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FEMS Microbiology Letters 43 (1987) 9-13
Published by Elsevier
9
FEM02789
Plasmid pMOL28-mediated inducible nickel resistance
in Alcaligenes eutrophus strain CH34
R o m a n A. Siddiqui a n d H a n s G. Schlegel
lnstitut fiir Mikrobiologie der Universitiz't Grttingen, GOttingen, F.R.G.
Received 19 January 1987
Accepted 10 February 1987
Key words: Alcaligenes eutrophus; Nickel resistance; Plasmid; Nickel uptake
1. SUMMARY
The effect of nickel salt on growth of the
nickel-resistant wild type strain Alcaligenes
eutrophus CH34, which harbours two plasmids,
and on its partially or totally cured derivatives as
well as of the wild type strain H16 was studied.
Plasmid pMOL28-mediated nickel resistance
turned out to be an inducible property. Full resistance is induced during growth in the presence of
0.03-3.0 mM NiCI 2. Induction requires growth.
While plasmid-free cells accumulate nickel at a
high rate, the pMOL28-harbouring-induced cells
accumulate only negligibly small amounts of
nickel. It is concluded that pMOL28 mediates a
protective mechanism preventing the cells to accumulate nickel ions intracellularly at toxic concentrations.
2. I N T R O D U C T I O N
The resistance to heavy metals in microorganisms can be caused by several mechanisms
including detoxification by binding and precipita-
tion, volatilization, efflux or exclusion [1-3].
Among potentially toxic metals nickel has so far
found little attention [4]. Nickel resistance is of
special interest in hydrogen-oxidizing bacteria as
nickel is required as a trace element for the
synthesis of hydrogenases [5]. However, at millimolar concentrations it inhibits growth of most
wild type bacteria.
We happened to receive an aquatic bacterium,
strain CH34, which arose attention due to its
resistance to zinc, cadmium and cobalt [6]. This
strain proved to be a hydrogen-oxidizing bacterium, resistant also to nickel, and due to its specific
characters was assigned to Alcaligenes eutrophus.
The wild type contains two plasmids [7]. Examination of partially or totally cured strains identified
pMOL28 (163 kb) as encoding resistance to nickel
and cobalt and pMOL30 (236 kb) as encoding
resistance to zinc, cadmium and cobalt [8]. The
availability of these strains prompted us to study
the effect of nickel salts on growth kinetics and on
inducibility of nickel resistance as well as the
uptake of 63Ni-labelled nickel salt.
3. MATERIALS A N D M E T H O D S
Correspondence to: H.G. Schlegel, Institut fiir Mikrobiologie
der Universitat G~Sttingen, Grisebachstrasse 8, D-3400
G~Sttingen, F.R.G.
3.1. Bacterial strains
The bacteria used in this study were A. eutrophus
0378-1097/87/$03.50 © 1987 Federation of European Microbiological Societies
10
strain H16, A. eutrophus CH34 harbouring both
plasmids and its partially cured derivatives AE126
and AE128 harbouring pMOL28 and pMOL30
respectively, and totally cured derivative AE104.
These strains are listed in Table 1 of [8].
3.2. Growth conditions
Growth and growth inhibition by nickel chloride were studied using a low-phosphate Tris/
mineral medium as described previously [8]. This
medium did not contain metal-chelating or -precipitating compounds and provided conditions of
maximum inhibitory effects exerted by heavy metal
salts. For heterotrophic growth 0.3% (w/v) sodium
gluconate was added.
Doubling times (td) were measured in 300-ml
Klett flasks containing 50 ml cell suspension on a
rotary shaker at 30 ° C. Turbidity was monitored
with a Klett-Summerson colorimeter (filter No.
54) and expressed as Klett units (KU). 100 KU
correspond to about 175 mg dry weight cells per
liter.
To examine induction of nickel resistance 10-ml
cultures were grown overnight without or in the
presence of 0.03-5 mM NiC12 in Tris/gluconate
medium, washed once and resuspended in the
same volume of Tris/mineral medium without
substrate. For inoculation 1.0 ml of this suspension was transferred into 50 ml Tris medium containing 3.0 mM NiC12.
3.3. Assay of 63Ni-nickel uptake
Cells were grown in 200 ml Tris/mineral
medium with 0.3% (w/v) gluconate, harvested in
the exponential growth phase and washed once
with 50 mM sodium phosphate buffer, pH 7.2.
Induced cells were grown in the presence of 0.5
mM NiC12. Washed pellets were resuspended in
ice-cold phosphate buffer to a protein concentration of approximately 0.25 mg/ml; the buffer
contained 0.02-0.2 g/1 chloramphenicol. Cell
suspensions (10 ml) in 100-ml Erlenmeyer flasks
were shaken for 5 min at 30 ° C. Experiments were
started by adding 63Ni-NiC12. Samples (0.2-0.5
ml) were withdrawn at time intervals, rapidly
filtered through membrane filters of 0.45 /~m
pore-size and washed twice with a 10-fold sample
volume of acid phosphate solution (50 mM
KH2PO4, pH 3.0) without NiCI 2 or with 4 mM
non-radioactive NiC12. The washing procedure removed about 98-99% of unspecifically bound
nickel ions. After washing the filters were dissolved
in 5 ml xylene-based scintillation fluid (Packard
Emulsifier Scintillator, special Mi-96) acidified by
50/tl 5 N HC1 and counted in a Beckman liquid
scintillation counter, model LS 7800.
3.4. Chemicals
Analytical grade NiC12.6H20 (E. Merck,
Darmstadt, F.R.G.) was prepared as !.0 M stock
solution and sterilized by autoclaving. Radioactive
63NIC12 with a specific activity of 0.73 mCi//~mol
was obtained from A m e r s h a m Buchler
(Braunschweig, F.R.G.). All other chemicals were
obtained from E. Merck, Darmstadt.
4. RESULTS
4.1. Effect of nickel chloride on growth kinetics
The wild type strain CH34 and its partially or
totally plasmid-cured derivatives AE126, AE128
and AE104 were characterized in a previous study
[8]. The minimal inhibitory concentrations (MIC),
at which no colony growth was observed, were
calculated from counts of colonies on agar plates
containing Ni, Co, Cd and Zn salts in 2:1 dilution steps. The kinetics of growth were not considered.
The present study aimed at elucidating the
effect of the metals on the growth of CH34 and its
derivatives. In particular, we concentrated on the
effect of nickel and the protective mechanisms
exerted by plasmid pMOL28. The growth-inhibiting effect of nickel became apparent when cells of
CH34, AE104 and the nickel-sensitive wild type
strain A. eutrophus H16, all grown in the absence
of nickel salt, were inoculated into liquid Tris/
gluconate media containing different concentrations of NiC12 (Fig. 1). Strain CH34 started to
grow immediately at its nickel concentration-dependent exponential growth rate when only 0.5 or
11
400
100
i
5 400
i
B
lOO
i
2 400
lO0
0
12
24
37
TIME (h)
Fig. 1. Growth of A. eutrophus CH34, of its plasmid-free
m u t a n t AE104 and of the wild type strain A. eutrophus H16.
The inoculum was grown without nickel. At zero time T r i s /
gluconate media containing no NiC12 (©), 0.5 m M NiC12 (e),
1.0 m M NiC12 (11) or 3.0 m M NiC12 (A) were inoculated with
suspensions of CH34 (A), AE104 (B), and H16 (C). Turbidity
of 100 Klett units corresponds to 175 m g dry weight per liter.
1.0 mM NiC12 were present, however, at 3 mM
NiC12 it did not grow for 12-24 h; only after this
lag period it reached its nickel concentration-dependent growth rate. In contrast the plasmid-free
derivative AE104 (Fig. 1B) and strain H16 (Fig.
1C) grew at 0.5 or 1.0 mM NiC12 with very low
rates and at 3 mM NiC12 not at all, even after 72
h (not shown).
The doubling times of the strains in the presence of different concentrations of NiC12 are listed
in Table 1. Comparison of the data allows the
conclusion that (i) the growth-retarding effect of
nickel ions is concentration-dependent and (ii)
strains lacking pMOL28 stop growth at concentrations higher than 1.0 mM NiC12, whereas the
strains harbouring the plasmid even tolerate 5 mM
NiC12 exhibiting a t a of 7 h, and (iii) the second
plasmid pMOL30, present in CH34, does not influence the expression of pMOL28.
The growth experiments documented in Fig. 1A
indicated that plasmid pMOL28-mediated resistance to nickel concentrations shortly below MIC
is an inducible property. When CH34 or AE126
were precultivated at a sub-inhibitory concentration (0.5 mM) or at 3 mM NiC12 and were then
inoculated into Tris/gluconate medium containing 3 mM NiC12 exponential growth commenced
immediately. The inductive effect proved to be
dependent on the nickel concentration during antecedent growth. At the concentration normally
added as a trace element (0.1 /LM) nickel did not
Table 1
Doubling times (td) in hours during exponential growth on gluconate medium of A. eutrophus H16 and A. eutrophus CH34 and
m u t a n t derivatives in the presence of varied concentrations of NiC12
Cells were shaken under air in Klett flasks at 30 ° C; Tris/mineral medium with 0.3% ( w / v ) gluconate. Data for wild type strains
N 9 A and G29 were very similar to those for H16. N.D., no growth detectable.
Concentration
Doubling time (h)
(mM) of NiC1:
H16
CH34
AE126
AE128
AE104
0
0.03
0.1
0.5
1.0
2.0
3.0
5.0
3.6
5.6
11.5
18.4
36.3
n:d.
n.d.
n.d.
3.2
3.5
3.6
4.2
4.9
5.7
5.7
7.5
3.2
3.7
3.4
4.1
4.5
5.6
5.8
6.8
3.5
3.9
5.5
15.0
33.9
n.d.
n.d.
n.d.
3.4
4.3
5.6
14.1
33.2
N.D.
N.D.
N.D.
12
exert an induction effect. Significant effects on the
decrease of the lag time were observed with nickel
salt concentration from 0.03 to 0.5 m M (Table 2).
As the lag times exhibited b y b o t h strains, C H 3 4
and AE126, are very similar, the presence of the
second plasmid, p M O L 3 0 , apparently does not
influence the induction or expression of p M O L 2 8 .
W h e n p M O L 2 8 - h a r b o u r i n g cells were grown in
the presence of 0.5 m M NiC12 they reached full
nickel resistance, i.e. the ability to grow at 3 m M
NiC12 with a high rate without lag period, within 5
h. This kind of induction occurred during growth
with gluconate as substrate or under autotrophic
conditions. Furthermore, induction of the expression of full nickel resistance required growth
conditions; nickel resistance was not expressed in
the absence of the energy source (gluconate) or the
N-source ( a m m o n i u m ) or when chloramphenicol
(200 /~g/ml) or nalidixic acid (200 ~tg/ml) was
added to the growth medium. These experiments
identified p M O L 2 8 - m e d i a t e d nickel resistance as
an inducible property, and nickel exerts its inductive effect at either sub-inhibitory or inhibitory
concentrations.
It was, furthermore, shown that the derivatives
of C H 3 4 such as AE128 and AE104 as well as
strain H16, which do not carry plasmid p M O L 2 8 ,
did not grow at all in the presence of 3 m M
NiCI2, neither after growth at sub-inhibitory nor
after incubation at inhibitory concentrations. In
spite of m a n y attempts we were not able to isolate
Ni-resistant
pMOL28.
mutants
from
strains
lacking
4.2. Uptake of nickel ions by strain CH34
W h e n cells of A. eutrophus C H 3 4 and its
plasmid-free derivative AE104 were resuspended
in p h o s p h a t e buffer and e x p o s e d to low
concentrations of nickel chloride, containing 63Ni
as a marker, the basic difference between both
strains became recognizable (Fig. 2). While the
wild type strain C H 3 4 took up the labelled nickel
ions at a slow, rapidly declining rate, the plasmid-
5
t~
.__q
O
'5
&
0
o
n 20
Z)
"~
C
15
1.1_
0
a
LI.J
',..b
Table 2
Lag times to reach the maximum exponential growth rate after
growth in the presence of sub-inhibitory concentrations of
NiC12
Preculture grown
in the presence of
NiC12 (mM)
0
0.03
0.06
0.1
0.2
0.3
0.4
0.5
Lag time (h) of strain
CH34
AE126
Z
LLI
0
n.-
5
LLI
Q.0
,
0
60
120
TIME (min)
26
9.2
8.5
5.0
4.9
2.5
1.2
0
27
10
12
10
4.5
4.0
2.5
0
Fig. 2. Uptake of 63Ni-nickel ions by uninduced and induced
cells of strain CH34 and by its plasmid-free derivative AE104
at three different concentrations of nickel chloride: (A) 0.1/.tM
(100% = 0.41 nmol Ni2+/mg protein); (B) 1.0 btM (100% = 4.05
nmol Ni2+/mg protein); (C) 10/~M NiC12 (100% ~ 39.5 nmol
Ni2+/mg protein). Symbols: D, strain CH34 uninduced; A,
strain CH34 induced; O, mutant AE104. Conditions and
techniques are described under METHODS.
13
free derivative accumulated nickel ions at a high,
only slowly declining rate. This difference appeared at 0.1, 1.0 and 10.0 /~M concentration of
Ni 2+ in the suspension medium. Cells of CH34,
which were grown in the presence of 0.5 mM
NiC12 and thus were induced for nickel resistance,
accumulated only negligibly small amounts of
Ni 2+. The present results suggest that the plasmid
exerts a constitutive effect and a much stronger
effect after induction of full nickel resistance during growth at sub-inhibitory concentrations of
nickel. On the basis of these experiments the
resistance to nickel ions of CH34 may be explained as a plasmid-mediated protective mechanism preventing the cells to accumulate nickel
ions intracellularly at toxic concentrations.
tion of the intracellularly accumulated nickel is
also dependent on the nickel concentration in the
medium. Whether the low rates of uptake and the
low extent of accumulation of nickel in the induced plasmid-harbouring cells are due to an exclusive or an efflux mechanism, and whether a
pmf- or ATP-dependent process is involved are
the questions of current experiments.
ACKNOWLEDGEMENTS
We thank C. Friedrich and M. Lohmeyer for
their advice in applying radioactive nickel. The
work was supported by grants from the Deutsche
Forschungsgemeinschaft and Frrderungsmittel des
Landes Niedersachsen.
5. DISCUSSION
REFERENCES
Bacterial resistance determinants are often
localized on plasmids or transposons [1,9]. This
facilitates their analysis by molecular genetic techniques. The first time that plasmid-linked nickel
resistance in a Gram-negative bacterium was reported concerned Escherichia coli [10]. Induction
of plasmid-determined metal resistance was reported for mercury resistance in Staphylococcus
aureus and Pseudomonas aeruginosa [11], arsenate
resistance in S. aureus [12], and copper resistance
in E. coli K12 [13]. Inducibility seems to be the
rule rather than the exception. In the case of
pMOL28 it was shown that full resistance, i.e. the
ability of the cells to grow at a high rate at 3 mM
NiC1 z, was induced not only by this growth-inhibitory concentration but also by sub-inhibitory
concentrations. The conditions required for inductive expression of resistance are those required for
the formation of an inducible enzyme and thus
indicate that a proteinaceous product is formed.
The experiments on the uptake of 63Ni-labelled
nickel salt provided evidence that the resistance of
the pMOL28-containing cells is based on the reduced uptake capacity for nickel ions. Plasmidharbouring cells accumulate only a fraction of the
nickel ions detected in plasmid-free cells; induced
cells accumulate even less nickel. The concentra-
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