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Journal of Bacteriology, October 2001, p. 5651-5658, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5651-5658.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cloning and Functional Analysis of the
pbr Lead Resistance Determinant of Ralstonia
metallidurans CH34
B.
Borremans,1
J. L.
Hobman,2
A.
Provoost,1
N. L.
Brown,2 and
D.
van der Lelie1,*
VITO, Vlaamse Instelling voor Technologisch Onderzoek,
Environmental Technology Centre, Boeretang 200, 2400 Mol,
Belgium,1 and School of Biosciences,
University of Birmingham, Edgbaston, Birmingham B15 2TT, United
Kingdom2
Received 14 May 2001/Accepted 11 July 2001
 |
ABSTRACT |
The lead resistance operon, pbr, of Ralstonia
metallidurans (formerly Alcaligenes eutrophus)
strain CH34 is unique, as it combines functions involved in uptake,
efflux, and accumulation of Pb(II). The pbr lead
resistance locus contains the following structural resistance genes:
(i) pbrT, which encodes a Pb(II) uptake protein; (ii)
pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii)
pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which
encodes a predicted prolipoprotein signal peptidase. Downstream of
pbrC, the pbrD gene, encoding a
Pb(II)-binding protein, was identified in a region of DNA, which was
essential for functional lead sequestration. Pb(II)-dependent inducible
transcription of pbrABCD from the PpbrA promoter is regulated by PbrR, which belongs to the MerR family of
metal ion-sensing regulatory proteins. This is the first report of a
mechanism for specific lead resistance in any bacterial genus.
| |
INTRODUCTION |
The presence of toxic heavy metals
in the environment has resulted in the development or acquisition by
bacteria of genetic systems that counteract their effects. Many
bacterial heavy metal resistance systems are based on efflux, and two
groups of efflux systems have been identified. These can be either
P-type ATPases, e.g., the Cu(II), Cd(II), and Zn(II) ATPases of
gram-negative bacteria (31), or chemiosmotic pumps, e.g.,
the three-component divalent-cation efflux systems cnr,
ncc, and czc of Ralstonia metallidurans (formerly Alcaligenes eutrophus) CH34
(35).
Lead resistance has been reported in both gram-negative and
gram-positive bacteria isolated from lead-contaminated soils, with
Pseudomonas marginalis showing extracellular lead exclusion and Bacillus megaterium demonstrating intracellular
cytoplasmic lead accumulation (28). Pb(II)-resistant
strains of Staphylococcus aureus and Citrobacter
freundii that accumulated the metal as an intracellular
lead-phosphate have also been isolated (15, 16), though
the molecular mechanism of detoxification remains to be elucidated.
Efflux of Pb(II) has also been reported for the CadA ATPase of S. aureus and the ZntA ATPase of Escherichia coli
(27).
R. metallidurans strain CH34 contains at least seven
determinants encoding resistances to toxic heavy metals, located on one of the two endogenous megaplasmids, pMOL28 and pMOL30 (for a review, see reference 35). One of these determinants, located on
pMOL30 (20), mediates resistance to Pb(II). In this paper
we describe the isolation and characterization of the Pb(II)
resistance determinant, pbr, from pMOL30.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
R.
metallidurans and E. coli were grown in 869 medium
(20) at 30 and 37°C, respectively. Antibiotic resistance
was selected on media supplemented with 20 µg of tetracycline or 100 µg of ampicillin per ml, as appropriate. To test for Pb(II)
resistance, cells were grown on RM medium, a modified 284 gluconate
minimal medium (20) in which Tris-HCl is replaced by 20 mM
morpholinepropanesulfonic acid (MOPS)-NaOH (pH 7) and Na is replaced
by 0.95 mM beta-glycerol-phosphate. These two components were filter
sterilized and added separately after the RM medium was autoclaved.
Pb(II) was added as
Pb(NO3)2 at the required concentrations.
Molecular cloning techniques, sequencing, and
electroporation.
Standard molecular cloning techniques and plasmid
DNA isolation from E. coli were performed as described by
Sambrook et al. (29). Electroporation was used to
introduce plasmid DNA into R. metallidurans and E. coli (36).
Subcloning, primer walking, and cycle sequencing strategies were used
for DNA sequencing. The sequences were analyzed using GCG software
(Genetics Computer Group, Inc., Madison, Wis.).
Cloning of the pbr lead resistance
determinant.
The pbr Pb(II) resistance determinant was
subcloned from an R. metallidurans CH34 cosmid library
constructed in pLAFR3 (8). The CH34 cosmid bank was
introduced by electroporation into the plasmid-free, heavy
metal-sensitive derivative of strain CH34, R. metallidurans
AE104 (20). Transformants were selected for resistance to
tetracycline and Pb (0.4 mM) on RM medium. The cosmids from 10 independent, Pb(II)-resistant transformants were isolated and digested
with EcoRI. Subclones in vector pRK415 (14)
were similarly introduced into R. metallidurans AE104 and
tested for the lead resistance phenotype conferred.
Pb(II) uptake experiments.
The uptake of Pb(II) was based on
a previously described protocol for Al(III) uptake (11).
All cultures were grown at 30°C in 200 ml of RM medium, in which no
precipitation of Pb(II) occurs. When the optical density at 660 nm reached a value of approximately 0.8, the cells were concentrated
fivefold by centrifugation and resuspended in fresh RM medium
containing 0.2 mM Pb(NO3)2.
As a negative control, the experiment was also performed in RM medium without Pb(NO3)2. After
2 h the cells were collected by centrifugation (2,000 × g, 10 min, Sorvall centrifuge, SS34 rotor) and subsequently washed with 50 ml of 0.2 M EDTA and 50 ml of sterile distilled water to
remove adventitiously bound Pb(II). The cells were freeze-dried and
weighed, and the amount of Pb(II) was determined using inductively coupled plasma analysis. The amount of accumulated Pb(II) per milligram
of biomass was calculated.
Transcript analysis.
The start point of transcription was
determined by primer extension analysis. Total RNA was extracted, using
the hot-acid phenol method (1) from exponential phase
R. metallidurans AE2473 grown in RM medium for 2 h with
or without 0.2 mM Pb(NO3)2.
The RNA preparation was treated with 10 U of RQ1 RNase-free DNase I for
30 min at 37°C (Promega, Southampton, United Kingdom). The primer
PbrApe, 5'-GCGCCAACCGTGCTCGGTTCTGGG-3', was labeled with 25µCi of [
-32P]ATP (NEN Life Sciences,
London, United Kingdom) by incubation with 10 U of T4 polynucleotide
kinase (Life Technologies, Paisley, Scotland) for 30 min at 37°C and
used with 10 µg of total RNA for primer extension analysis. Labeled
primer and RNA were heated to 85°C for 5 min and then at 45°C
overnight prior to ethanol precipitation. Primer-RNA hybrids were
resuspended in the reaction mixture for Superscript II reverse
transcriptase (Life Technologies) and incubated at 42°C for 60 min
and then at 72°C for 10 min after the addition of 400 U of
Superscript II. The labeled cDNA was run on a 6% (wt/vol) denaturing
polyacrylamide gel alongside a sequencing reaction mixture
generated using the PbrApe primer.
The reverse transcription (RT)-PCR method described by Gupta et al.
(
9) for transcript analysis of the
sil operon
was adapted
for this study of
pbr transcription. RNA was
prepared as described
above from AE2473 and AE104 grown for 2 h
with or without 0.2
mM
Pb(NO
3)
2. RNA was annealed
in the presence of deoxynucleoside
triphosphates to the primer pbrD-RT,
5'-CTACAGGCGTAGGCACCGTCG-3'
(positions 7231 to 7211; see
Results for numbering), for 5 min
at 65°C and then cooled on ice.
First-strand buffer, dithiothreitol,
and RNasin (Promega) were added to
the annealed primer-RNA hybrids,
and the reaction mixture was incubated
at 42°C for 60 min and
then at 72°C for 10 min after the addition
of Superscript II reverse
transcriptase. cDNA (2 µl) from the RT
reaction was added to a
PCR with the primers pbrA-N term,
5'-ATGAGCGAATGTGGCTCGAAG-3'
(positions 2730 to 2750), and
pbrA-C term, 5'-TCATCGACGCAACAGCCTCAA-3'
(positions 5126 to
5106). PCR conditions were 96°C for 3 min (step
1), followed by
96°C for 60 s (step 2), 58°C for 60 s (step 3),
and
72°C for 3 min (step 4). Steps 2 to 4 were repeated 39 times
followed
by a further incubation at 72°C for 5 min. Appropriate
controls
were run (see Results). RT-PCR products were run on a
0.7%
(wt/vol) agarose Tris-borate-EDTA
gel.
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RESULTS |
Restriction map of pMOL1027 and identification of the minimal
Pb(II) resistance region.
A cosmid library of R. metallidurans total DNA (8) was screened for Pb(II)
resistance clones in the heavy metal-sensitive strain R. metallidurans AE104 (20). Ten identical
Pb(II)-resistant clones were identified, and the restriction map of one
of the cosmid clones, pMOL1027, is shown in Fig.
1. No resistance to other divalent metal
salts, including Zn(II), Cd(II), Co(II), Cu(II), Ni(II), and Hg(II),
was observed, even after induction by Pb(II). The minimal region of DNA
required for Pb(II) resistance was determined by subcloning from
pMOL1027 into the vector pRK415 (14) and testing R. metallidurans AE104 transformants for Pb(II) resistance. The
Pb(II) resistance locus was localized initially to a 12-kb
BamHI fragment in clone pMOL1504. Several subclones of
pMOL1504 were made (Fig. 1). The 8.9-kb
EcoRI-EcoRI-BamHI fragment of pMOL1558
conferring Pb(II) resistance was completely sequenced (EMBL
accession no. AJ278984). pMOL1576 contains the 6.4-kb EcoRI-EcoRI-NsiI fragment (positions 1 to 6383) and also conferred Pb(II) resistance. Strains carrying
plasmids pMOL1544, containing the 5.6-kb
EcoRI-EcoRI-PstI fragment (positions 1 to 5598), and pMOL1540, containing the 3.8-kb
EcoRI-EcoRI fragment (positions 1 to 3852), were
Pb(II) sensitive.
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FIG. 1.
Restriction map of pMOL1027 and identification of the
minimal region required for Pb(II) resistance. The bars indicate the
positions of the subcloned fragments and are flanked by the restriction
sites used, as follows: BamHI (B), EcoRI
(E), HindIII (H), NsiI (N), and
PstI (P). On the right side of each bar, the plasmid and
strain number for the corresponding subclone are indicated as well as
the lead resistance phenotype, defined as MIC in millimolar
Pb(NO3)2 compared with the MIC of 0.15 mM for
the plasmid-free strain AE104. The physical map of the 8,890-bp
EcoRI-EcoRI-BamHI fragment
required for Pb(II) resistance is shown in more detail. The positions
of the pbrT, pbrR, pbrA,
pbrB, pbrC, and pbrD genes
plus the sizes of their corresponding ORFs (in numbers of amino acids),
the incomplete transposon (Tnxxxx, similar to
Tn5271 [22]) flanking the
pbr operon and its presumed transposase gene
(tnp*), and the pbr-promoter-operator
region (pbr P/O) as well as the important restriction
sites used for subcloning are indicated.
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|
Analysis of the Pb(II) resistance region.
Within the 8.9-kb
DNA sequence, seven predicted open reading frames (ORFs) were
identified in an operon-like structure, encoding proteins with the
predicted sizes of 652 (C), 145 (C), 798, 112, 206, 241, and 303 amino
acids (aa), where C represents the sequence encoded by the
complementary strand. ORF652, ORF145, ORF798, ORF112, and ORF206 were
located on the 6.4-kb EcoRI-EcoRI-NsiI
fragment of pMOL1576, which was the minimal Pb(II) resistance clone.
Both database search analysis and functional analysis were used to determine the roles of the different ORFs of the pbr operon.
ORF652 (positions 127 to 2085, complement) encodes a predicted membrane
protein that contains 8 to 10 potential transmembrane
regions. The
N-terminal region of the ORF652 protein (aa 106 to
218) shows sequence
similarity with the C-terminal cytochrome
c6 domain of the diheme C-type cytochrome
FixP from
Azorhizobium caulinodans (30% identity over the
last 113 aa) (
18), while
the C-terminal region (aa 223 to
619) shows sequence similarity
with a large family of integral membrane
proteins, including the
FTR1 plasma membrane iron permease of
Saccharomyces cerevisiae (30% identity) (
32).
Expression of ORF652 in the absence of
the other structural genes of
the
pbr operon resulted in Pb(II)
hypersensitivity in
R. metallidurans AE2451 (Fig.
1). For this
strain, the MIC
of Pb(NO
3)
2 was 0.05 mM
compared to 0.15 mM determined
for the plasmid-free strain AE104.
ORF652 is therefore suggested
to encode a Pb(II) uptake protein,
located in the inner membrane.
As it is suggested to be functionally
analogous to
merT, encoding
the Hg uptake protein of the
mer operon (
21), ORF652 was named
pbrT.
The predicted ORF145 gene product (positions 2206 to 2643, complement)
showed strong similarity to members of the MerR family
(for reviews,
see references
10 and
34). MerR is the
regulator
of the Hg resistance
mer operon. The ORF145
protein was named
PbrR.
The predicted protein encoded by ORF798 (positions 2730 to 5126) showed
strong similarity to members of the P-type ATPase
family involved in
heavy metal efflux, such as CadA of
S. aureus (
23), and was named PbrA. Although plasmid pMOL1544 (Fig.
1)
did not encode full Pb(II) resistance, a Pb(II) hypersensitivity
phenotype like that found for pMOL1540 was not observed. This
suggests
that the PbrA protein is a functional Pb(II) efflux ATPase,
able to
counteract Pb(II) uptake by
PbrT.
The DNA fragment that contained the
pbrR-
pbrA
intergenic region (positions 2644 to 2729) showed striking similarity
to the
merO/P and
zntO/P regions (
5,
24) (Fig.
2B). The transcription
start site of the P
pbrA promoter was determined by primer
extension
on RNA isolated from strain AE2473 grown with and without 0.2
mM Pb(NO
3)
2. Transcription
started at C
2701 and was located at
the same
distance (7 bp) from the
10 sequences as the transcription
start
sites in the P
merT and P
zntA promoters (Fig.
2A
and B).
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FIG. 2.
Comparison of the PzntA,
PmerTPAD, and PpbrA promoters. (A)
Primer extension analysis of the PpbrA promoter region.
Autoradiograph of a 6% acrylamide sequencing gel showing the
PpbrA promoter region DNA sequence aligned with the
primer extension product generated as described in Materials and
Methods. Total RNA was isolated from log-phase R.
metallidurans AE2540 cells grown for 2 h with (+) and
without () 0.2 mM Pb(NO3)2. (B) Mapping of
the transcriptional start sites of the PzntA
(5), PmerTPAD (17), and
PpbrA (shown in panel A) promoters. The positions of the
35 and 10 regions of the promoters are underlined. The +1
nucleotide is indicated in bold.
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Analysis of the predicted ORF112 protein reveals characteristics of a
membrane lipoprotein. These include the presence of
an N-terminal
leader sequence with the processing site Ile-Ala-Ser-Cys,
which fits
well with the consensus sequence for recognition by
prolipoprotein
signal peptidases (
4), and two hydrophobic regions
which
are potential transmembrane sequences. Inactivation of ORF112,
as in
plasmid pMOL1544, results in a Pb(II)-sensitive phenotype,
indicating
its essential role in Pb resistance. The gene was designated
pbrB. PbrB shows amino acid sequence similarity to a family
of
membrane-bound components of bacterial transport systems of which
BcrC, which is required for bacitracin resistance in
Bacillus licheniformis (
25), is the best characterized. The
ORF206 protein
showed strong similarity (30 and 46% identity) with
prolipoprotein
signal peptidases from
Pseudomonas
fluorescens (
12) and
E. coli (
37). The ORF206 protein was named
PbrC.
Downstream from the minimal region required for Pb(II) resistance, two
further ORFs, ORF241 and part of ORF303, were identified.
ORF241
(positions 6645 to 7232) encodes a predicted protein containing
a
hypothetical metal binding site with the sequence
Cys-7X-Cys-Cys-7X-Cys-7X-His-14X-Cys,
with a high proportion of Pro and
Ser residues between the cysteines.
RT-PCR (see below) showed that
Pb-dependent transcription of ORF241
is coupled to that of
pbrABC.
Since the predicted ORF241 protein contains a region with a potential
heavy metal binding site, its role, if any, in Pb(II)
management was
investigated, even though it did not seem to be
absolutely required for
Pb(II) resistance. Pb(II) accumulation
levels in the strains AE104
(plasmid free), AE2451, AE2472, AE2473,
and AE2540 were compared after
2 h of incubation in the presence
of 0.2 mM
PbNO
3. The results are presented in Fig.
3. For strains
AE2451, AE2472, and
AE2540, which contained plasmids with the
pbrTRA',
pbrTRAB', and
pbrTRABC regions, respectively, no
increase
in Pb(II) accumulation was observed compared to the
Pb(II)-sensitive
control strain AE104. However, with strain AE2473
(
pbrTRABCD),
a significant increase in Pb(II) accumulation
(4.6 instead of
1.2 pg/µg [dry weight]) was observed. This suggests
that the region
downstream of
pbrTRABC, which encodes
ORF241, is involved in Pb(II)
accumulation, and ORF241 was renamed
pbrD.
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FIG. 3.
Pb(II) accumulation in the strains AE104 (plasmid free),
AE2473 (pbrTRABCD), AE2472 (pbrTRAB'),
AE2451 (pbrTRA'), and AE2540 (pbrTRABC)
after 2 h of incubation in the presence of 0.2 mM
PbNO3. Lead accumulation is expressed in nanograms of
Pb/microgram (dry weight ([DW]).
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The pbrABCD genes form one transcriptional
unit.
RT-PCR was used to test whether the transcription of
pbrB, pbrC, and pbrD was Pb dependent
and coupled to that of pbrA. Total RNA was extracted from
strain AE2473 grown in the presence or absence of 0.2 mM
Pb(NO3)2, and cDNA
synthesis was initiated from primer pbrD-RT (position 7211 at the 3'
end of pbrD). Subsequently, primers directed against the
pbrA gene were used to obtain RT-PCR products. For cells
grown in the absence of lead no RT-PCR product was obtained. However,
when cells were induced with 0.2 mM
Pb(NO3)2, an RT-PCR product
with the expected size of approximately 2,400 bp was found. This
experiment shows that pbrA and pbrD, and
consequently pbrB and pbrC, are transcribed from
one large mRNA, the synthesis of which is Pb(II) dependent.
| |
DISCUSSION |
This paper describes the first bacterial resistance determinant
that is specific for lead. It has recently been shown that heavy metal
resistance systems, such as the cad Cd(II) resistance of
S. aureus and the znt Zn efflux system of
E. coli, can also confer Pb(II) resistance in E. coli (26, 27). In these cases resistance to several
metals, including Pb(II), involves a metal-translocating P-type ATPase.
These constitute a highly conserved and widely distributed family of
efflux ATPases, which contain heavy metal-associated metal binding
domains (6) that are also found in other proteins interacting with heavy metals, including the prokaryotic MerA and MerP
(Hg) proteins and the eukaryotic Menkes and Wilsons (Cu) proteins
(30, 31). The PbrA Pb(II) ATPase is phylogenetically grouped with the CadA-type Cd ATPases and the ZntA Zn/Cd ATPase (Fig.
4), and they form a group distinct from
the Cu/Ag-type ATPases. However, unlike CadA and ZntA, PbrA possesses
two heavy metal-associated motifs with the amino acid sequence
Cys-Pro-Thr-Glu-Glu instead of the consensus sequence Cys-X-X-Cys
(Table 1). This difference, where one Cys
is replaced by two Glu, might reflect the preferential coordination of
Pb(II) to oxygen rather than to sulfur (2, 13).
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FIG. 4.
Phylogenetic tree of P-type heavy metal transport
ATPases. Scale designation and bootstrap values are indicated for the
branches. The sequence ID numbers are shown between brackets. The
sequence of the R. metallidurans CopF Cu-ATPase was
recently determined (Borremans and van der Lelie, EMBL accession no.
AJ278983). Bar represents 0.1 nucleotide substitution per 10 nucleotides.
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A MerR-like regulator, PbrR, controls transcription of the
pbr structural genes, with the first gene downstream of the
pbr operator-promoter encoding a P-type ATPase, like the
zntA operon (5) and copA
(33). However, zntR and cueR are
physically distant on the chromosome from their cognate promoters,
whereas pbrR is divergently transcribed from pbrA
with an operator-promoter (O/P) structure similar to that found in the
gram-negative mer operons, Tn501 and
Tn21 (10).
In contrast to the cad and znt operons, which
both appear to comprise a regulatory gene plus an efflux-ATPase only,
additional proteins are required for maximal Pb(II) resistance in
R. metallidurans CH34. These are PbrT, PbrB, PbrC, and PbrD.
Our model for pbr-encoded Pb(II) resistance is presented in
Fig. 5 and involves a Pb(II) uptake
system encoded by PbrT. Expression of PbrT in the absence of PbrABCD
results in Pb(II) hypersensitivity, probably due to increased Pb(II)
uptake into the cytoplasm. The result of this Pb(II) uptake would be to
reduce the interaction of free Pb(II) with side chains of membrane and
periplasmic proteins, which would cause extensive cellular damage. Once
Pb(II) has entered the cytoplasm, it can be exported by the PbrA Pb(II)
efflux ATPase or be bound by the PbrD protein, which may function as a
chaperone for Pb(II). PbrD is not absolutely required for Pb(II)
resistance, but cells lacking PbrD show a decreased accumulation of
Pb(II) compared to that observed for wild-type cells, and this
accumulation may protect against free exported Pb(II) and the futile
cycle of Pb(II) uptake and export.
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FIG. 5.
Model for the pbr Pb(II) resistance
operon-encoded lead resistance of R. metallidurans CH34.
The model involves the following proteins: PbrT, which transports
Pb(II) into the cytoplasm; PbrA, the Pb(II) efflux ATPase; PbrB and
PbrC, a Pb(II) transport-facilitating lipoprotein and its
prolipoprotein signal peptidase, respectively; and PbrD, a protein
involved in Pb(II) sequestration. The PbrR protein, which mediates
Pb(II)-inducible transcription from its divergent promoter, regulates
the pbr operon.
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|
The PbrA Pb(II) efflux ATPase has been shown to be functional and able
to compensate for the Pb(II) uptake driven by PbrT. However, for full
Pb(II) resistance PbrB and PbrC are required. PbrB and related proteins
may be part of a new family of transporter-assisting resistance
proteins. Comparison of PbrB with the EMBL database resulted in 16 hits. However, with the exception of the BcrC protein of B. licheniformis, all others were hypothetical membrane
(lipo)proteins. The BcrC gene of B. licheniformis
encodes a hydrophobic membrane protein; this protein and the BcrB
membrane protein function as membrane components of the bacitracin
resistance ABC transporter (25). Inactivation of BcrC
results in bacitracin sensitivity, and inactivation of PbrB results in
Pb(II) sensitivity. The PbrB lipoprotein may promote transfer of Pb(II)
from the periplasm to the outer membrane. This would result in a
decreased Pb(II) uptake by PbrT.
At increased concentrations of Pb(II), metal removal from the solution
was observed during the late log phase and the stationary phase, during
which a progressive increase of the pH (up to 9) was observed (results
not shown). At this increased pH the formation of lead complexes with
hydroxide and carbonates will be strongly favored and should play an
important role in avoiding reentry of Pb(II). A similar phenomenon has
been described for the czc cadmium-zinc-cobalt resistance
system of R. metallidurans CH34 (7).
The presence of pbrC, encoding a predicted prolipoprotein
signal peptidase, in the Pb(II) resistance determinant of pMOL30 is the
first identification of a prolipoprotein signal peptidase gene as part
of a heavy metal resistance operon. Since pbrB and pbrC are part of a single transcriptional unit, we
hypothesize that the PbrC prolipoprotein signal peptidase is required
for the processing of the PbrB prolipoprotein. So, either PbrC is specifically required for the processing of PbrB or, alternatively, the
availability of chromosomally encoded prolipoprotein signal peptidases
is insufficient for the processing of PbrB, as has been suggested for
the ORF1 proteins encoded by pTA1015 and pTA1040 (19),
which were found in an operon-like organization with signal peptidase
genes. The organization of pbrABCD in one transcriptional unit would result in fine-tuning of both the transcription and posttranslational processing of these proteins.
| |
ACKNOWLEDGMENTS |
We are grateful to Tatiana Vallaeys for having provided the
phylogenetic analysis of the ATPases. Philippe Corbisier, Safieh Taghavi, Max Mergeay, and Ludo Diels are acknowledged for fruitful and
cooperative discussion.
This collaboration between VITO and The University of Birmingham was
supported by grant ENV4-CT95-0141 from the European Commission and by
grant B10333 to N.L.B. from the Biotechnology and Biological Sciences
Research Council.
| |
FOOTNOTES |
*
Corresponding author. Present address: Brookhaven
National Laboratory (BNL), Biology Department, Upton, NY 11973-5000. Phone: (631) 344-5349. Fax: (631) 344-3407. E-mail:
vdlelied{at}bnl.gov.
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Journal of Bacteriology, October 2001, p. 5651-5658, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5651-5658.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
