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Journal of Bacteriology, December 1999, p. 7381-7384, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Negative Regulation of the Gene for Fe-Containing
Superoxide Dismutase by an Ni-Responsive Factor in
Streptomyces coelicolor
Hye-Jung
Chung,
Jae-Hyun
Choi,
Eun-Ja
Kim,
You-Hee
Cho, and
Jung-Hye
Roe*
Department of Microbiology, College of
Natural Sciences, and Research Center for Molecular Microbiology,
Seoul National University, Seoul 151-742, Korea
Received 19 May 1999/Accepted 13 September 1999
 |
ABSTRACT |
In Streptomyces coelicolor, transcription of the
sodF genes, encoding Fe-containing superoxide dismutases,
is negatively regulated by nickel. Gel mobility shift assays with
sodF1 promoter fragments and cell extracts from the A3(2)
strain indicate the presence of a nickel-responsive DNA-binding
protein, most likely a transcriptional repressor. The boundary for the
Ni-responsive cis-acting region was identified both in
vitro and vivo. Ni does not regulate the level of the putative
repressor but only the binding competence of this protein.
| |
TEXT |
All aerobically growing organisms
encounter toxic derivatives of molecular oxygen and thus are equipped
with defense systems against oxidative stress (5, 8).
Superoxide dismutase (SOD) is an important component of this protective
system, disproportionating superoxide anion into dioxygen and hydrogen
peroxide (6). Based on the metal ions present in active
sites, four groups of SODs have been distinguished;
CuZnSOD, MnSOD, FeSOD, and NiSOD (6, 13, 20). Many
organisms possess more than one type of SOD. For example, aerobically
grown Escherichia coli contains MnSOD and FeSOD in the
cytosol and CuZnSOD in the periplasm (1, 17). The regulation
of sod gene expression has been best demonstrated for the
MnSOD gene (sodA), which is under the control of a number of
transcription factors, including SoxRS, Fur, ArcA, Fnr, and IHF
(3, 17).
Streptomyces coelicolor Müller contains two types of
SOD: NiSOD, encoded by the sodN gene, and FeSOD, encoded by
the sodF gene (11, 12). In S. coelicolor A3(2), two FeSOD polypeptides are produced from two
separate genes: sodF1, which is identical to the
sodF gene of the Müller strain, and sodF2,
which differs from sodF1 by about 12% of its nucleotide
sequence (2). Expression of these sod genes is
differentially regulated by nickel, which increases the expression of
the sodN gene at both the transcriptional and
posttranscriptional levels and represses the transcription of the
sodF genes (2, 11, 12). The details of the
regulation of SOD gene transcription by various metals have been
studied primarily in E. coli (regulation by manganese and
iron) and in yeasts (regulation by copper) (7, 16). However,
the antagonistic regulation of two sod genes by a single
metal is most pronounced in S. coelicolor.
Ni-dependent transcriptional regulation has been reported in the
expression of the hydrogenase gene (hup) in
Bradyrhizobium japonicum (14, 15), and a
nickel-specific transport system (encoded by nikABCDE) in
E. coli (4), in which the nikA operon has been suggested to be repressed by a nickel-responsive regulator, NikR (4). In this study, we investigated the metal
specificity of the sodF1 gene regulation in S. coelicolor A3(2) and report the involvement of a nickel-responsive
DNA-binding protein, most likely a repressor, in the regulation of
sodF1 gene expression.
Effects of various transition metals on sodF1 gene
expression.
To examine whether transition metals other than nickel
regulate SOD expression, the amount of FeSODs in S. coelicolor A3(2) cells grown on a nutrient agar (NA) plate
supplemented with various metals was analyzed by immunoblotting (Fig.
1A). Nickel effectively repressed the
production of SodF1 even at 1 µM and that of SodF2 at 100 µM (Fig.
1A, lanes 2 and 3). Cobalt suppressed SodF1 production partially at 100 µM and SodF2 production only marginally (Fig. 1A, lane 4). Other
transition metals did not affect the production of either SodF1 or
SodF2. In all cases, the level of FeSOD activity correlated well with
the amount of SodF polypeptides (data not shown). In contrast,
production of NiSOD was increased at 1 µM NiCl2 and 100 µM CoCl2 but was not affected by other metals (Fig. 1B).
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FIG. 1.
Effects of various transition metals on the expression
of FeSODs. S. coelicolor A3(2) M145 cells were grown for 4 days on NA plates containing various transition metals
(NiCl2, CoCl2, FeCl2,
MnCl2, and ZnCl2) at the indicated
concentrations. (A) Cell extract containing 20 µg of proteins was
analyzed for the amount of SodF1 and SodF2 proteins by Western blotting
using antibodies against the SodF protein of the Müller strain as
described previously (2). (B) The amount of SodN proteins
was analyzed in parallel by using antibodies against the SodN protein
of the Müller strain (12). (C) RNAs prepared from the
above-described cells were analyzed for sodF1 mRNA by S1
nuclease mapping using a sodF probe labeled at the 5'
position of the BglII end at position +479 relative to the
transcription start point (11). (D) The sodN mRNA
was analyzed in parallel by using a sodN probe labeled at
the 5' position of the BglII end at position +402 relative
to the transcription start point (12).
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|
The change in levels of sodF1 transcripts in these cells was
analyzed by S1 nuclease mapping (Fig. 1C), and the effects of various
metals on transcription correlated reasonably well with those on
polypeptide production. Therefore, we suggest that sodF1 transcription is very sensitive to inhibition by nickel and less sensitive to inhibition by cobalt. As a comparison, changes in sodN mRNA were analyzed in parallel, and this gene exhibited
regulation by nickel and cobalt opposite to that of the sodF
genes (Fig. 1D). Comparison of the concentrations of these two metals
required for regulation indicated that the minimum concentrations at
which SodF1 started to decrease and SodN started to increase were 10 nM
NiCl2 and 1 µM CoCl2 (data not shown). The
total amount of SodF1 and SodN polypeptides from cells grown at 100 µM CoCl2 was comparable to that at 100 nM
NiCl2, implying that nickel is more effective than cobalt
by at least 2 orders of magnitude in regulating sodF1 and
sodN gene expression.
Ni-responsive protein binding to sodF1 promoter.
To search for the presence of transcriptional regulators responsive to
nickel, gel mobility shift assays were performed with cell extracts and
sodF1 promoter fragments. Cell extracts were prepared from
A3(2) cultures grown with or without added NiCl2 in YEME
medium (9). Two different sodF1 promoter
fragments of different lengths were generated by PCR using two sets of
primers: SODF1N1 (5'-GCG GCA CCA AGC TTT CCG AAC AAC-3'
[the HindIII site at position 
130 relative to the
transcription start site is underlined]) and SODF1Bam (5'-CAT GGC
GGA TCC CTC CGG-3' [the BamHI site at position
+30 is underlined]) were used to generate the longer fragment (130
to +30), and SODF1N2 (5'-CCG TGC GGG GAA GCT TCG TGT GCG-3'
[the HindIII site at position 60 relative to the
transcription start site is underlined]) and SODF1Bam were used to
generate the shorter fragment (60 to +30).
Using the two sodF1 promoter fragments, two distinct
complexes were formed by extracts from an Ni-supplemented culture (Fig. 2A, lanes 5 to 7 and 11 to 13). The
proportion of the slower-migrating complex increased with greater
amounts of cell extract, suggesting that the bound protein is the
multimeric form of that in the faster-migrating complex. The binding
patterns of the longer and shorter DNA fragments were almost the same,
indicating that the binding site is located within the boundary of the
shorter fragment. Given that Ni represses sodF1 gene
transcription, the Ni-sensitive binding pattern suggests that the bound
factor functions as a repressor for the sodF1 promoter. The
sodF1 complexes were strong enough to resist 60-fold molar excess of nonspecific competitors, while they began to be competed out
by a 5-fold molar excess of unlabeled sodF1 promoter
fragments (Fig. 2B). The slower-migrating complex was competed out more easily, consistent with the proposal that it is a multimeric form of
the faster-migrating complex.
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FIG. 2.
Binding of Ni-responsive protein to sodF1
promoter region. (A) Gel mobility shift assays used cell extracts
prepared from S. coelicolor A3(2) M145 cells grown for 2 days in YEME with (lanes 5 to 7 and 11 to 13) or without (lanes 2 to 4 and 8 to 10) 100 µM NiCl2. The indicated amount of cell
extract was incubated with the sodF1 DNA probe spanning the
region between nt 130 (lanes 1 to 7) or 60 (lanes 8 to 14) and nt
+30 relative to the transcription start point. Each binding reaction
mixture contained about 10 fmol (0.6 to 1.0 ng) of
32P-labeled DNA fragment (about 104 cpm) in 4 mM Tris · HCl (pH 8.0)-1 mM EDTA-4 mM dithiothreitol-5 mM
MgCl2-20 mM KCl-bovine serum albumin at 0.3 µg/µl-10% (vol/vol) glycerol-1 µg of poly(dI-dC). FP denotes
free probe DNA, and arrows indicate retarded bands sensitive to nickel.
(B) Specificity of binding to sodF1. The shorter promoter
probe was incubated with 50 µg of protein from M145 cells grown in
the presence of 100 µM NiCl2 as for panel A. The binding
reaction was challenged with a 5- to 60-fold molar excess of either
unlabeled promoter fragments (Specific; lanes 3 to 5) or
HpaII restriction fragments of pGEM-3Zf DNA (Non-specific;
lanes 7 to 9) in the binding buffer.
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Presence of Ni-responsive regulatory site as monitored by a
sodF1-xylE fusion in vivo.
The promoter fragments used
in the gel mobility shift assays were also tested for the presence of a
functional Ni-sensitive regulatory region in vivo. Each fragment was
fused with the xylE reporter gene in low-copy plasmid pXE4
(10), generating pXEF130 and pXEF60, containing 130 and 60 nucleotides (nt) upstream of the sodF1 transcription start
site. Cells harboring each plasmid were grown for 20 h on NA
plates with or without supplementation with 100 µM NiCl2
and assayed for catechol dioxygenase activity as described previously
(10). The level of sodF1 promoter-driven XylE
activity decreased about fivefold in the presence of 100 µM
NiCl2 (Fig. 3). The
repression of sodF1-xylE by 100 µM NiCl2 was
elevated up to 10-fold when cells were grown for 2 days instead of
20 h (data not shown). This observation confirms that the critical Ni-responsive negative regulatory site resides between nt 60 and +30
in the sodF1 gene, consistent with the results of the gel
mobility shift assay.
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FIG. 3.
Negative regulation of sodF1-xylE expression
by nickel. The same sodF1 promoter fragments used in Fig. 2
were cloned into plasmid pXE4 upstream of the xylE coding
region, generating pXEF130 (130) and pXEF60 (60). Recombinant
plasmids were introduced into S. lividans TK24; this was
followed by selection on R5 plates with thiostrepton at 10 µg/ml.
Cell extracts were prepared from transformants grown for 20 h on
NA plates with (solid bars) or without (open bars) 100 µM
NiCl2 and assayed for catechol dioxygenase activity as
described by Ingram et al. (10).
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Regulation of sodF1 promoter-binding activity by
nickel.
We next examined whether nickel enhances the synthesis or
activity of the sodF1 promoter-binding protein. A cell
extract from a nickel-deficient culture was incubated with a
sodF1 fragment in the absence or presence of nickel in the
binding buffer. Retarded complexes appeared when more than 1 µM
NiCl2 was added in the buffer (Fig.
4A, lanes 3 to 6). This suggests that the
putative sodF1-binding repressor is synthesized and present
as an inactive form in Ni-deficient cells and turns into the active
binding form in the presence of Ni. Whether Ni exerts its role by
direct binding or via a mediator remains to be studied. The effect of
other transition metals was also examined, and consistent with the
results in Fig. 1, only cobalt allowed in vitro activation of the
sodF1-binding protein, although much less efficiently than
did nickel (Fig. 4B).
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FIG. 4.
Activation of the sodF1 promoter-binding
activity by nickel in vitro. Extracts (50 µg of protein) of A3(2)
M145 cells from a nickel-deficient culture in YEME medium were
incubated with the shorter sodF1 promoter fragment (from nt
60 to nt +30) as described in the legend to Fig. 2 in the absence or
presence of NiCl2 at the indicated concentrations in the
binding buffer (A). The same extract was incubated with the
sodF1 promoter in the presence of various transition metals
as chloride salts at the indicated concentrations (B).
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|
Our results demonstrate the presence of a sodF1
promoter-binding protein, most likely a transcriptional repressor,
whose binding activity was enhanced greatly in response to low levels
of nickel and also in response to much higher levels of cobalt. The
cis-acting negative regulatory site located between nt 60
and +30 of the sodF1 promoter and the nickel-sensitive
binding of a trans-acting factor to this region constitute
the nickel-dependent negative regulatory system of sodF1
gene transcription. Various reports suggest that not only the
deficiency but also the overexpression of SOD is toxic to cells
(18, 19). The antagonistic production of FeSOD and NiSOD
regulated by nickel could therefore be a kind of homeostatic regulatory
mechanism to maintain the total SOD activity in S. coelicolor within an optimal range. The modulation of the
DNA-binding activity of a pre-existing regulator by Ni ensures a rapid
response, keeping the total SOD activity relatively constant.
| |
ACKNOWLEDGMENTS |
We thank J. S. Hahn for helpful discussions.
This work was supported by a grant from the Korea Science and
Engineering Foundation to the Research Center for Molecular Microbiology, Seoul National University.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea. Phone: 82-2-880-6706. Fax: 82-2-888-4911. E-mail:
jhroe{at}plaza.snu.ac.kr.
Present address: Laboratory of Pathology, DCS, NCI, National
Institutes of Health, Bethesda, Md.
Present address: Department of Molecular and Experimental
Medicine, The Scripps Research Institute, La Jolla, CA 92037.
| |
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Journal of Bacteriology, December 1999, p. 7381-7384, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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