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Appl Environ Microbiol, February 1998, p. 453-458, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Alcaligenes eutrophus as a Bacterial
Chromate Sensor
Nicola
Peitzsch,1
Günther
Eberz,2 and
Dietrich H.
Nies1,*
Institut für Mikrobiologie, D-06099
Halle,1 and
Department of Central
Research/Biotechnology, Bayer AG, D-51368
Leverkusen,2 Germany
Received 16 September 1997/Accepted 12 November 1997
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ABSTRACT |
In Alcaligenes eutrophus CH34, determinants encoding
inducible resistance to chromate (chr) and to cobalt and
nickel (cnr) are located adjacent to each other on plasmid
pMOL28. To develop metal-sensing bacterial strains, a cloned part of
plasmid pMOL28, which contains both determinants, was mutated with
Tn5-lacZ. The chr::lacZ
fusions were specifically induced by chromium; cnr was induced best by Ni2+ but was also induced by
Co2+, Mn2+, chromate, Cu2+,
Cd2+, and Zn2+. The broad-host-range IncP1
plasmid pEBZ141, which contains a chr::lux fusion, was constructed.
A. eutrophus AE104(pEBZ141), carrying a
chr::lux transcriptional fusion,
could be used as a biosensor for chromate when cultivated in glycerol
as an optimal carbon source. Chromate and bichromate were the best
inducers; induction by Cr3+ was 10 times lower, and other
ions induced only a little or not at all. Interactions among induction
of the chr resistance determinant, chromate reduction,
chromate accumulation, and the sulfate concentration of the growth
medium were demonstrated.
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INTRODUCTION |
Alcaligenes eutrophus
CH34 and related bacteria are adapted to survive in environments with
high concentrations of heavy metal ions (8). Strain CH34
contains at least seven determinants encoding resistances to toxic
heavy metals; these determinants are located either on the bacterial
chromosome or on one of the two indigenous large plasmids, pMOL28 (180 kilobase pairs [kb] [35]) and pMOL30 (238 kb
[9, 22]). A. eutrophus has recently been
reclassified as Ralstonia eutropha (38). However,
strain CH34 and related strains are, though closely related to R. eutropha, different from its type strain. Since classification of
all the metal-resistant, CH34-like organisms is under way
(18a), strain CH34 should remain classified as A. eutrophus until this work is done.
On plasmid pMOL28, two inducible metal resistance determinants are
located adjacent to each other: the cnr determinant encodes resistance to Co2+ and Ni2+ (17) and
physiologically is based on metal cation efflux; the chr
determinant gives resistance to chromate (22).
Interestingly, in serpentine soils, nickel, chromium, and cobalt are
present in high concentrations (1). The adjacent locations
of the resistance determinants cnr and chr may be
the result of the adaptation of A. eutrophus to such an
environment.
The mechanism of chromate resistance is reduced accumulation of
chromium (26), but chromate efflux has not been
demonstrated. There are three open reading frames in the sequence of
the 2.6-kb EcoRI fragment encoding chr:
chrB, chrA, and ORF3, which is not essential for
chromate resistance and not complete within the 2.6-kb EcoRI
fragment (22). ChrA is a membrane-bound protein and probably
responsible for the resistance. The function of ChrB, however, is
unclear.
In this investigation, regulation of chr and cnr
was studied with lacZ fusions. To deepen our understanding
of chr induction, a lux-coupled chromate sensor
was developed. When the sensor was characterized, interactions among
chr-dependent chromate resistance, sulfate metabolism, and
chromate reduction were revealed. This shed some light on the
unexpected complexity of the chromate metabolism of A. eutrophus.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
The
bacterial strains and plasmids used in this study are listed in Table
1; some plasmid constructs are depicted
in Fig. 1. Tris-buffered mineral salts
medium (19) was used for growing A. eutrophus.
Additionally, a phosphate-buffered mineral salts medium (29)
and a HEPES-buffered mineral salts medium were used. The HEPES-buffered
medium contained the following (per liter of H2O): 0.3 mM
Na2KPO4, 0.2 mM K2HPO4,
50 mM HEPES buffer (pH 7.0), 2 g of NH4Cl, 0.2 g
of MgSO4 · 7H2O, 10 mg of
CaCl2 · 2H2O, and 5 mg of
FeCl3 · 6H2O. Analytical-grade salts of
CdCl2 · H2O, ZnCl2, CoCl2 · 6H2O, NiCl2 · 6H2O, AlCl3, MnCl2 · 6H2O, MnSO4 · H2O, CrCl3, CuCl2 · 6H2O,
K2CrO4,
K2Cr2O4,
Na2WO4, Na3VO4,
Na3AsO4, and Na2MoO4
were used to prepare 1 M stock solutions, which were sterilized by
filtration. Solid Tris-buffered media contained 2.0% (wt/vol) agar.
Nutrient broth (Difco) was used as complex medium for A. eutrophus, and Luria broth (28) was used for
Escherichia coli. Metal resistance was tested on solid
Tris-buffered mineral salts medium containing 0.2% (wt/vol) sodium
gluconate and either 0.2 mM chromate, 1 mM Ni2+, or 1 mM
Co2+.
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FIG. 1.
Insertion points of Tn5-lacZ transposons in
the cnr-chr region of plasmid pMOL28. Plasmid pDNA206
(21), containing the cnr-chr region of plasmid
pMOL28, was mutated with  ::Tn5-lacZ B20
(33), and derivatives which had lost metal resistance were
selected. The points of insertion of the transposons were determined by
digestion with EcoRI, BamHI, or
HindIII and are indicated by the positions of the
circles. The promoterless lacZ gene on the transposon was
transcribed either from left to right (circles below the line) or from
right to left (circles above the line). Grey circles indicate induction
by chromate; the filled triangle indicates the position of the fusion
in plasmid pDNA265. Black circles indicate induction by
Ni2+; numbers with the circles indicate plasmids pDNA255,
pDNA238, pDNA228, and pDNA229. The number 257 and the square give the
position of the transposon insertion in plasmid pDNA257, which did not
lead to a loss of metal resistance. Physical maps of the cnr
determinant (17) with the cnrYXHCBA genes and of
the chr determinant (21) with the
chrBA genes, ORF3, and the newly proposed ORF13 are drawn to
scale above the transposon insertion map.
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Sewage water.
Artificial sewage water was composed of the
following components (per liter): 10 mg of aniline, 5 mg of
nitrobenzol, 10 mg of phenol, 2 mg of toluene, 50 mg of acetone, 50 mg
of ethanol, 100 mg of isopropanol, 300 mg of methanol, 29 mg of urea,
6.5 g of chloride anions, 138 mg of nitrate anions, 1.7 mg of
phosphate anions, and 3.5 g of sulfate anions. This artificial
sewage water resembles in its composition real sewage water from an
industrial plant.
Enzymatic activities.
-Galactosidase activity in
permeabilized cells was determined (24), with 1 U defined as
the activity forming 1 nmol of o-nitrophenol per min at
30°C. The bioluminescence of slow-growing cells was measured with a
Lumac/3M M2010A biocounter. Cells were grown for 48 h at 28°C in
HEPES medium plus tetracycline (7.5 µg ml
1), harvested,
and adjusted to 109 CFU/ml. A volume of 0.5 ml of the cell
suspension was used for each determination and incubated in polystyrene
tubes. The light output was integrated for 10 s. The
bioluminescence of fast-growing cells was measured with a Lumistox
luminometer (Dr. Lange, Berlin, Germany) in Tris-buffered mineral salts
medium containing 9 mM sodium gluconate as the carbon source.
Chromate reduction and uptake.
The cells were cultivated for
18 h at 30°C in Tris-buffered mineral salts medium containing 30 µM or 3 mM disodium sulfate and 46 mM sodium gluconate as the carbon
source. The cells were harvested by centrifugation, washed, and
suspended in 10 mM Tris HCl, pH 7.0, containing 46 mM sodium gluconate,
and 50 µM [51Cr]chromate (specific activity, 17.6 GBq/g; Du Pont de Nemours, Bad Homburg, Germany) was added. Samples (1 ml) were removed and centrifuged. The cells were discarded. The total
chromium concentration in the supernatant was determined with a Beckman
LS6500 scintillation counter with 100-µl samples. The total chromate
in the supernatant was measured with diphenylcarbazide as described
previously (10). The amount of reduced chromium in the
supernatant was the difference between the total chromium in the
supernatant and the total chromate in the supernatant. Since the cells
previously removed by centrifugation were responsible for chromate
reduction, this value was divided by the dry weight of the cells at the
time the sample was taken. The uptake of chromium was determined by
filtration as previously described (26).
Genetic techniques.
Standard molecular genetic
(28) and previously published (23) techniques
were used. For conjugal gene transfer, overnight cultures of donor
strain E. coli S17/1 (32) and of the A. eutrophus recipient strains grown at 30°C in complex medium were
mixed (1:1) and plated onto nutrient broth agar. After overnight
growth, the bacteria were suspended in saline and plated onto selective
media as previously described (23). For transposon
mutagenesis, E. coli S17/1(pDNA206) was infected with
::Tn5-lacZ B20 (33) and plated onto
Luria broth agar containing 50 µg of kanamycin per ml. The resulting
transposon mutants were conjugated by replica plating with A. eutrophus AE104 on Tris-gluconate-tetracycline agar (2 g of sodium
gluconate per liter, 12.5 µg of tetracycline per ml). AE104
transconjugants were replica plated onto nutrient broth agar containing
3 mg of kanamycin and 0.1 mg of X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside) per
ml. Kanamycin-resistant strains which were sensitive to 1 mM
Ni2+ or to 0.2 mM chromate were purified and analyzed.
Construction of the broad-host-range chromate sensor
plasmid.
By using the XhoI site, which is a single site
in the kanamycin resistance genes of both plasmids, plasmid pRME1
(11) was cloned into the broad-host-range plasmid pVK102
(15). A hybrid plasmid encoding kanamycin resistance was
isolated, and the cos site was deleted with PstI
and BglII. The resulting plasmid, pEBZ112, consists of
plasmid pVK102 with the multiple-cloning site of pRME1 replacing the
cos site. By using a BamHI site, the Vibrio
fischeri luxCDABE operon of Tn4431 from plasmid pUCD623
(30) was cloned into plasmid pEBZ112, resulting in plasmid
pEBZ116. Finally, the 1.6-kb chrBA'
EcoRI-XbaI fragment of plasmid pECD352 was cloned as an EcoRI blunt-end fragment into pEBZ116 upstream of
lux, resulting in plasmid pEBZ141 (Fig.
2). Plasmid pECD352 carries the 5' end of
chr from the EcoRI site at position 1 up to bp
1697 in the middle of chrA (22).
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FIG. 2.
Map of the chromate sensor plasmid pEBZ141. The genes
encoding resistances to tetracycline (tet) and kanamycin
(kan) and the chrBA'::lux
gene fusion are indicated. Restriction endonucleases were
SalI (S), XbaI (A), BamHI (B),
PstI (P), KpnI (K), SacI (Sc),
EcoRI (E), and XhoI (X).
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RESULTS |
Induction of chr and cnr.
To develop
metal-sensing bacterial strains and to study the regulation of
chr and cnr, plasmid pDNA206 (21),
which contains both determinants on a 30-kb fragment of pMOL28, was
mutated with ::Tn5-lacZ B20 (33).
Thirty-eight Co2+-, Ni2+-, and/or
chromate-sensitive derivatives of pDNA206 were identified in
plasmid-free A. eutrophus AE104. The mutant strains were
either chromate sensitive (10 strains) or sensitive to Co2+
and Ni2+ (28 strains). No strains were sensitive to all
three metal ions.
By means of digestion with
EcoRI,
BamHI, and
HindIII, the orientation and insertion point of each
transposon were determined.
Of the 28 insertions in the
cnr
determinant, only 4 were in the
proposed direction of
cnr
transcription (pDNA228, pDNA229, pDNA238,
and pDNA255) (Fig.
1); three
of these fusions were in the
cnrA gene, and the remaining
one (pDNA229) was in
cnrB. Only these
four fusions could be
induced with 100 µM Ni
2+; strains with the other 24 fusions (Fig.
1) displayed constitutive
expression of
-galactosidase
at various levels (data not shown).
Of the 10 insertions in the
chr determinant, 8 were inducible
by chromate and in the
proposed direction of
chr transcription
(Fig.
1). Two (Fig.
1) could not be induced with chromate and
had the opposite orientation.
Induction of
chr in strain AE104(pDNA265) (Fig.
1) with a
chr::
lacZ transcriptional fusion was
highly specific for chromate
(Table
2). Arsenate, molybdate (Table
2), and a 100 µM concentration
of
either Ni
2+, Co
2+, Zn
2+, or
Cd
2+ (data not shown) did not induce
chr. When
various
chr transposon
insertions were compared,
chrA insertions gave the strongest chromate-dependent
increase in
-galactosidase activity. Fusions located in the
intergenic
region between
chrB and
chrA or in the
ORF3 region were clearly
inducible by chromate; however, the induction
rate was lower than
with
chrA::
lacZ
fusions (data not shown).
AE104(pDNA229) with a transposon insertion in
cnrB showed
the highest
-galactosidase activity induced by 100 µM
Ni
2+ of all strains with
cnr transcriptional
fusions (data not shown).
To compare inductions of
cnr in
strain AE104(pDNA229) by various
heavy metal salts, the strain was
cultivated for 8 h in the presence
of various concentrations of
metal cations and chromate (Table
2). Again, induction was strong and
specific with Ni
2+; the maximum induction occurred in the
presence of 128 µM Ni
2+ (Table
2). At higher
concentrations, activity decreased due
to the toxic action of
Ni
2+ (data not shown). Although there was some induction by
most other
metal ions tested (Table
2),
cnr seemed to be
induced best by
nickel.
A chromate sensor strain.
Of all the fusions in the
chr-cnr region tested, fusions in chrA were the
most specific and gave the strongest responses (data not shown). To
measure chr induction in whole cells, plasmid pEBZ141 (Fig.
2), with a chrBA'::luxCDABE
transcriptional fusion, was constructed. The plasmid was transferred
into A. eutrophus CH34(pMOL28, pMOL30) and into its
plasmid-free derivative AE104. To compare the influences of various
carbon sources on the light emissions of the resulting transconjugants,
CH34(pMOL30, pMOL28, pEBZ141) and AE104(pEBZ141) were cultivated at
28°C in HEPES medium containing 10 µM chromate, 7.5 µg of
tetracycline per ml, and 4 g of a carbon source per liter. Poor or
no growth and low light emission were observed for both strains in
acetate, citrate, formate, pyruvate, and succinate (data not shown). In
gluconate, light emission increased during the exponential phase of
growth to about 300 relative light units and decreased again during the
stationary phase. In glycerol, growth of both strains was slow. Light
emission by CH34(pMOL30, pMOL28, pEBZ141) was poor (about 20 relative
light units); however, AE104(pEBZ141) achieved light emissions of about
6,000 relative light units (data not shown). Therefore, glycerol as the
carbon source and strain AE104(pEBZ141) were considered optimal for the study of chr induction in whole cells.
When the temperature was raised from 28 to 32°C, light emission by
AE104(pEBZ141) in the presence of 10 µM chromate decreased
strongly
(data not shown). Thus, 28°C was used as the temperature
for further
studies. The sulfate concentration in the medium used
to precultivate
the cells strongly influenced the light emission
induced afterwards by
10 µM chromate: while light emission was
strong at sulfate
concentrations above 500 µM (results for 500
µM and 1 mM are shown
in Fig.
3A; results for sulfate
concentrations
of 2, 4, 8, 16, and 32 mM were identical to those for 1 mM and
are not shown), light emission decreased at 250 µM sulfate and
was nearly zero at 125 µM (Fig.
3A). Very little light emission
was
induced by 10 µM chromate when the cells were precultivated
at
sulfate concentrations lower than 63 µM (data not shown). Thus,
precultivation of AE104(pEBZ141) at low sulfate concentrations
decreased the inducibility of
chr::
lux
by chromate.
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FIG. 3.
AE104(pEBZ141) as a chromate sensor. Strain
AE104(pEBZ141) was precultivated in glycerol-HEPES medium containing
various sulfate concentrations (A) or 1 mM sulfate (B). When the early
stationary phase was reached, the cell density was adjusted to
109/ml. A volume of 0.5 ml of this cell suspension was
tested with various concentrations of chromate and other metal ions,
and light emission (in relative light units [RLU]) by the products of
the chr::lux operon fusion was
determined at 28°C. (A) The cells were precultivated in the presence
of 1,000 ( ), 500 ( ), 250 ( ), or 125 ( ) µM sulfate, and
light emission was measured after induction with 10 µM chromate. (B)
Inductions by 1 () and 10 () µM chromate were compared with
inductions by () and 10 () µM dichromate or by 1 ( ) and 10 ( ) µM Cr3+ and with that in cells incubated without
chromium ( ).
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As seen with the
lacZ fusion, induction of
chr::
lux was highly specific for
chromate. No light emission above the control
level could be induced by
10 or 100 µM molybdate, 10 or 100 µM
tungstate, 10 µM vanadate,
or 100 µM MnSO
4 (data not shown). However,
100 µM
vanadate induced the
chr::
lux fusion
with about 0.2% (twice
the control value) of the maximal light
emission obtained by induction
with 0.1 µM chromate (data not shown).
Other chromium species
induced
chr::
lux
as well. At 0.1 µM, induction by dichromate or
Cr
3+
yielded the maximal light emission, which was for both compounds
about
4% of the maximal light emission obtained with 0.1 µM chromate
(data
not shown). At higher concentrations (Fig.
3B), however,
the difference
between the chromium compounds decreased. Here
1 and 10 µM dichromate
induced as strongly as the respective chromate
concentrations, and 10 µM Cr
3+ induced as strongly as 1 µM chromate (Fig.
3B).
Light emission by AE104(pEBZ141) could already be induced by 1 nM
chromate. The increase in relative light units per minute
depended on
the inducing chromate concentration (up to 50 µM);
however, due to
the toxic effect of chromate, light emission decreased
at 100 µM
(data not shown). For chromate concentrations from 1
nM to 50 µM, the
increase in relative light units per minute was
linear between 1 and
2 h (data not shown).
When the chromate sensor strain AE104(pEBZ141) was tested in artificial
sewage water, induction by chromate was similar to
that in mineral
salts medium (data not shown); however, with all
chromate
concentrations used (10 nM to 50 µM) except 1 µM, the
induction
process took about 1 h longer in artificial sewage water
than in
mineral salts medium alone (data not shown).
Interaction between chr induction and the sulfate
concentration.
Since sulfate starvation derepresses the transport
systems responsible for sulfate and chromate uptake (25),
sulfate starvation should have increased the sensitivity of the
chromate sensor, but the opposite was observed (Fig. 3A). Growth of
A. eutrophus CH34 in glycerol was very slow (0.02 h1) compared to growth in gluconate (0.2 h1
[data not shown]). To characterize the influence of sulfate on induction of chr in Tris-buffered mineral salts medium with
gluconate as the carbon source (conditions normally used to cultivate
the strain) plasmid pEBZ141 was transferred into A. eutrophus AE126(pMOL28). The control plasmid pEBZ116, which
contains a promoterless lux operon but not chrBA'
upstream of it, was transferred into strains AE104 and AE126(pMOL28).
Cells of all four strains, AE104(pEBZ141), AE104(pEBZ116),
AE126(pMOL28, pEBZ141), and AE126(pMOL28, pEBZ116), were cultivated
in Tris-buffered mineral salts medium, and induction of
chr::lux was determined in
exponentially growing cells (shown in Fig.
4 for pEBZ141; data not shown for
pEBZ116).
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FIG. 4.
Induction of chr::lux in
growing cells. Cells of strain AE104(pEBZ141) ( and ) and of
strain AE126(pMOL28, pEBZ141) ( and ), both containing a
chr::lux fusion on plasmid pEBZ141,
were cultivated in gluconate-Tris medium containing either 3 mM or 30 µM sulfate in the presence ( and ) or absence ( and ) of
10 µM chromate, and the light intensities (in relative light units
[RLU]) were determined.
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With fast-growing cells, the light emission did not reach the high
levels achieved with slow-growing glycerol cells (Fig.
4A). Three
different levels of light production were observed.
In all control
cells containing plasmid pEBZ116 with no
chr upstream
of
lux, light production did not depend on the presence of 10
µM chromate, the sulfate concentration used to grow the cells,
or the
particular strain used (data not shown). A level of about
400 relative
light units was reached in 30 µM sulfate with both
chr::
lux-containing strains,
AE104(pEBZ141) and AE126(pMOL28,
pEBZ141), and in 3 mM sulfate with
AE126(pMOL28, pEBZ141) if the
cells were induced with 10 µM
chromate. A level of 2,000 relative
light units was reached only in 3 mM sulfate with strain AE104(pEBZ141),
induced with 10 µM chromate.
Therefore, sulfate starvation reduced
induction of
chr by
chromate also in Tris-gluconate-cultivated,
fast-growing cells of
strain AE104. However, in AE126(pMOL28)
grown at high and low sulfate
concentrations, induction of
chr::
lux by chromate was as low as in AE104 cells cultivated at low sulfate
concentrations. Thus, the presence of the
chr or
cnr gene, or
of other genes on plasmid pMOL28, decreases the
induction of the
chr operon substantially in cells grown at
high but not low sulfate
concentrations.
Interaction between chr and chromate reduction.
To
find a reason for the unexpected effect of sulfate on chr
induction, cells of the plasmid-free strain AE104 were cultivated without chromate in the presence of 3 mM or 30 µM sulfate, and the
levels of chromate accumulation and chromate reduction were determined
(Fig. 5). Strain AE104 was able to reduce
chromate (Fig. 5A). Sulfate starvation led to increased chromate uptake but also to increased chromate reduction. Other sulfur sources (cysteine, methionine, taurine, sulfite, and thiosulfate) were also
tested, but due to interference with the chromate determination assay,
these experiments yielded no results (data not shown). Thus, A. eutrophus reduced chromate, and the sulfate concentration in the
growth medium influences chromate uptake and reduction.
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FIG. 5.
Chromate uptake and chromate reduction in strain AE104.
Cells of the plasmid-free A. eutrophus strain AE104 were
cultivated 18 h with shaking at 30°C in gluconate-Tris medium
containing 1% sodium gluconate and 3 mM () or 30 µM ()
sulfate. The cells were washed once and suspended in 10 mM Tris HCl
buffer, pH 7.0, containing 1% sodium gluconate. Potassium
[51Cr]chromate (50 µM) was added, and incubation was
continued with shaking at 30°C. Samples were removed and used to
determine the amount of chromate reduced by the cells (A) and the
amount of cell-bound chromium (B). Each point is the mean for three
experiments; the bars give the standard deviations. d.w., dry weight.
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DISCUSSION |
A. eutrophus AE104(pEBZ141) may be readily used as a
biosensor for chromate, even in unnatural environments, such as
industrial sewage water. The chromate-sensing process is highly
specific. The main inducers are chromate and dichromate;
Cr3+ is 10-fold less active as an inducer. Other oxyanions
show activities which are less than 1% of the activity obtained with
chromate. The data gathered during development and characterization of
the chromate sensor also shed some light on regulation of the
chr and the cnr resistance determinants and on
the chromium metabolism of strain CH34, which is the wild-type
counterpart of the plasmid-free strain AE104 (19).
Cells of A. eutrophus starved for sulfur are known to
derepress the sulfate and chromate uptake systems (25).
Therefore, it was expected that incubation of the sensor strain
AE104(pEBZ141) with sulfate starvation would lead to enhanced
sensitivity of the sensor bacteria. Surprisingly, the opposite was the
case: sulfate starvation repressed induction of the
chr::lux reporter by chromate. To
explain this result, the interaction between chr and
chromate reduction was investigated. Many bacteria reduce chromate
(3, 5, 6, 12, 13, 18, 31, 34, 36), and now it has been shown
that A. eutrophus does so also. The product of the reduction
should be Cr(III) or Cr(II), since metallic chromium, Cr(IV), and Cr(V)
should not be stable in aqueous environments at neutral pH values
(37).
In strain AE104, sulfate starvation induced uptake and reduction of
chromate (Fig. 5). Thus, chromate reduction by strain AE104 might be
catalyzed by the sulfate reduction pathway. Since chromate is rapidly
reduced by sulfate-starved AE104, the intracellular concentration of
chromate, probably the inducer of chr, might be lower in
sulfate-starved cells than in sulfate-saturated cells. This may explain
the repression of chr induction in sulfate-starved cells.
This result could indicate a connection between a chromosomally encoded
sulfate reduction pathway and the plasmid-borne chr chromate
resistance system. Two known products of chr are essential for chromate resistance, which is based on reduced accumulation of
chromium (26). The first is ChrA, a membrane-bound protein with various transmembrane-spanning alpha-helices (16),
which is encoded at the 3' end of chr. Deletion of
chrA leads to chromate sensitivity and loss of the mechanism
leading to reduced accumulation of the metal ion (22). Thus,
ChrA is probably a chromate efflux protein. Genes with products
homologous to ChrA have been found in Synechococcus
(20), Synechocystis (14),
Methanococcus jannaschii (2),
Pseudomonas (4), and Vibrio cholerae
(27). Expression of the ChrA-homologous SrpC protein of
Synechococcus is induced by sulfate starvation
(20); therefore, regulation is different from ChrA
regulation in A. eutrophus. Gene products homologous to
ChrB, the second product of chr, have not been found in
other organisms until now. ChrB is also essential for chromate
resistance: expression of a chr derivative with a deletion
in the 5' end of chrB led to hyperaccumulation of chromium
(22).
Chromate might be rapidly transported into sulfate-starved cells of
A. eutrophus but also reduced very quickly to a less toxic form inside the cells. This makes sense, because effluxed chromate comes back into the cell while reduced chromium is detoxified permanently. The chr resistance system is not induced under
these conditions and may not be required. In sulfate-saturated cells, however, chromate is not reduced because the sulfate reduction pathway
is repressed; chromate accumulates and becomes toxic, the
chr system is induced, and ChrA pumps out the chromate. In this case, ChrA would be a safety valve for chromate, which would explain the high specificity of chr induction by chromate.
The data currently available do not justify further speculations, but
chromate reduction seems to be part of the chromate detoxification system in A. eutrophus.
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ACKNOWLEDGMENTS |
D.H.N. thanks Anke Nies and Grit Becker and G.E. thanks Susanne
Ecker and Kerstin Schwindel for skillful assistance. We thank Simon
Silver for destroying an early version of the manuscript with his
productive criticisms.
This work was supported by the Bundesministerium für Forschung
und Technologie as a project of the Gene Centre of Berlin, by
Forschungsmittel des Landes Sachsen-Anhalt, and by Fonds der Chemischen Industrie.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie, Kurt-Mothes-Str. 3, D-06099 Halle, Germany.
Phone: (49)-345-5526352. Fax: (49)-345-5527010. E-mail:
D.Nies{at}mikrobiologie.Uni-Halle.DE.
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Appl Environ Microbiol, February 1998, p. 453-458, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
