

system provides a contrasting example where nickel-specific uptake and efflux

435

systems are both known, and are regulated in a widely different way in vivo. Our

436

finding provides a basis to investigate further about the mechanisms that achieve 20


DOI: 10.1039/C4MT00318G

Page 21 of 36


DOI: 10.1039/C4MT00318G

437

nickel-homeostasis and nickel-tolerant physiology in this soil-inhabiting bacterium as

438

well as in other nickel-tolerant bacteria.

439

440

Acknowledgements

441

We thank Dr. T Song for providing M. smegmatis mc2-155 strain, Dr. BK Cho for

442

sharing a genome-wide RNAseq data, and Dr. Kang-Lok Lee and Seung-Hwan Choi

443

for helpful discussions. This work was supported by the Intelligent Synthetic Biology

444

Center of Global Frontier Project funded by NRF. JH Lee was supported by BK21-

445

Plus graduate scholarship to Life Sciences at SNU.

446

447

Notes and references

448

School of Biological Sciences, and Institute of Microbiology, Seoul National

449

University, Seoul 151-742, Korea.

450

‡

451

(MIT) 77 Massachusetts Ave., Cambridge, MA 02139

452

* corresponding author, email: [email protected]

453

1.

S. B. Mulrooney and R. P. Hausinger, FEMS Microbiol Rev, 2003, 27, 239-261.

454

2.

Y. Li and D. B. Zamble, Chemical reviews, 2009, 109, 4617-4643.

455

3.

S. W. Ragsdale, J Biol Chem, 2009, 284, 18571-18575.

456

4.

H. Kaluarachchi, K. C. Chan Chung and D. B. Zamble, Nat Prod Rep, 2010, 27, 681-694.

457

5.

L. Macomber and R. P. Hausinger, Metallomics : integrated biometal science, 2011, 3,

current address: Department of Biology, Massachusetts Institute of Technology

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Metallomics


DOI: 10.1039/C4MT00318G

458

1153-1162.

459

6.

C. Navarro, L. F. Wu and M. A. Mandrand-Berthelot, Mol Microbiol, 1993, 9, 1181-1191.

460

7.

S. P. Ballantine and D. H. Boxer, J Bacteriol, 1985, 163, 454-459.

461

8.

P. T. Chivers and R. T. Sauer, J Biol Chem, 2000, 275, 19735-19741.

462

9.

L. F. Wu, M. A. Mandrand-Berthelot, R. Waugh, C. J. Edmonds, S. E. Holt and D. H. Boxer,

Mol Microbiol, 1989, 3, 1709-1718.

463 464

10.

A. Rodrigue, G. Effantin and M. A. Mandrand-Berthelot, J Bacteriol, 2005, 187, 2912-2916.

465

11.

J. S. Iwig and P. T. Chivers, Nat Prod Rep, 2010, 27, 658-667.

466

12.

J. S. Iwig, J. L. Rowe and P. T. Chivers, Mol Microbiol, 2006, 62, 252-262.

467

13.

D. Koch, D. H. Nies and G. Grass, Biometals, 2007, 20, 759-771.

468

14.

H. L. Mobley, R. M. Garner and P. Bauerfeind, Mol Microbiol, 1995, 16, 97-109.

469

15.

J. K. Hendricks and H. L. Mobley, J Bacteriol, 1997, 179, 5892-5902.

470

16.

K. J. Nolan, D. J. McGee, H. M. Mitchell, T. Kolesnikow, J. M. Harro, J. O'Rourke, J. E. Wilson, S. J. Danon, N. D. Moss, H. L. Mobley and A. Lee, Infect Immun, 2002, 70, 685-691.

471 472

17.

T. Eitinger, J. Suhr, L. Moore and J. A. Smith, Biometals, 2005, 18, 399-405.

473

18.

F. D. Ernst, E. J. Kuipers, A. Heijens, R. Sarwari, J. Stoof, C. W. Penn, J. G. Kusters and A. H. van Vliet, Infect Immun, 2005, 73, 7252-7258.

474 475

19.

L. Wolfram, E. Haas and P. Bauerfeind, J Bacteriol, 2006, 188, 1245-1250.

476

20.

F. N. Stahler, S. Odenbreit, R. Haas, J. Wilrich, A. H. Van Vliet, J. G. Kusters, M. Kist and S. Bereswill, Infect Immun, 2006, 74, 3845-3852.

477 478

21.

J. Hinds, N. G. Stoker, N. J. Robinson and J. S. Cavet, J Biol Chem, 2007, 282, 32298-32310.

479 480

22.

J. S. Cavet, W. Meng, M. A. Pennella, R. J. Appelhoff, D. P. Giedroc and N. J. Robinson, J Biol

Chem, 2002, 277, 38441-38448.

481 482

D. R. Campbell, K. E. Chapman, K. J. Waldron, S. Tottey, S. Kendall, G. Cavallaro, C. Andreini,

23.

S. T. Cole, R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier,

483

S. Gas, C. E. Barry, 3rd, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R.

484

Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K.

485

Jagels, A. Krogh, J. McLean, S. Moule, L. Murphy, K. Oliver, J. Osborne, M. A. Quail, M. A.

486

Rajandream, J. Rogers, S. Rutter, K. Seeger, J. Skelton, R. Squares, S. Squares, J. E. Sulston, K.

487

Taylor, S. Whitehead and B. G. Barrell, Nature, 1998, 393, 537-544.

488

24.

D. A. Rodionov, P. Hebbeln, M. S. Gelfand and T. Eitinger, J Bacteriol, 2006, 188, 317-327.

489

25.

Y. Zhang, D. A. Rodionov, M. S. Gelfand and V. N. Gladyshev, BMC Genomics, 2009, 10, 78.

490

26.

H. D. Youn, E. J. Kim, J. H. Roe, Y. C. Hah and S. O. Kang, The Biochemical journal, 1996,

491

318 ( Pt 3), 889-896.

492

27.

E. J. Kim, H. J. Chung, B. Suh, Y. C. Hah and J. H. Roe, Mol Microbiol, 1998, 27, 187-195.

493

28.

C. L. Dupont, K. Neupane, J. Shearer and B. Palenik, Environ Microbiol, 2008, 10, 1831-1843.

494

29.

A. Schmidt, M. Gube and E. Kothe, J Basic Microbiol, 2009, 49, 109-118. 22



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 23 of 36


DOI: 10.1039/C4MT00318G

495

30.

496 497

12185-12196. 31.

498 499

C. Greening, M. Berney, K. Hards, G. M. Cook and R. Conrad, Proc Natl Acad Sci U S A, 2014, 111, 4257-4261.

32.

500 501

H. Merkens, R. Kappl, R. P. Jakob, F. X. Schmid and S. Fetzner, Biochemistry, 2008, 47,

P. Constant, S. P. Chowdhury, J. Pratscher and R. Conrad, Environ Microbiol, 2010, 12, 821829.

33.

W. Lin, V. Mathys, E. L. Ang, V. H. Koh, J. M. Martinez Gomez, M. L. Ang, S. Z. Zainul Rahim, M. P. Tan, K. Pethe and S. Alonso, Infect Immun, 2012, 80, 2771-2779.

502 503

34.

H. J. Chung, J. H. Choi, E. J. Kim, Y. H. Cho and J. H. Roe, J Bacteriol, 1999, 181, 7381-7384.

504

35.

B. E. Ahn, J. Cha, E. J. Lee, A. R. Han, C. J. Thompson and J. H. Roe, Mol Microbiol, 2006, 59,

505 506

1848-1858. 36.

Y. J. An, B. E. Ahn, A. R. Han, H. M. Kim, K. M. Chung, J. H. Shin, Y. B. Cho, J. H. Roe and S. S. Cha, Nucleic Acids Res, 2009, 37, 3442-3451.

507 508

37.

H. M. Kim, J. H. Shin, Y. B. Cho and J. H. Roe, Nucleic Acids Res, 2014, 42, 2003-2014.

509

38.

H. Reyes-Caballero, C. W. Lee and D. P. Giedroc, Biochemistry, 2011, 50, 7941-7952.

510

39.

L. S. Busenlehner, M. A. Pennella and D. P. Giedroc, FEMS Microbiol Rev, 2003, 27, 131-143.

511

40.

J. L. Rowe, G. L. Starnes and P. T. Chivers, J Bacteriol, 2005, 187, 6317-6323.

512

41.

P. T. Chivers and R. T. Sauer, Chem Biol, 2002, 9, 1141-1148.

513

42.

S. C.-H. Wang, M. Sc., University of Toronto, 2004.

514

43.

J. S. Iwig, S. Leitch, R. W. Herbst, M. J. Maroney and P. T. Chivers, J Am Chem Soc, 2008,

515

130, 7592-7606.

516

44.

R. P. Hausinger, Biochemistry of nickel, Plenum Press, New York, 1993.

517

45.

M. J. Amoroso, D. Schubert, P. Mitscherlich, P. Schumann and E. Kothe, J Basic Microbiol,

518 519

2000, 40, 295-301. 46.

520 521

A. Mengoni, R. Barzanti, C. Gonnelli, R. Gabbrielli and M. Bazzicalupo, Environ Microbiol, 2001, 3, 691-698.

47.

M. Lu, Y. L. Jiang, S. Wang, H. Jin, R. G. Zhang, M. J. Virolle, Y. Chen and C. Z. Zhou, PLoS

One, 2014, 9, e109660.

522 523

48.

T. Kieser, Practical streptomyces genetics, John Innes Foundation, Norwich, 2000.

524

49.

B.-E. Ahn, Doctor of Philosophy Thesis(doctoral), Seoul National Unversity, 2006.

525

50.

B. Gust, T. Kieser and K. Chater, The John Innes Centre, Norwich, United Kingdom, 2002.

526

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

527

Fig 1. Sequence information around the nmtR and nmtA gene. The genetic context

528

(synteny) and sequence information in the intergenic region of the nmtR (SCO6459)

529

and nmtA (SCO6460) gene of Streptomyces coelicolor M145 was given; SCO6458,

530

hypothetical protein; SCO6461, possible ADA-like regulatory protein; SCO6462,

531

methylated-DNA-protein-cysteine methyltransferase. A palindromic sequence that

532

matches the core SmtB/ArsR-binding motif suggested by 19Busenlehner et al (2003)

533

was indicated

534

red, and the palindromically-pairing sequences were presented in capital letters. The

535

5’ ends of the nmtR and nmtA transcripts were presented by bend arrows. The

536

predicted position of -10 and -35 element of each promoter was indicated. For nmtA

537

promoter, the position of -10 and -35 was predicted based on the location of

538

transcription start site (+1) at the G nucleotide of the initiation codon. The uncertainty

539

in the initiating nucleotides in nmtA transcription was indicated by a broken line

540

above the sequence CCGCG.

39

. Nucleotides that match with the core sequence were presented in

541 542

Fig. 2. Effect of nmtR mutation on the expression of nickel-responsive genes.

543

wild type and ΔnmtR cells were growth in YEME to early exponential phase (OD600

544

of 0.2-0.3), followed by treatment with various concentrations of NiSO4 up to 1 mM

545

for 40 min before cell harvest. The RNA samples were analyzed by S1 mapping to

546

monitor the level of nmtA, nmtR, sodF, s-SodF, and sodN transcripts.

The

547 548

Fig 3. Sensitivity of nmtA mutant to nickel and cobalt. Serial dilution of spore

549

suspensions (10 ~ 105 spores) from the wild type, ∆nmtA, and ∆nmtR mutants were 24


DOI: 10.1039/C4MT00318G

Page 25 of 36


DOI: 10.1039/C4MT00318G

550

spotted on NA plates that contain no added metal, 1 mM NiSO4, or 0.5 mM CoCl2.

551

Photos were taken after 48 hrs incubation at 30°C.

552 553

Fig 4. NmtR binding to the nmtA promoter region. (A) Ni(II) and Co(II)-specific

554

inhibition of NmtR binding at the nmtA promoter probe. Cell extracts (40 µg) from the

555

wild type cells were incubated with 0.1 mM (final concentration) each of NiSO4,

556

FeSO4, ZnCl2, CoCl2, MnSO4 or CuSO4, followed by EMSA. (B) Inhibition of NmtR

557

binding to nmtA promoter DNA by Ni(II) and Co(II) in vitro. Purified NmtR (250 nM

558

final) was incubated in the binding buffer with or without increasing amounts of

559

NiSO4 or CoCl2 for 10 min, followed by further incubation with the nmtA promoter

560

DNA fragment for 30 min.

561 562

Fig 5. Effect of nickel on the growth and induction of nickel-regulated genes in S.

563

coelicolor and Mycobacterium smegmatis. (A) The wild type S. coelicolor cells were

564

grown in liquid YEME media containing 0, 50, 500, and 1000 µM NiSO4.The cell

565

growth was monitored by following OD600 after inoculation of spores (4x107) to 200

566

ml media and incubation at 30°C.

567

NmtR-regulated (nmtA) and Nur-regulated (sodF, s-SodF, sodN) genes. RNA

568

samples were prepared from cells grown to OD600 of 0.7-0.8, demonstrated in panel

569

A. Ribosomal RNAs in each sample were shown as a control to indicate the quality

570

of RNA samples. (C) Effect of nickel on the growth and induction of the nmtA gene in

571

M. smegmatis. The wild type M. smegmatis cells were grown in liquid YEME media

572

containing 0, 50, 100, 250 and 500M NiSO4 by inoculating the seed culture to 100

(B) S1 mapping analysis of transcripts from

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

573

mL media and incubated at 30°C for 15 hr before cell harvest. OD600 measured at

574

this time was indicated in the figure. The amount of nmtA RNA was determined by

575

qRT-PCR and presented as a relative value to the non-induced sample. The amount

576

of nmtR RNA was also indicated as a relative value. The data represent average

577

values and standard error measurements from three independent experiments.

578 579

Fig 6. Nickel titration to determine sensitivities of Nur and NmtR in vivo in S.

580

coelicolor. (A) Wild type cells were growth in YEME to OD600 of 1.0, and then treated

581

with NiSO4 for 40 min at 10 nM to 1 mM. Transcripts from nmtA, sodF and nikA

582

genes were analysed by S1 mapping. Ribosomal RNAs in each sample were shown

583

as a control to indicate the quality of RNA samples. (B) The change in the amount of

584

nmtA, sodF and nikA transcripts in response to added nickel was presented as %

585

expression relative to the fully induced levels. The concentrations of nickel that

586

caused 50% changes in the activities of Nur and NmtR were estimated to be 1.2 x

587

10-8 M and 7.5 x 10-4 M Ni(II), respectively, from fitting curves.

588

26


DOI: 10.1039/C4MT00318G

Page 27 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

173x83mm (150 x 150 DPI)


DOI: 10.1039/C4MT00318G

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

157x101mm (150 x 150 DPI)


DOI: 10.1039/C4MT00318G

Page 29 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

149x41mm (132 x 132 DPI)


DOI: 10.1039/C4MT00318G

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

77x54mm (150 x 150 DPI)


DOI: 10.1039/C4MT00318G

Page 31 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

123x62mm (150 x 150 DPI)


DOI: 10.1039/C4MT00318G

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

86x92mm (150 x 150 DPI)


DOI: 10.1039/C4MT00318G

Page 33 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

68x90mm (150 x 150 DPI)


DOI: 10.1039/C4MT00318G

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

145x97mm (144 x 150 DPI)


DOI: 10.1039/C4MT00318G

Page 35 of 36



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

145x77mm (150 x 150 DPI)


DOI: 10.1039/C4MT00318G

Metallomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

146x112mm (150 x 150 DPI)


DOI: 10.1039/C4MT00318G

Streptomyces coelicolor SCO4226 is a nickel binding protein.

The Coordinated Positive Regulation of Topoisomerase Genes Maintains Topological Homeostasis in Streptomyces coelicolor.

Streptomyces antioxidans sp. nov., a Novel Mangrove Soil Actinobacterium with Antioxidative and Neuroprotective Potentials.

Sequence of a tRNAGly from Streptomyces coelicolor.

Streptomyces gilvigriseus sp. nov., a novel actinobacterium isolated from mangrove forest soil.

A terD domain-encoding gene (SCO2368) is involved in calcium homeostasis and participates in calcium regulation of a DosR-like regulon in Streptomyces coelicolor.

Draft Genome Sequence of Streptomyces sp. M1013, a Close Relative of Streptomyces ambofaciens and Streptomyces coelicolor.

Transcriptomic analysis of a classical model of carbon catabolite regulation in Streptomyces coelicolor.

Genetic analysis of absB, a Streptomyces coelicolor locus involved in global antibiotic regulation.

Salternamides A-D from a Halophilic Streptomyces sp. Actinobacterium.

Characterization of p-hydroxycinnamate catabolism in a soil Actinobacterium.

Characterization of a plasmid from Streptomyces coelicolor A3(2).

Identification of Streptomyces coelicolor genes involved in regulation of antibiotic synthesis.

Fertility properties and regulation of antimicrobial substance production by plasmid SCP2 of Streptomyces coelicolor.

A2 two-component system in Streptomyces coelicolor and the potential of its deletion strain as a heterologous host for antibiotic production.

6S RNA modulates growth and antibiotic production in Streptomyces coelicolor.

Genetic and Proteomic Analyses of Pupylation in Streptomyces coelicolor.

The purification and characterization of 3-dehydroquinase from Streptomyces coelicolor.

Identification and cloning of a umu locus in Streptomyces coelicolor A3(2).

The regulatory role of Streptomyces coelicolor TamR in central metabolism.

Plasmid-determined antibiotic synthesis and resistance in Streptomyces coelicolor.

Factors affecting recombinant frequency in protoplast fusions of Streptomyces coelicolor.

antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity.

Bioactive DOPA melanin isolated and characterised from a marine actinobacterium Streptomyces sp. MVCS6 from Versova coast.