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Journal of Bacteriology, April 2000, p. 2345-2349, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cloning and Sequence Analysis of the Mercury
Resistance Operon of Streptomyces sp. Strain CHR28 Reveals a
Novel Putative Second Regulatory Gene
Jacques
Ravel,
Jocelyne
DiRuggiero,
Frank T.
Robb, and
Russell T.
Hill*
Center of Marine Biotechnology, University of
Maryland Biotechnology Institute, Baltimore, Maryland 21202
Received 8 October 1999/Accepted 26 January 2000
 |
ABSTRACT |
A DNA library of pRJ28, a large linear plasmid encoding mercury
resistance, was constructed, and the mercury resistance genes were
cloned. The 5,921-bp sequence was analyzed and showed a high degree of
similarity to the Streptomyces lividans 1326 mercury resistance operon. Genes merR, merT,
merP, and orfIV were found in a similar order
and in a single transcription unit. merA and merB were found to be transcribed in the opposite direction
to genes merR, merT, merP, and
orfIV, as in S. lividans 1326. A novel putative
regulatory gene, orfX, was found 22 bp downstream of merA. orfX encodes a 137-amino acid protein
with a potential helix-turn-helix motif in the N-terminal domain,
characteristic of the MerR family of transcriptional regulators.
Transcriptional studies showed that orfX is cotranscribed
with merA and merB. It is hypothesized that
orfX plays a role in the regulation of the mercury
resistance operon, probably by binding at the MerR operator site.
| |
TEXT |
Mercury resistance is widespread
among prokaryotes, and resistance genes are often found on plasmids or
transposons (11). The major mechanism of resistance is
reductive detoxification of Hg(II) to elemental mercury Hg(0), which is
extremely volatile and leaves the cell by diffusing through the cell
membrane. The process is mediated intracellularly by a mercuric
reductase (MerA). Mercuric ions are transported from outside the cell
by a series of transporter proteins. MerP is an extracellular mercuric
ion binding protein, and MerT is a membrane-anchored protein
responsible for transporting Hg(II) into the cell. All gram-positive
and some gram-negative systems are resistant to a broad range of
mercuric compounds, including organomercurials like phenylmercuric
acetate (PMA) (7). This ability is due to the presence of an
organomercurial lyase (MerB) which cleaves the carbon-mercury bonds and
releases Hg(II). Narrow-spectrum resistance is observed when the
merB gene is missing (17). The systems are
regulated by transcriptional regulator MerR. In all cases studied, with
the exception of in Streptomyces lividans where MerR is a
repressor (14, 16), MerR is an activator/repressor
transcriptional regulator. In the presence of Hg(II), MerR binds Hg(II)
and activates its own transcription as well as that of the other
mer genes. In the absence of Hg(II), MerR binds tightly to
an operator and represses the system (7). In a few mercury
resistance operons, a second regulator gene, merD, is
present and binds weakly to the MerR operator site. MerD has been shown
to down-regulate the system (9, 10).
We have previously described mercury-resistant Streptomyces
strain CHR28, in which mercury resistance genes are encoded by the
large linear plasmid pRJ28 (330 kb) (13). CHR28 is an
environmental strain isolated from a heavily polluted site in the
Baltimore Harbor and might have developed resistance and/or regulation
mechanisms adapted to its environment which differ from those of
S. lividans 1326. Mercury resistance genes of the laboratory
strain S. lividans 1326 have previously been cloned and
sequenced (2, 16), and recently, the negatively regulated
repressor MerR has been purified and characterized (14). In
this study, we successfully constructed a DNA library of plasmid pRJ28
and cloned the mercury resistance genes. We report the analysis of a
5,921-bp sequence of the CHR28 mercury resistance operon and the
discovery of a novel putative regulatory gene, orfX.
Transcriptional analysis with a nuclease protection assay and reverse
transcription-PCR (RT-PCR) is reported.
Cloning and sequence analysis of the Streptomyces sp.
strain CHR28 mer operon.
Plasmid pRJ28 DNA was
purified by electroelution from pulsed-field electrophoresis agarose
gels. A library of plasmid pRJ28 was constructed in pBKSII. Screening
of about 900 clones with insert sizes ranging from 2 to 4 kb by using
probes MER-A, MER-B, and MER-RTP (12) allowed identification
of five overlapping clones encoding mercury resistance genes. Each
clone was sequenced on both strands by primer walking. The fragments
were assembled into a 5,921-bp contiguous stretch of sequence, which is
840 bp longer than the sequence of S. lividans 1326 mercury
resistance operon (16).
Seven open reading frames (ORFs) were found, and sequence comparison to
the mercury resistance operon genes merA, merB,
merR, merT, merP, and orfIV
(a putative transporter gene) of S. lividans 1326 (16) and other mercury resistance genes permitted
attribution of putative functions to each ORF. The analysis showed that
all the genes found in the S. lividans 1326 mercury
resistance operon were present in the same order in CHR28 (Fig.
1). The S. lividans 1326 mercury transporter genes, orfIV, merP, and
merT, the regulator gene merR, the mercuric
reductase gene merA, and the organomercurial lyase gene
merB were aligned to the CHR28 sequences and were found to
be highly similar (between 80 and 96% similarities at the nucleotide level and between 73 and 94% identities at the amino acid level). As
in S. lividans 1326, merR and merT, as
well as merP and orfIV, have overlapping
start-stop codons. A 500-bp sequence was obtained downstream of
merB, but no ORF was found in this region. Sequence comparison of the merA-merB region with S. lividans 1326 sequence revealed a 594-bp insert between the
merA and merB genes (Fig. 1). In this insert, a
new ORF was identified and termed orfX. This ORF is 411 bp
and encodes a putative 137-amino-acid protein.
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FIG. 1.
Structure of the mercury resistance operon of S. lividans 1326 (A) and Streptomyces sp. strain CHR28
(B). The promoter region is shown, and regulatory motifs are indicated,
deduced by homology with those of the S. lividans 1326 mer operon (2, 14).
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|
The mer operon in Streptomyces sp. strain CHR28
comprises two divergently oriented sets of four and three genes,
respectively. Genes merR, merT, merP,
and orfIV are transcribed leftward, and merA,
orfX, and merB are transcribed from left to
right. The promoter/operator region is 127 bp long and is located
between genes merR and merA (Fig. 1). Upstream of
merB, a 14-bp palindromic region identical to that in
S. lividans 1326 indicated the presence of a transcriptional terminator (1). Another palindromic sequence was found in
the region between merR and merA (Fig. 1). This
region is also found in S. lividans 1326 and is probably
involved in regulation (5). No specific structures were
found in the 201-bp region between orfX and merB.
The promoter/operator region is highly conserved when compared with the
S. lividans 1326 promoter region. Two 8-bp direct repeats
are also conserved in position but not in sequence between the two
promoter regions and have no known function, although they are thought
to be involved in regulation (Fig. 1). Delic et al. (4)
described two Streptomyces promoters with 10-bp direct
repeats and have implicated these repeats in regulation of chitinase genes.
Transcriptional analysis by RT-PCR and nuclease protection
assay.
Transcriptional studies using RT-PCR to locate
cotranscribed sets of genes indicated that merR and
merA are not cotranscribed and that merA,
orfX, and merB formed a contiguous transcript
(Fig. 2). Primer sets spanning intergenic
regions from merA to orfX and orfX to
merB gave amplification products with cDNA synthesized from
RNA prepared from cultures grown with HgCl2 and PMA (Fig. 2). No product was obtained for the primer set covering the divergently transcribed merR and merA genes (Fig. 2). The
number of mRNA transcripts in cells grown without mercury compounds may
be very low, explaining the lack of product with all primer sets in
this case (Fig. 2).
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FIG. 2.
RT-PCR analysis of RNA prepared from cultures grown with
0.05 mM HgCl2, 0.005 mM PMA, or without mercury with primer
sets R2F-A1R, A2F-R3R, and R4F-B1R. +, thermoscript reverse
transcriptase added;  , no Thermoscript reverse transcriptase added;
C+, positive control (PCR performed with 100 ng of
Streptomyces CHR28 genomic DNA); C, negative control (PCR
performed with no DNA added). A schematic representation of the
Streptomyces CHR28 mercury resistance operon is shown with
amplified regions indicated by thick lines.
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|
A nuclease protection assay was performed by using the Multi-NPA kit
(Ambion, Inc., Austin, Tex.). Primer probes were designed from the
merA, merB, and merR genes to allow
quantification of merA, merB, and merR
mRNA amounts in cells grown in the presence or absence of
HgCl2 and PMA and are as follows: MER-A (38 mer, position 3181 to 3216),
5 ' - GC TCCAGGC C GAC G TAG C C GCCGCCGAGAACCAGCCAA-3'; MER-B (48 mer, position 4989 to 5033),
5'-GCGTGCCCAGGATGGCGGGGAAGATCAGGGTGTCCAGGGCGCACCCAA-3'; and MER-R (31 mer, position 2324 to 2351):
5'-CACGCGGGGCTGGAGATACCGGCGTGCCAA-3' (the
underlined sequence is nonhomologous to the target gene sequence in
each case). Each oligonucleotide (10 pmol) was end labeled with T4
polynucleotide kinase with 10 pmol of [
-32P]ATP (6,000 mCi/mmol) using the Ready-to-Go kit (Pharmacia BioChem). Each probe was
gel purified to eliminate shorter-than-full-length oligonucleotides and
was eluted in Probe Elution Buffer (Ambion). The amount of radioactive
labeling of each probe was determined by scintillation counting, and
specific activities were determined: MER-A, 1.0 × 106
cpm/pmol; MER-B, 9.8 × 105 cpm/pmol; and MER-R,
5.0 × 105 cpm/pmol. Total RNA extracts (5, 10, and 30 µg) from cultures grown with 0.05 mM HgCl2, 0.005 mM PMA,
or no mercury were mixed with 5 fmol of each labeled probe (5- to
10-fold molar excess of probe over target mRNA) in a final reaction
volume of 100 µl. RNA and probes were ethanol precipitated, and
pellets were resuspended in 10 µl of Hybridization Buffer (Ambion).
RNA and probes were incubated overnight at 37°C. Nuclease digestion
treatment was carried out according to the manufacturer's
recommendations (Ambion) for 45 min at room temperature with a nuclease
mix diluted 1:100 in 1× Nuclease Digestion Buffer (Ambion). Nuclease
consisted of a mixture of S1 nuclease-RNase A-RNase T1.
Protected fragments were ethanol precipitated, dried pellets were
resuspended in 10 µl of Gel Loading Buffer (Ambion), and protected
fragments were separated on a 12% polyacrylamide gel. After
electrophoresis, the gel was dried and exposed to a Phosphoscreen
(Eastman-Kodak) for 48 h. The screen was scanned by using a Storm
PhosphorImager (Molecular Dynamics). Digitized images were analyzed
with the computer software ImageQuant (Molecular Dynamics).
Figure 3A shows that the signal intensity
increased with the amount of total RNA, indicating that an excess of
probe was present and validating the assay. The amount of
merA mRNA was equal to that of merB mRNA under
all conditions tested, thus confirming that merA and
merB are cotranscribed (Fig. 3A). The amount of merR mRNA was three to four times higher than the amounts of
merA or merB mRNA (Fig. 3A). The total mRNA
levels of mer genes were two to three times higher when
cells were grown in the presence of 0.05 mM HgCl2, compared
with growth in 0.005 mM PMA (Fig. 3A). Numbers of mer
transcripts were very low when cells were grown without mercuric
compounds. In the absence of mercury, the amount of merR
mRNA was four times higher than merA and merB but
six times lower than merR mRNA from cells grown in the
presence of 0.05 mM HgCl2. Analysis of these data reveals
that the proportion of each transcript (merR,
merA, or merB) remained constant under each
condition tested (Fig. 3B). For each growth condition and amount of RNA
tested, the proportion of merA and merB
transcripts is consistently about 30% (Fig. 3B) and that of
merR is about 70%. These results suggest that both sets of
genes are coregulated. We hypothesize that as mercuric ion
concentration increases, Hg(II) binds to the repressor MerR,
conformational changes make MerR unable to bind to the promoter region,
and transcription starts in both directions. The higher transcript
levels of merR-orfIV may be due to that promoter being more
efficient than the merA-merB promoter. Quantities of each of
the three mRNAs were lower when cells were grown in the presence of
0.005 mM PMA than in the presence of 0.05 mM HgCl2. It
appears that the system maintains a high basal level but that an
increased concentration of Hg(II) increases transcription rates. The
organic moiety of PMA is cleaved by MerB, and Hg(II) is released in the
cell. However, the amounts of Hg(II) generated by this route are lower
than the amounts of Hg(II) in cells grown in the presence of 0.05 mM
HgCl2.
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FIG. 3.
(A) Nuclease protection assay with probe MER-R (light
gray bars), MER-A (dark gray bars), and MER-B (black bars), with 5, 10, and 30 µg of total RNA in each case. Quantitative analysis in
relative intensity units (RIU) was done with the ImageQuant Software
(Molecular Dynamics). (B) Intensity relative to a total intensity of
100%.
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|
orfX: a putative second regulator gene.
Comparison
of the CHR28 mercury resistance operon sequence to that of S. lividans 1326 revealed a 594-bp insert between gene merA and merB. An ORF encoding a putative
137-amino-acid protein named OrfX was found in the 5' end of this
insert. The start codon is located 22 bp downstream of the MerA stop
codon. A putative ribosomal binding site is located in this region.
Sequence analysis of OrfX showed homology with the MerR transcriptional
regulator family (NolA, MerD, MerR [Bacillus and
Tn501], and TipA) (Fig. 4).
One major characteristic derived from amino acid alignments between
members of this family is the high homology found at the N terminus of
these proteins. This region is the DNA binding domain and contains a
helix-turn-helix motif as indicated in Fig. 4. OrfX has the highest
identity (42.2%) in this N-terminal region with NolA, a
transcriptional regulator of nodulation in Bradyrhizobium japonicum (15). The function of OrfX is unknown, and
analysis of the C-terminal domain indicates that it is unlikely to bind mercuric ions. Two cysteine residues are present in the predicted protein and only one is located in the C-terminal domain, the Hg(II)
binding domain in MerR regulators, in which three highly conserved
cysteine residues are located. However, it cannot be excluded that OrfX
may be involved in the binding of organomercurial compounds. Figure 4
shows that OrfX has 32.6% identity with MerD, a protein which
down-regulates the mercury resistance operon in Serratia
marcescens (9, 10). Using a DNase footprinting assay, MerD has been shown to bind to a palindromic region located in the
promoter/operator region (9). It is likely that OrfX also binds at the promoter/operator of CHR28 mer genes.
orfX is located between merA and merB
and is cotranscribed with these genes, as shown by RT-PCR (Fig. 2).
This implies that as binding of Hg(II) to MerR derepresses the system,
the amount of OrfX is likely to increase. OrfX then has the potential
to bind to the promoter/operator region and activate or down-regulate
the operon, as is the case with S. marcescens MerD
(9). In this case, binding of MerD to the MerR operator was
weak compared to the binding of MerR. It is possible that binding of
OrfX at the promoter/operator represents a feedback inhibition
mechanism for gene expression.
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FIG. 4.
(A) Amino acid alignment of the N-terminal region of
B. japonicum NolA (M58360), Bacillus cerus MerR
(MerRb) (Y09027), Pseudomonas sp. Tn501 MerR
(MerRt) (K02503), S. lividans TipA (M24524), and S. marcescens MerD (M15049) with OrfX from CHR28. The dashed line
represents the helix-turn-helix (HTH) motif of the putative DNA binding
site. (B) Table shows the similarity and identity between several
proteins and OrfX over the region shown above.
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Other transcriptional regulators of the MerR family have been
described. However, like OrfX, their functions are unknown. These
include a merR gene found on Tn5467, a
Tn21-like transposon located on the Thiobacillus
ferrooxidans plasmid pTF-FC2 (3). In this case, no
other mer genes were found on the plasmid. However, a
mercury resistance system is present on the chromosome and includes duplicated merR genes (3, 7, 8). This is also the
case with gene yhdM in Escherichia coli, where
adjacent ORFs have no similarity to any of the proteins usually
associated with mercury resistance. Analysis of the complete sequence
of the Haemophilus influenzae genome has revealed several
candidate ORFs belonging to the MerR transcriptional regulator family
(6). Our finding of OrfX in the mercury resistance operon
present on a giant linear plasmid in Streptomyces sp. strain
CHR28 adds an additional putative transcriptional regulator to this family.
Nucleotide sequence accession number.
The sequence of the
Streptomyces sp. strain CHR28 mer operon has been
deposited in GenBank under accession no. AF222792.
| |
ACKNOWLEDGMENTS |
This study was supported by the Schering-Plough Research Institute
and a NASA Biotechnology Grant (to J.D. and F.T.R.).
We thank Ann Horan for helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center of Marine
Biotechnology, Columbus Center, Suite 236, 701 East Pratt St.,
Baltimore, MD 21202. Phone: (410) 234-8883. Fax: (410) 234-8896. E-mail: hillr{at}umbi.umd.edu.
Contribution no. 517 from the Center of Marine Biotechnology.
Present address: Chemistry Department, The Johns Hopkins
University, Baltimore, MD 21218.
| |
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Journal of Bacteriology, April 2000, p. 2345-2349, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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