ELSEVIER
FEMS Microbiology Ecology 20 (1996) 185-194
Mercury resistance in aromatic compound degrading
Pseudomonas strains
Paola Barbieri
*, Giuseppina
Bestetti, Daniela Reniero, Enrica Galli
Dipartimerzto di Genetica e di Biologia dei Micrurganismi,
UniL,ersitir degli Studi di Milano. Via Celoria. 26. 20133 Milan, Ital?
Received 7 August 1995; revised 27 March 1996; accepted 29 March 1996
Abstract
Six of ten Pseudomonas
strains selected from environmental samples for their ability to degrade aromatic compounds
were found to be mercury resistant. Mercury detoxification proceeded through Hg’+ volatilization and the genes involved
were chromosomally located. All the mercury resistant strains proved able to degrade aromatic compounds in the presence of
Hg’+.
Keywords:
Mercuric chloride; Organomercurial: Detoxification: Aromatic compound; Degradation
1. Introduction
Bacteria
of the genus Pseudomonas
are known
for their versatility in the degradation of a variety of
aromatic compounds derived from recalcitrant materials such as petroleum or lignin. Aromatic compound degrading bacteria can easily be isolated from
polluted soils or wastewater treatment plant sludges
and exploited for bioremediation
purposes. Their
exploitation,
however, involves problems such as
competition
with prevailing indigenous
microflora,
predation. substrate accessibility [I], or the presence
of inhibitory compounds.
Heavy metals are known to be powerful inhibitors
of biodegradation
activities [2,3], thus their presence
may impair biodegradation
of aromatic compounds
in polluted sites. Situations where simultaneous con-
* Corresponding author. Tel: +39 (2) 266 05227: Fax: f39
(2) 266 4551; E-mail: [email protected]
tamination by heavy metals and organic compounds
are present can be expected, and have been detected
in industrial areas [4-61. For this reason, there is
increasing interest [7,8] in bacterial strains that degrade aromatic compounds and tolerate toxic metals
and reports on strains naturally endowed with both
abilities have sporadically appeared [7,9-l 11. Even
though studies on metal tolerant bacteria have pointed
out that this characteristic
is not uncommon,
this
property is not often examined in aromatic degrading
bacteria, and mostly not considered.
We were particularly interested in bacterial mercury resistance since an investigation [I’_] carried out
in the district of Milan evidenced that 39% of samples from natural water courses and 59% from artificial ones were above the 0.5 p.g I-’ (2.5 nM) limit
suggested for mercury concentration
in the lowest
quality waters, mercury being the highest metal pollutant. Unfortunately,
in that study no data on the
levels of organic pollutants were made available.
0168.6496/96/$15.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier
PII SO168-6496(96)00029-3
Science
B.V
P. Burbieri
186
et ~1. / FEMS Microbiology
The aim of this work is to evaluate metal, and in
particular mercury resistance in a small, but representative, group of aromatic compound-degrading
Pseudomonas strains and to obtain preliminary information about the suitability of such strains for possible applications to bioremediation
purposes.
2. Materials and methods
2.1. Bacterial
strains and growth conditions
The Pseudomonas strains listed in Table 1 were
isolated
by enrichment
cultures
from different
wastewater sludges for their ability to use a given
aromatic compound as the only carbon and energy
source. The strains were isolated over a 10 year
period [ 13- 191. For this long-term storage, the strains
were maintained
at - 80°C in 25% glycerol, and
then grown at 30°C on mineral medium M9 [20],
supplemented
each with its own appropriated substrate. Hydrocarbons
were supplied in the vapor
phase and soluble substrates were supplied at a final
concentration of 10 mM.
2.2. Screening for metal resistance
Pseudomonas strains were tested for tolerance to
several different metal salts. The salts were incorporated into LB (Luria-Bertani medium) [20] agar plates
at various concentrations according to their solubility
and toxicity previously published [2 I]. 10 p,l droplets
containing about lo5 bacteria were streaked on the
plates, and after 24 and 48 h incubation at 30°C
Table I
Bacterial
Ecology 20
C1996)
185-l
94
their ability to form confluent growth on the metal
containing media was evaluated. When, among the
tested strains, remarkable growth differences were
found in the presence of increasing metal salt concentrations, the plating efficiency was calculated as
the rate between the number of colony forming units
in the presence of a given metal concentration
and
the number of colony forming units in the absence of
metal salts.
2.3. Injluence of mercury chloride and organomercurial compounds on bacterial growth
The levels of resistance to inorganic and organic
mercury were evaluated in NEM medium: 2 g l- ’ ,
D-glucose; 5 g l-l, Casamino acids; 1 g 1-l) yeast
extract; and 0.1 g 1~ ’ , MgSO,. 100 t.~l of an overnight
culture grown in NEM in the presence of a sublethal
concentration
of Hg for enzyme induction (5 p,M
HgCl,) were inoculated in 10 ml of fresh medium
containing
increasing
concentrations
of the compound. After 18 h of incubation at 30°C with shaking
the growth was evaluated by measuring the OD at
600 nm.
M9 medium was used to evaluate the levels of
HgCl, tolerated by the Pseudomonas strains when
grown on aromatic compounds as the only source of
carbon and energy. A series of flasks with M9
lacking any organic compound other than the growth
substrate and containing increasing concentrations of
HgClz were inoculated with a 1:20 dilution of an
overnight culture grown in M9 supplemented
only
with 5 g 1-l yeast extract. The substrates were
supplied as described above; growth conditions and
strains
Strain
P. jluorescens
c2
P. jluorescens
ST
CINNS
Fe2
MST
TMB
N3
A
F
BF
P. stutzeri
P. jluorescens
P. putida
P. putida
P. fluorescens
P. putida
P. jluorescens
Pseudomonas
SD.
Isolation substrate
Relevant growth characteristics
Ethylbenzene
Styrene
Cinnamic acid
Ferulic acid
tx-Methylstyrene
1.2.4.Trimethylbenzene
Naphthalene
Styrene
Phenylacetic acid
Biahenvl
Propyl- and butylbenzene
2-Phenylethanol,
phenylacetic
Phenylpropionic
acid
p-Hydroxycinnamic
acid
Styrene, toluene, benzene
nr-Xylene, p-xylene, toluene
Biphenyl
Phenylacetic acid
1-Phenylethanol
Reference
acid
1131
[I41
1151
1161
[171
[IsI
[191
Unpublished
Unpublished
Unpublished
data
data
data
P. Barbieri et al. / FEMS Microbiology Ecology 20 (19961 185-I94
187
monitoring were performed as described above, and
cultures were checked after 36 h of incubation.
causing the Hg’+-dependent
NADPH per min at 25°C.
2.4. Preparation
reductase assay
2.5. DNA extraction
of cell-free
extracts
and mercuric
Cells grown in LB broth were inoculated into the
same medium with or without the inducer (20 pM
HgCl,) and harvested in the late exponential growth
phase. The harvested cells were washed twice by
centrifuging
in 0.01 M potassium phosphate buffer
(pH 7.0) and resuspended in the same buffer supplemented with 4 mM 2-mercaptoethanol.
The cells
were disrupted at 4°C by passage through a French
pressure cell (American Instruments Co., Inc., Washington, DC). After treatment with 100 pg DNAse,
broken cell suspension was clarified by centrifuging
twice at 26000 X g at 4°C for 30 min. The clear
supernatant solution was used as the source of soluble enzymes. Total protein content was determined
by the Layne method [22] with bovine serum albumin (Sigma Chemical Co., St. Louis, MO) as a
standard.
Mercuric reductase activity [23] was assayed in
cell-extract by following the HgCl ?-dependent oxidation of NADPH (Boehringer Mannheim) at 340 nm
in a 3 ml reaction mixture which contained 100 pM
NADPH, 100 p,M HgCl,, 20 mM potassium phosphate (pH 7.0), 2 mM 2-mercaptoethanol,
and 10 ~1
cell extract. Assays were carried out at 25°C in a
Beckman DU20 spectrophotometer.
One unit of mercuric reductase was defined as the amount of enzyme
Table 2
Concentrations
Strain
Ag+
c2
ST
CINNS
Fe2
MST
TMB
N3
A
F
BF
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
oxidation
and Southern analysis
Bacterial DNA was extracted by lysing the cells
with SDS. The lysate was then treated with pronase
and RNase and purified by phenol extraction and
dialysis as described by Ljungquist and Bukhari [24].
Plasmid DNA was extracted by a procedure based
on the method of Hansen and Olsen [25]; for large
scale preparation
it was purified by an ethidium
bromide-CsCl density gradient. Endonuclease digestions, DNA analysis by gel electrophoresis and transfer to Hybond N filter were performed by standard
procedures [20].
Completely sequenced TnSOI [26] was used as
the source of mer and tnp gene probes. Tn501 was
digested with AuaI to generate a 2.7 kb fragment
carrying merTPAD genes and with AJYII-EcoRI to
generate a 3.5 kb fragment carrying tnpRA genes.
merB probes were from plasmid pDU13.58 of Serrutia marcescens, carried by a 0.57 kb fragment obtained by AvaII-EcoRV
digestion of pHG106 [27],
and from plasmid pPB of P. stutzeri OX, carried by
a 0.9 kb Hind111 fragment [28]. The fragments were
extracted from low melting agarose gel, purified by
phenol-chloroform
extraction and 32P-labelled with
03*P-dATP using the Random Primer Labelling kit
(Bethesda Research Laboratories, Inc.) according to
the supplier’s instructions. Hybridization was carried
out in 2 X SSC and 50% (v/v> formamide at 42°C
(mh4) of metals which allowed confluent growth of different Pseudomonas strains on LB plates
HAsO’Nil+
pb?+
Sn?+
B o?Ba?+
Cd?+
Co?+
Cr&
Cu?+
f_@+
TeOf4
4 7
13
13
13
45
45
45
45
13
13
13
nd: not determined.
7.8
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
0.4
0.4
16
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
< 0.4
0.4
0.4
0.4
1
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
< 0.35
0.35
< 0.35
0.35
0.35
< 0.35
0.35
0.35
< 0.35
< 0.35
of 1 pmol of
1
1
1
1
1
I
1
1
1
1
0.04
0.04
< 0.02
0.04
< 0.02
< 0.02
< 0.02
0.04
0.04
0.04
1.5
1.5
1.5
1.5
nd
1.5
nd
1.5
1.5
1.5
5.2
5.2
5.2
5.2
nd
5.2
nd
5.2
5.2
5.2
0.8
0.8
0.8
0.8
nd
0.8
nd
0.8
0.8
0.8
0.02
0.02
0.02
0.02
nd
< 0.02
nd
0.02
0.02
0.02
Tl+
zfl?+
< 0.2
0.2
< 0.2
< 0.2
nd
0.2
nd
0.2
0.2
< 0.2
0.35
< 0.35
1.75
7.0
0.35
0.35
0.35
< 0.35
< 0.35
< 0.35
P.Barbieri
188
et al. / FEMS Microbiolugy
overnight.
After hybridization,
the filters were
washed several times in 2 X SSC. 0.1% SDS at
65°C. For autoradiography
the dried filters were
exposed to Fuji X-ray film next to an intensificator
screen at - 80°C.
3. Results
3.1. Screening for metal tolerance
In a preliminary screening we evaluated the ability of the Pseudomonas strains listed in Table 1 to
form confluent growth in the presence of increasing
concentrations of different metal salts. In Table 2 are
reported the highest tested concentration
of each
metal which allowed confluent growth. The tested
strains did not exhibit significant differences as regards the tolerance to AgNO,, CoCl,, K2Cr207,
CuSO,, NiSO,, (CH,COO),Pb,
SnCl,, Na,TeO,,
and CH,COOTl; on the contrary significantly different tolerance levels were found in the presence of
HgCIL, NazHAsO,,
Na,B,O,.
CdCl,, ZnSO,, and
BaC12. Six strains, namely Fe2, BF, A, ST, F, and
C2, were found to grow in the presence of 40 p_M
HgCl : . Two of these strains showed, when compared with the others, a higher tolerance to other
metal salts in addition to HgCl,. C2 was also able to
grow in the presence of 7.8 mM Na,B,O,,
a concentration 3-fold higher than that tolerated by the
other strains, and Fe2 was found able to grow in the
Table 3
Plating efficiency of Psezrdorwnas
preliminary screening
Strain
in the presence
20 (IYY6)
185-103
presence of 7 mM ZnSO,, a concentration
4- to
20-fold higher than that tolerated by the other strains,
and 45 mM Na,HAsO,.
Tolerance to Na,HAsO,
was also found in the Hg-sensitive
strains TMB,
MST, and N3. Strain TMB was also found to be the
only one able to tolerate 1 mM CdCl,. Only one
strain, CINNS, was found to tolerate 16 mM BaCl,.
The metal-tolerant
strains were further analyzed for
their plating efficiency in presence of the highest
metal concentrations
where they showed growth in
the preliminary
screening (Table 3). It was found
that the plating efficiency of the resistant strains
grown in the presence of a given metal concentration
was approx. 5 order of magnitude higher than that of
the sensitive strains.
3.2. Evaluation
compounds
qf growth in the presence ofmercury
The ability to grow in increasing concentrations
of different mercurial compounds was evaluated as
described in Materials and methods for strains Fe2.
BF, A, ST, F, and C2; strain TMB. which was
unable to grow in the presence of 40 p,M HgCl,,
was used as the negative control. Although almost all
the screenings for Hg resistance proceed in rich
media, the possible formation of complexes with
organic compounds, and in particular with sullhydryl
groups, can lead to overestimation
of the MIC. In
these growth assays we used NEM medium. which
was developed by Nelson et al. [29] to minimize the
of the metals
for which
differences
in the growth
were observed
in the
Metal concentration
Hg’+
ST
A
c2
Fe2
F
BF
CINNS
TMB
MST
N3
strains
Ecology
Zn’+ (7 mM)
B,O$-
I
(0.04 mM)
< 10-6
< 10-b
1
9 x 10-I
8.6 x 10-l
5 x 10-l
4 x 10-l
< IO-b
< 10-b
< lomh
< 10-h
<
<
9x
<
<
<
<
<
<
< lomb
6 x IO- '
< lomh
< IO-b
< 1O-h
< 10-O
< 1o-6
i IOmh
< IO-b
10-6
lo-h
10-l
lomb
10-h
lomh
10-O
10-h
1om6
(7.8 mM)
Ba’+ (I6 mM)
Cd’+
< 10-b
< 10-h
< 1om6
< l0-h
< lo-b
< 10-h
< lomh
< IO-h
1.8X IO-’
< 10-O
< l0-h
< 10-h
<10-e
< 10-h
< 10-b
< lo-”
8.3 x 10-l
< 10-h
< 10-h
< 10-h
(I mM)
HASO:IO-b
IO-h
IO-h
8.8 x loIO-h
< 10-b
< IO-h
1
I
6.7 X IO-
(45 mM)
’
’
P. Barhieri et al. / FEMS Microbiology
titration of Hg’+ by potential binding to sulfur-containing compounds.
As reported in Fig. 1, the MIC of HgCl, for the
sensitive strain TMB was 20 FM; on the contrary,
for the other tested strains. with the only exception
of strain BF which showed a MIC of 4.5 FM. the
MICs were higher than 45 ~_LLM
(see also Table 5,
under NEM entry). Strain ST exhibited the highest
level of resistance to HgC12, showing at 45 PM
HgCl? 70% of growth with respect to the control
culture grown in the absence of Hg; on the contrary,
strains F and BF seemed to be less tolerant, although
their resistance level was much higher than that of
the sensitive TMB strain.
In Pseudomonas two phenotypic classes of mercury resistance are known: (i) narrow spectrum resistance to HgCl?, and (ii) broad spectrum mercury
resistance to some specific organomercurial
compounds such as phenylmercuric
acetate (PMA),
methylmercuric
chloride (MMC), and thimerosal
Ecology 20 ( 1996) 185- 194
189
in addition
to HgCl?.
Resistance
to
(TH).
organomercurial
compounds is due to the activity of
organomercurial
lyase, which hydrolizes the C-Hg
bond before Hg” reduction [30].
To verify which class our strains could fall into,
we assayed their ability to grow in the presence of
PMA, MMC. and TH. The results, reported in Fig. 1,
showed that the differences among the MICs of
organomercurial
compounds for the six Hg resistant
strains were less remarkable than those observed in
the presence of inorganic mercury, especially when
the strains were grown in the presence of PMA.
However, only strain Fe2 was able to grow in the
presence of organomercurial
concentrations
significantly higher than those tolerated by even a Hg-sensitive strain like TMB. In fact, Fe2 showed 50% and
85% of growth in the presence of 45 pM TH and
3.75 p,M MMC, concentrations
which were inhibitory or allowed less than 30% growth respectively for all the other strains. Therefore. Fe2 seemed
PHENVLMERCURIC
Fig. 1. Effect of mercuric chloride, phenylmercuric
acetate, thimerosal. and methylmercuric
chloride on the growth of Pseudomonas A (H ),
ST (0). BF (V ), C2 (A ), Fe2 (0). F (0). TMB (A ). Data are expressed as percentage of growth in the presence of the given
concentration of mercuric compounds with respect to the control culture grown in the absence of mercury.
190
P. Barbieri et al. / FEMS Microbiology
to be the only one to display
resistance phenotype.
3.3. Mercuric
reductase
a broad
spectrum
assays
Bacterial mercury resistance is due to the mercuric reductase catalyzed conversion of inorganic,
and in some cases, organic mercury compounds to
elemental mercury (Hg”). which is less toxic and
more volatile and released from the cells [23,30].
To verify if the mercury resistance observed was
due to a real detoxification, the presence of mercuric
reductase activity was investigated in cell-free extracts of strains BF, C2, A, ST, F, and Fe2, grown in
the presence or in the absence of HgCl,. When the
cells were grown in the presence of HgCl? comparable levels of mercuric reductase were measured in
the extracts of strains C2, A, St, F. and Fe2, while in
strain BF the level was lower (Table 4). No activity
kb
7.0 6.4 -
Ecology 20 ClYY6) 185-194
Table 4
Mercuric reductase activity in cell extracts of the Hg resistant
Preudomorzas strains after induction in the presence of HgCl,
Strain
Specific activity
(nmol of NADPH oxidized
min- ’ rng- ’ of protein)
ST
A
C2
Fe2
F
BF
86
95
69
70
68
43
was detected in uninduced cell-extracts. HgCl,-dependent NADPH oxidation was not detectable in
cell-free extracts of sensitive strain TMB grown in
the presence of a sublethal concentration of HgCl? (5
FM).
1
1 2 3 4 5 6 7 8 9 10
2
3
4
5 6
7
8
9 10
kb
12.5 10.0 7.0 6.0 -
2.9 -
2.4 -
Fig. 7. Southern hybridizations
of total and plasmid DNA from Pseudomonas strains probed with Tn501 nzerTPAD genes (A) and with
Tn501 tnpRA genes (B). Lane I, BF (total DNA); lane 2. C2 (plasmid pC2); lane 3, C2 (total DNA): lane 4. Fe2 (plasmid pFe2); lane 5,
Fe2 (total DNA); lane 6. F (total DNA); lane 7, A (total DNA); lane 8, TMB (total DNA); lane 9. ST (plasmid PEG); lane IO. ST (total
DNA). All DNA samples were digested with EcoRI. Hind111 digested A DNA was used as the molecular weight marker (not shown). kb.
kilobases.
P. Barbieri et al. / FEMS Microbiolog! Ecology 20 C19961 185-l 94
3.4. Southern analyses
We previously showed that three of the six Hg
plasmid,
resistant
strains harbour an indigenous
namely pC2 [ 131, PEG [ 141, and pFe2 [ 161, involved
in aromatic compound catabolism.
To investigate the presence and the location of
mer genes in the genome of the Hg resistant strains,
Southern analysis was performed using a Tn501 mer
gene probe, as these genes are highly homologous
with mer genes isolated from different Gram-negative bacteria [30].
None of the plasmids cited was found to contain
DNA homologous with Tn501 nzer genes but sequences that hybridized to this probe were found in
the chromosome of all the Hg resistant strains (Fig.
2A). Hybridization
with tnpRA genes (Fig. 2B) revealed that, with the only exception of strain BF, the
tested strains, including the Hg-sensitive strain TMB,
carry in their genome sequences homologous to this
probe; sequences sharing homology with tnpRA were
also detected in PEG, but not in pC2 and pFe2.
Strain Fe2, which was the only one that displayed
a certain level of resistance to organomercurials,
did
not show any hybridization
signal when probed
against both the S. marcescens and the P. stutzeri
merB (data not shown).
3.5. Growth
of mercury
on aromatic compounds
in the presence
We investigated the levels of mercury tolerance in
strains A, ST, F, BF, C2, and Fe2, when grown on
Table 5
MICs of HgClz for different
cultural conditions
Strain
ST
A
c2
Fe2
F
BF
TMB
Pseudomonas strains
MIC of HgCI, (FM) in:
Isolation substrate
Styrene
Styrene
Ethylbenzene
Ferulic acid
Phenylacetic acid
Biphenyl
1,2.4-Trimethylbenzene
TMB was used as negative
in different
control.
M9+
isolation
substrate
M9+
NEM
20
25
30
25
20
25
10
30
25
> 30
30
20
30
10
> 50
> 50
> 50
50
50
45
20
191
an aromatic compound, namely the isolation substrate, as the only carbon and energy source (Table
5). Hg sensitive strain TMB was used as negative
control. As expected, in minimal medium the MICs
were lower than those observed in NEM medium.
In general, the MICs in the presence of aromatic
compounds are only slightly lower than those evaluated in the presence of malate. Such differences do
not seem to correlate with the chemical properties of
the compound used as carbon source. However, in
all the cases the MICs were 2 to 3-fold higher than
those of the sensitive strain TMB grown in the same
cultural conditions.
4. Discussion
Interest in bacterial resistance to metal salts, especially when associated with degradative activities,
has been increasing since in industrial areas both
metal and organic compound pollution is of environmental concern. Studies of these two bacterial abilities, namely the tolerance to toxic metals and the
degradative capacities, have been extensively carried
out, but the two features are often considered separately, not taking into account that in industrial areas
pollution by organic compounds, i.e. fossil fuels or
derivatives, is often accompanied
by pollution by
heavy metals, such as mercury.
We tested the metal tolerance of ten different
Pseudomonas strains from our collection. They were
previously isolated from environmental
samples for
their ability to utilize aromatic compounds as the
only carbon and energy source. Few of them were
found to tolerate metal salts such as ZnSO,,
Na,B,O,,
BaCl?, Na,HAsO,,
and CdCl,, while six
strains out of the ten tested were found to be able to
grow in the presence of HgCl,. The HgCl, detoxification mechanism was found to involve an inducible
mercuric reductase activity. However, although the
levels of mercuric reductase measured in the cell-free
extracts of the six mercury resistant strains were
al ways significant after induction with HgC12, differences were found among the different strains. According to its low activity level, strain BF displayed
the lowest tolerance.
On the basis of growth data (detailed above),
strain Fe2 was the only one which might be sup-
192
P. Barbieri
et al. / FEMS Microbiology
posed to fall into the broad spectrum resistance class.
Nevertheless,
we could not detect any homology
between its genome and two different versions of
merB gene. However. recent papers [28,31] suggest
that there may exist different versions, sharing no
homology, of this gene. To verify if strain Fe2 really
falls into broad spectrum
resistance
class, Hg
volatilization
in organomercurial
compounds should
be assayed.
Plasmid involvement
in single or even multiple
metal resistance has been widely reported [32]. In
particular, mercury resistance is often plasmid encoded, and, in different catabolic strains. it was
found that both the catabolic and the mercury detoxification genes are plasmid-borne, as is the case of the
OCT plasmid in the octane degrader P. oleovorans
[ 1 I], pWW 17 in the phenylacetate degrader P. putidu
MT14 [lo]. pJP4 in the 2,4-dichlorophenoxyacetate
degrader Alcaligenes eutrophus JMP 134 [33.34], and
other degradative plasmids of the IncPP group [35].
Moreover, mer genes are often part of transposons
[26,36], a localization which can facilitate their horizontal transfer among different bacterial species and
also between plasmid and chromosome.
Indeed,
chromosomal location of mercury resistance genes is
not unknown [37,38], although their evolutive origins
are poorly known.
As opposed to what has been previously observed
in P. stnt;eri OX, where mer genes are plasmid
encoded and catabolic genes chromosomally
located
[9], in the six Hg resistant strains hereby described,
three of which carry a plasmid involved in aromatic
compound catabolism [ 13,14.16], Hg resistance is
chromosomally
encoded by genes homologous with
those of Tn501. Three strains, A. ST, and F. which
degrade styrene or related molecules (Table 1) and
had been isolated from different sources, showed an
identical hybridization
profile, suggesting that they
are very closely related and that they may have
shared a common source of mercury resistance encoding genetic material. These observations
might
suggest that in these three strains mer genes could
have been acquired by horizontal gene transfer, taking into account that they carry in their genome
sequences homologous to Tn501 tnpRA. However,
the presence of such sequences is not necessarily
related to mercury resistance, as they were found
even in the Hg sensitive strain TMB: on the other
Ecology
20
f 1996)
185-194
hand also m-xylene and p-xylene catabolic genes are
known to be carried by transposons [39].
It is worth noting that the composition
of the
medium used to carry out screenings for metal resistance in bacteria affects the estimation of the MICs,
as many metals can be complexed by organic compounds leading to overestimation
of the levels of
tolerance. On the other hand, it must be taken into
account that, in natural environments,
the formation
of complexes may reduce the amount of bioavailable
metals. Clearly, increasing concentrations of mercury
does inhibit biodegradation
of organic matter, but it
does not seem possible to correlate lower resistance
levels with a particular substrate. However, the ability of the Hg resistant strains to grow in minimal
medium with the isolation substrate supplied as the
only carbon and energy source and in the presence of
activities can be
HgC12. suggests that degradative
carried out even in the presence of mercury and that
these two abilities,
namely
aromatic compound
degradation
and mercury resistance,
can be expressed simultaneously,
at least at the levels normally found at sites polluted by mixed mercury and
organic contaminants,
such those detected in the
district of Milan.
On the whole, our data suggest that bacterial
strains endowed with the capacity of both degrade
aromatic compounds and tolerate toxic metals are
not uncommon
in polluted environments,
as also
reported by other authors [7.35]. Indeed, most such
strains were isolated from polluted environments,
where also metal resistance,
in addition to the
degradative abilities. can be of adaptative relevance.
Thus. such metal resistant and aromatic degrading
strains are suitable for further studies, aimed at
exploiting their characteristics
in the treatment of
polluted sites or wastes, especially in the cases of
non-contained environmental
applications. i.e. in situ
bioremediation
or treatment plants, where the use of
genetically engineered bacteria is unadvisable.
Acknowledgements
This work was supported
by the Minister0
dell’UniversitB della Ricerca Scientifica e Tecnologica. U. Pasotti collaborated in the experimental work.
P. Barbieri
et al. / FEMS Microbiology
Ecology
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