FEMS Microbiology Letters 83 (1991) 283-290
© 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50
Published by Elsevier
ADONIS 037810979100450Z
283
FEMSLE 04643
Exocellular and intracellular accumulation of lead
in Pseudomonas fluorescens ATCC 13525 is mediated
by the phosphate content of the growth medium
Ala. A1-Aoukaty, Vasu D. A p p a n n a and John H u a n g
Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada
Received 10 July 1991
Accepted 17 July 1991
Key words: Lead detoxification; Bioaccumulation; Bioprecipitation; Citrate; Phosphate;
Pseudomonas fluorescens
1. SUMMARY
Pseudomonas fluorescens appears to elicit disparate lead detoxification mechanisms in phosphate-rich and phosphate-deficient media. When
grown in the presence of 0.1 mM Pb 2+ complexed to citrate, the sole source of carbon, only a
slight diminution in cellular yield was observed in
the former medium. However, in a phosphate-deficient milieu, lead imposed approximately a 30%
reduction in bacterial multiplication. At stationary phase of growth, 72% of the metal was found
in the bacterial cells from the phosphate-deficient
medium, while that from phosphate-rich broth
contained only 12.5% The latter medium was
characterised by an insoluble pellet that accounted for 73.5% of the lead. Although no citrate was detected in the phosphate-rich media
after 40 h of incubation, only 72% of citrate was
Correspondence to: V.D. Appanna, Department of Chemistry
and Biochemistry, Laurentian University, Ramsey Lake Road,
Sudbury, Ontario, Canada P3E 2C6.
consumed even after 70 h of growth in the phosphate-deficient cultures. The inclusion of lead
did not appear to enhance the production of
either extracellular proteins or carbohydrates.
2. INTRODUCTION
Metallic elements are indispensable for cellular growth and for the maintenance of metabolic
functions. In trace amounts, they constitute an
essential requirement for almost all living organisms as metal-dependent processes play a determinant role in normal cellular functioning. Enzymatic activities are effected by metals as diverse
as calcium and selenium [1,2]. However, when
available in either high or very low quantities
metals exert an inhibitory influence on all life
forms and may also arrest cellular multiplication.
Owing to acid rain and industrial wastes, high
levels of bioavailable forms of metals have become a major concern. This situation has further
accentuated the already alarming concentration
of fuel-combustion-induced lead in the biosphere.
284
Metals like lead may mediate their toxic properties by displacing native metals from the normal
binding sites, inducing conformational changes in
proteins and nucleic acids and by perturbing
m e m b r a n e permeability [3]. In humans lead intoxification is known to perturb the nervous system
and the biosynthesis of heme [4]. E. coli exposed
to this metal showed decrease in growth, respiration and ATPase activity [5]. However, some microorganisms are known to initiate a wide array
of strategies in an effort to circumvent the presence of high levels of lead ions in their environment. These mechanisms include biomethylation,
reduced uptake and bioprecipitation of the metallic element. While some Gram-negative aerobic
bacteria have been shown to biomethylate lead,
insolubilization of this element as its sulphide
derivative has been observed in Klebsiella aerogenes [6].
As part of our study to evaluate the influence
of abnormal levels of metals on microbes, we
have studied the impact of lead on Pseudomonas
fluorescens, a microorganism known to inhabit a
wide variety of natural environments. The defined medium containing citrate as the sole source
of carbon, and to which the test metal was added,
afforded an interesting system to study microbelead interaction. In this report, the toxicity and
the biotransformation of lead citrate are described. The influence of phosphate on lead
detoxification mechanisms is also discussed.
3. M A T E R I A L S A N D M E T H O D S
Folin Ciocalteu's phenol reagent, serum albumin, D-glucose and citric acid were from Sigma.
Agar was purchased from Difco and citrate assay
kit was from Boehringer. All chemicals were
reagent grade.
3.1. Organism, media and growth conditions
Pseudomonas fluorescens A T C C 13525 was obtained from American Type Culture Collection.
It was maintained at 4 ° C by monthly subculture
on a defined citrate medium (DCM) solidified by
the inclusion of 2% agar. The liquid D C M
medium (phosphate-rich) contained citric acid
(4.0 g 1 1), N a 2 H P O 4 (6.0 g 1 1), KH2PO4 (3.0 g
1 i), ( N H 4 ) 2 S O 4 (1.0 g 1-1) and MgSO 4 • 7 H~O
(0.2 g 1 1). In the phosphate-limited medium
N a 2 H P O 4 (2.4 mg 1-1) and K H 2 P O 4 (1.2 mg l - t )
were used. The pH was adjusted to 6.8 with
dilute N a O H . In the metal-enriched medium, citrate was amended with the appropriate amounts
of Pb(NO3) 2. The medium without lead-complexed citrate served as a control. The media
were dispensed in 200 ml amounts in 500-ml
Erlenmeyer flasks and inoculations were made
with 1 ml of stationary phase cells grown in a
control phosphate-rich medium. The cultures
were aerated on a gyratory water-bath shaker
model G76 (New Brunswick Scientific) at 26 ° C.
3.2. Assays
Bacterial growth was monitored at different
time intervals by the method of Lowry (1951).
Cells were harvested by centrifugation at 5000 × g
for 15 rain at 4 ° C and treated with 0.5 M N a O H
in boiling water for 10 rain. The soluble protein
was estimated using Folin Ciocalteu's phenol
reagent, with bovine serum albumin as the standard. This technique was also applied in the
estimation of exocellular protein [7]. The total
carbohydrate content of the spent fluid devoid of
bacterial cells was determined by phenol-sulfuric
acid assay with i>glucose as standard [8]. The
concentration of citrate with or without lead in
the spent fluid at different growth phases was
determined enzymatically with the citrate assay
kit obtained from Boehringer. All assays were
done in triplicate. At various growth intervals,
cells were removed by centrifugation and the p H
of the broth recorded with the aid of a Fisher pH
meter model 610A.
3.3. X-ray fluorescence analysis"
Samples (50-ml) were collected at different
time intervals of growth and the supernatant and
bacterial cells obtained after centrifugation were
freeze dried. A water insoluble pellet was isolated at stationary phase of growth in both phosphate-rich and phosphate-deficient media. These
fractions were examined for their lead contents
by x-ray fluorescence spectroscopy. They were
placed in the 31-mm disposable x-ray cells and
285
spectra were recorded using a Philips PW1404
automatic, sequential s p e c t r o m e t e r following
standard procedures. P10 gas (10% methane, and
90% argon) was used in the flow proportional
counter. A dual M o / S C x-ray tube was used
throughout this investigation. Peak shifts are given
in two theta degrees (20) and the intensities in
kilocounts per second (kcps) • LIF200 was used as
the analysing crystal. U n d e r these conditions the
k a peak characteristic of lead was observed at
33.93, 2O.
50(
4.1. Effect of metals on growth
When cultured in a phosphate-rich medium
without any added lead, Pseudomonas fluorescens
reached stationary phase in 24 h. The cell yield
was 485 /,g of protein per ml of broth. In a
lead-supplemented culture, slightly lower growth
rate was observed and a 10% diminution in cellular yield was recorded. In phosphate-limited media, a marked variation in growth pattern waas
discerned. Bacterial multiplication was slower and
stationary-phase growth was obtained after 36 h
of innoculation. Lead a p p e a r e d to be inhibitory
in this instance. Pseudomonas fluorescens in phosphate starved culture with 0.1 m M lead was characterised by a 30% diminution of cell yield compared to bacterial yield observed in the same
medium u n a m e n d e d with the test metal (Fig. 1).
4.2. Influence of lead on extracellular protein synthesis
At different time intervals of growth the spent
fluid, following removal of the bacterial cells, was
analysed for its protein content. Approximately
185 > g of protein per ml of culture was observed
at stationary phase of growth in the phosphaterich medium with no added test metal. A slight
diminution in exocellular protein was recorded in
the same culture supplemented with 0.1 mM
Pb 2+. In phosphate deficient medium, Pseudomonas fluorescens a p p e a r e d to show a marked
difference in extracellular-protein-production
pattern. A four-fold increment in protein-positive
moieties was detected in the spent fluid in the
•
•
A
--5
300
c
~ D
/.!/
200
n-
[]
,5/-__
S lOO
0
4. R E S U L T S
~I
o
,o
2o
i
30
Incubation t i m e
go
7'0
(h)
Fig. 1. Effect of lead on growth of Pseudomonas fluorescens
ATCC 13525. I - - I
Phosphate-rich medium unamended with test metal. • • Phosphate-rich medium
supplemented with 0.1 mM lead. [] - [] Phosphate-deficient medium without test metal, t,
/, Phosphate-deficient medium enriched with 0.1 mM lead.
control phosphate-starved broth. Enrichment of
this medium with 0.1 mM Pb z+, induced a drastic
decrease in the elaboration of exocellular protein.
In this instance only about 55 /a.g of protein per
ml of culture was detected (Fig. 2).
4.3. Exocellular carbohydrate and metal stress
The spent fluid was also analysed for its carbohydrate content. At stationary phase of growth
the high-phosphate medium contained 126/~g of
glucose equivalent per ml of carbohydrate per ml
of culture. A drastic reduction in exocellular carbohydrate production was detected in the high
phosphate media amended with 0.1 m M Pb 2+.
The carbohydrate moieties at stationary phase of
growth in the phosphate-deficient medium without added lead had 100/~g of glucose equivalent
of carbohydrate per ml of culture. Inclusion of
0.1 m M Pb z+ in this phosphate-limited medium
appeared to completely arrest exocellular carbohydrate production (Table 1). No detectable level
of carbohydrate positive compounds was observed. The p H of the spent fluid showed a
gradual increase in all the different culture conditions. At stationary phase of growth the p H values of control and metal stressed media ranged
from 8.4-8.9.
286
500'
700
[]
a
600
[3
4oc
/
3u
~ 500
'~
3oo
/
~ 2oo
8
400
c 300
/
A
a
k9
o
CL
"~
A
u
46
m 1.00
E
n
2oc
rl
/
I
/
0
0
I
10
20
30
40
tncubotion time
50
i
i
60
70
(h)
Fig. 2. Biosyntheses of exocellular proteins in Pseudomonas
fluorescens exposed to lead in phosphate-containing media.
•
• Control phosphate-rich medium. • - - •
Phosphate-rich medium with lead (0.1 mM). 1 : 3 - - 1 : 3
Control phosphate-deficient medium, zx
zx Phosphatedeficient medium with lead (0.1 mM).
4.4. Citrate mineralisation in control and metalstressed media
T h e r a t e of c i t r a t e i n t e r n a l i s a t i o n is i l l u s t r a t e d
in Fig. 3. A t 24 h o f growth, no c i t r a t e was
d e t e c t e d in t h e s p e n t fluid o b t a i n e d f r o m t h e
Table 1
Exocellular carbohydrate at stationary phase of growth
Growth conditions
, i, ' ~ I
20
30
Incubation time
0
0
~
10
/zg of carbohydrate ml 1 of
supernatant (expressed as
D-glucose equivalent)
1 Phosphate-rich
126
medium with test metal
2 Phosphate-rich
4.5
medium supplemented
with 0.1 mM lead
3 Phosphate-deficient
100
medium unamended with
test metal
No detectable amounts
4 Phosphate-deficient
medium with
0.1 mM lead
40
,
50
t
60
|
70
(h)
Fig. 3. Citrate utilisation in Pseudomonasfluorescens stressed
with lead. •
• Phosphate-rich medium without
lead. • - - A
Phosphate-rich medium with lead (0.1
mM). 1 : 3 - - 1 : 3
Control phosphate-deficient medium.
A
zx Phosphate-deficient medium enriched with lead
(0.1 mM).
b a c t e r i a c u l t u r e d in the p h o s p h a t e - r i c h m e d i u m .
A slightly i n c r e a s e d r a t e of c i t r a t e d e c o m p o s i t i o n
was d i s c e r n e d in the p b 2 + - s u p p l e m e n t e d phosphate-rich medium.
In p h o s p h a t e - d e f i c i e n t m e d i u m , the s p e n t fluid
s h o w e d no d e t e c t a b l e t r a c e of c i t r a t e a f t e r 40 h of
growth. T h e a d d i t i o n of Pb 2+ to a p h o s p h a t e s t a r v e d c u l t u r e d i m i n i s h e d the r a t e of c i t r a t e
u p t a k e a n d even a f t e r 120 h of growth, 28% of
t h e c i t r a t e was still p r e s e n t in t h e s p e n t fluid.
4.5. Lead uptake: an x-ray fluorescence examination
T h e l e a d c o n t e n t o f the s p e n t fluid at various
time of growth was a n a l y s e d with t h e aid of x-ray
f l u o r e s c e n c e s p e c t r o m e t e r . A t z e r o time, in the
p h o s p h a t e - r i c h m e d i u m s u p p l e m e n t e d with l e a d
a p e a k c h a r a c t e r i s t i c of K a s p e c t r a l line o f led
was o b s e r v e d at 33.93, 20. S a m p l e s (50 ml) obt a i n e d at l o g a r i t h m i c p h a s e of growth r e v e a l e d a
62.2% d e c r e a s e in the intensity of the l e a d peak.
T h e s p e c t r u m d e r i v e d from i s o l a t e d s u p e r n a t a n t
at s t a t i o n a r y p h a s e o f growth s h o w e d the l e a d
a t t r i b u t a b l e p e a k with only 16% of t h e intensity
o b s e r v e d at z e r o time. T h e b a c t e r i a l cells harv e s t e d f r o m t h e l e a d - s u p p l e m e n t e d c u l t u r e at the
c e s s a t i o n of c e l l u l a r m u l t i p l i c a t i o n s h o w e d only
12.5% o f t h e total l e a d f o u n d at t h e t i m e o f
287
inoculation. The insoluble pellet that was discernable at late-logarithmic and stationary phases of
growth had an x-ray fluorescence spectrum indicative of lead. This insoluble residue contained
73.5% of the lead present in the initial medium
(Fig. 4). A phosphorus peak (not shown) was also
detected in this pellet.
The profile of lead uptake in the phosphatestarved medium is illustrated in Fig. 5. After 24 h
of growth approximately 28% of the lead was
present in the supernatant. At the stationary
phase of growth, metabolism of this heavy metal
continued and only 6% of the initial lead was
detected extracellularly in soluble form(s). Very
20.00
16.20
12,80
9.800
7.200
tO
a. 5 . 0 0 0
0 3.200
1.800
O.BO0
0.200
O. OOC
3O 0 0 0
20.00
16.20
12,80
I
I
Two-theta
20.00
16.20
12.80
I
32.000
I
34.000
to
n
L)
(degrees)
33.93
Pb
B
9.BOO
to
a_
(J
7. 2 0 0
5.000
3.200
1. 8 0 0
20.00
C
16.20
12.80
9.8OO
7.200
u9
o_ 5 , 0 0 0
L)
3.200
.r
1.800
0.800
0.200
0,000
30.000
9.800
7.200
5.000
3.200
1.800
0.800
0.200
0.000
30.000
I
I
I
I
32.000
I
34.000
20. O O r
16.20L E
0.1500
0,200
0.000
30;000
D
I
I
I
32.000
I
I
9.8°°1%
72oo1-
34.000
to
O_
0 5.0001_
3
.
2
1
B
0
0.8001 0
.
2
0,0001
I
30.000
I
I
32.000
I
I
/
\
0
0
0
~
0
32.000
~,
~
-
0
J
k
~
t
i
i
34.000
I
34.000
Fig. 4. X-ray fluorescence spectra of lead obtained from Pseudomonas fluorescens cultured in lead-supplemented phosphate-rich
medium at various time of growth (50 ml samples were analysed). A: supernatant at incubation time. B: supernatant at logarithmic
phase of growth. C: supernatant at stationary phase of growth. D: Bacterial cells at cessation cellular multiplication. E: Insoluble
pellet isolated at cessation of cellular multiplication.
288
small amounts of insoluble residue were discerned in this medium at stationary phase of
growth. The spectrum from this pellet indicated
the presence of lead. Approximately 22% of the
lead present at incubation time was found in this
insoluble residue. The bacteria harvested at stationary phase of growth contained 72% of the
lead.
5. DISCUSSION
The foregoing data illustrate the phosphatedependent lead citrate mineralisation in Pseudomonas fluorescens. In a phosphate-rich culture,
the bacterium predominantly precipitates lead,
while in a phosphate-deficient medium the metallic element was concentrated within the microbe.
20.00 r
1 2o A
128°I-
I
98°°K
7.2001--
t
5ooo F
3
1
0
0
,
,
.
.
,/
't
"k
-----,
2
8
8
2
0
0
0
0
0.0001
I
30.000
0
0
0
0
I
I
32.000
I
I
32.000
I
(degrees)
I
I
34.000
20.00
16.20
12.80
9.800
7.200
5.000
¢)
x
I
34,000
Two-theta
2QO0
B
16.20
12.80
9.800
7.200
~9 5 . 0 0 0
o_ 3 . 2 0 0
o, /
1.800
0.800
0,200
O,OOC
30,000
,~.
~
~
~
~
I
2 0 . 0 0 r- D
16.20
12.80
9. 8 0 0
7.200
5.000
3.200
1.800
0.800
0.200
0,000
30.000
20.00
16.20
12,80
9.800
7.200
5.000
U 3.200
-~ 1 . 8 0 0
0.800
0.200
0.000
30.000
I
I
I
32.000
I
I
32.000
I
I
34.000
I
I
l
34.000
3.2o0
1.800
0,800
0,200
O.OOC
30.000
I
I
~
32.000
m
I
34,000
Fig. 5. The fate of lead as revealed by x-ray fluorescence spectroscopy in Pseudomonas fluorescens grown in a defined
phosphate-deficient m e d i u m enriched with lead (50 ml samples were analysed). A: lead in supernatant at incubation time. B: lead
in supernatant in exponentially grown bacteria. C: lead in supernatant at stationary phase of growth. D: lead in bacterial cells at
stationary phase of growth. E: lead in insoluble pellet obtained at stationary phase of growth.
289
Bioprecipitation as a detoxification mechanism is
not uncommon in the microbial world. In Klebsiella aerogenes lead is known to trigger the formation of insoluble lead sulphide [9]. The exocellular precipitation of lead and cadmium as phosphates has also been reported [6,10]. And the
insoluble pellet isolated at stationary phase of
growth contained 73.5% of the metal apparently
associated with phosphorus moieties. Although a
marked diminution in cellular yield was observed
in phosphate-deficient medium, lead was predominantly localised in the bacterial cells. The
anionic nature of microbial cell walls provide a
favourable surrounding fo~ cationic metal deposition [11]. Metals like lanthanides have recently
been shown to be concentrated predominantly in
the periplasmic space [12]. To circumvent high
levels of non-metabolic metals, microorganisms
have evolved intracellular metal depositories. Intracellular metal-binding cyanophycin and metallothionein have been reported [13,14]. The nature
and the distribution of a lead sequestered within
the Pseudomonas fluorescens have to be further
delineated.
Citrate, the sole source of carbon to which
lead was complexed was rapidly metabolised in
the phosphate-rich medium. 28% of the total
citrate appeared unutilised in the phosphate deficient medium enriched with lead. Citrate, a tricarboxylate is known to be internalised via outermembrane proteins in some Gram-negative bacteria. It dissociates into the periplasmic space and
is then shuttled by specific proteins to the inner
membrane where it is consumed [15]. In Pseudomonas aeroginosa, while a 41-kDa and 19-kDa
protein in the outer membrane apparently induced by citrate were involved in the translocation of this tricarboxylic acid, other specific receptors were instrumental in shuttling iron-bound
citrate to the inner membrane [16]. In both the
phosphate-rich and -deficient media the pH of
the spent fluid increased. Thus it would appear
that the trianionic citrate would be bound to the
lead. This situation would indeed assure the interaction of citrate-lead complex with the bacterial cells. Whether disparate or similar protein
channel(s) in the outer membrane may be con-
tributing to the uptake of citrate and citrate-lead
complex, it is quite evident that in the
phosphate-rich medium, lead is converted into a
phosphorus-containing derivative and is predominantly externalised. Microintracellular protonation most likely in the periplasm may provide a
route for the removal of lead from its citrate
ligand. While the carbon source may be further
translocated to the inner membrane for energy
production, the lead may be removed from the
cellular milieu following its biotransformation into
an insoluble residue. In the phosphate-deficient
medium, subsequent to the facilitated intake of
lead citrate by outer-membrane protein(s) and
the freeing of citrate from the metallic element,
lead is sequestered in the bacterial cells. Intracellular ptoein in metal homeostasis has been reported in prokaryotes [13]. The nature and the
distribution of lead in the microbe needs to be
further investigated.
Extracellular biopolymers like carbohydrates
and proteins have been shown to play a determinant role in metal detoxification. The production
of cadmium-binding protein has been demonstrated in Pseudomonas putida [14]. We have
shown both qualitative and quantitative variations
in exopolysaccharide production in rhizobia [17].
These altered biomolecules may be either augmenting the affinity of these exopolymers in metal
sequestration a n d / o r mediating the adaptation
of the organism to non-ideal situation [18]. In this
study such a possibility seems unlikely as no major stimulation in exocellular protein and carbohydrate biosyntheses were observed in metal supplemented cultures.
Although the exact mechanisms involved in
lead detoxification has to await further delineation, it is evident that in a phosphate-rich culture, Pseudomonas fluorescens primarily insolubilizes lead and in a phosphate-deficient medium,
lead is predominantly sequestered within the microbe. Thus variation of nutrient concentration
promotes either intracellular or extracellular
bioaccumulation of lead. These findings have the
potential of being exploited in the removal of
lead from polluted areas.
290
ACKNOWLEDGEMENTS
This work was supported by grants from
Canada Employment and Immigration, Ministry
o f Mines and Northern development and Laurentian University.
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