Previous Article | Next Article 
Applied and Environmental Microbiology, February 2001, p. 1015-1019, Vol. 67, No. 2
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.2.1015-1019.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Nickel-Resistance-Based Minitransposons: New Tools
for Genetic Manipulation of Environmental Bacteria
Safieh
Taghavi,1
Hilde
Delanghe,1
Cindy
Lodewyckx,1,2
Max
Mergeay,1 and
Daniël
van der
Lelie1,*
Vlaamse Instelling voor Technologisch
Onderzoek (Vito), Environmental Technology Centre, Boeretang 200,
2400 Mol,1 and Limburgs Universitair
Centrum, Environmental Biology, Universitaire Campus, B3590
Diepenbeek,2 Belgium
Received 5 October 2000/Accepted 27 November 2000
 |
ABSTRACT |
The ncc and nre nickel resistance
determinants from Ralstonia eutropha-like strain 31A were
used to construct mini-Tn5 transposons. Broad host
expression of nickel resistance was observed for the nre
minitransposons in members of the 
, 
, and 
subclasses of the
Proteobacteria, while the ncc minitransposons
expressed nickel resistance only in R. eutropha-like strains.
| |
TEXT |
Several nickel resistance
determinants have been identified in Ralstonia eutropha
(Alcaligenes eutrophus) (24) strains isolated from
different biotopes heavily polluted with heavy metals. The cnrYXHCBA operon of R. eutropha CH34 plasmid
pMOL28 (12), which mediates medium levels of nickel
resistance (up to 10 mM) and cobalt resistance, is the most thoroughly
studied determinant (3, 11, 17, 18, 20). The resistance
mechanism mediated by cnr is inducible and is due to an
energy-dependent efflux system driven by a chemo-osmotic
proton-antiporter system (6, 18, 22, 23). A 14.5-kb
BamHI fragment of plasmid pTOM9 from R. eutropha-like strain 31A (Alcaligenes xylosoxidans 31A)
(10) and a similar BamHI fragment of plasmid
pGOE2 from R. eutropha-like strain KTO2 were also found to
encode Ni resistance. On both fragments a locus mediating high-level
nickel resistance (up to 20 to 50 mM) and a locus mediating low-level
nickel resistance (3 mM) were identified and designated ncc
and nre, respectively (15, 16). The
nccYXHCBAN determinant, which except for the
nccN gene is very similar to cnr, causes high
levels of nickel and cobalt resistance and a low level of cadmium
resistance in R. eutropha. Neither cnr nor
ncc is expressed in Escherichia coli. On the
other hand, the 1.8-kb nre locus causes low levels of nickel
resistance in both Ralstonia and E. coli
(16). An nre-like determinant, which could be expressed in E. coli and Citrobacter
freundii, was also found in Klebsiella oxytoca
CCUG15788 (19, 20).
Recently, amplified ribosomal DNA restriction analysis was used to
determine the phylogenetic position of zinc- and nickel-resistant Ralstonia-like strains (2). The ncc
operon was found in many nickel-resistant R. eutropha-like
strains and in environmental strains in the direct vicinity of the
genus Burkholderia (2), a member of the subclass of the class Proteobacteria like the genus
Ralstonia. This might indicate that ncc has range
of expression broader than the genus Ralstonia.
Heavy metal resistance markers with broad host expression ranges have
been shown to be useful for genetic manipulation of Pseudomonas strains potentially designated for environmental
release (14). Broad-host-range expression of
ncc-nre was recently confirmed by Dong et al.
(7), who found ncc-nre-based Ni resistance in Comamonas, Sphingobacterium heparinum, flavobacteria, and
even gram-positive bacteria related to Arthrobacter.
However, it was not clear from this study which of the Ni resistance
determinants was responsible for the broad-host-range Ni resistance. In
addition, plasmid instability problems were encountered with some of
the transconjugants. In order to study the range of expression of ncc and nre and to develop new tools for genetic
manipulation of environmental bacteria, which are not based on
antibiotic resistance markers, the Ni resistance markers were
introduced into mini-Tn5 transposon vectors. The new
nre-based minitransposons were found to have a broad
expression range and were successfully used for constructing
Ni-resistant transconjugants of plant-associated bacteria belonging to
families of the , , and subclasses of the class
Proteobacteria, including plant-associated endophytic bacteria with potential to improve phytoremediation strategies (C. Lodewyckx, S. Taghavi, M. Mergeay, J. Vangronsveld, H. Clijsters, and
D. van der Lelie, submitted for publication).
Construction of Ni resistance minitransposons.
The
ncc operon of pTOM9 was cloned in pUC18/NotI as a
8.1-kb BamHI-PstI fragment, resulting in
pMOL1522 (E. coli CM2395). Plasmid pMOL1522 was digested
with NotI, and the ncc-containing NotI fragment was subsequently cloned in the unique
NotI site of pUTmini-Tn5-Km1
(4). This resulted in plasmid
pUTminiTn5-Km1/ncc (pMOL1524 in E. coli CM2428).
In order to construct an nre-based mini-Tn5
transposon vector, it was necessary to inactivate the NotI
site in nreB. PCR mutagenesis performed with primers
nre-PstI (sense) and nre-NotI
(antisense) and with primers nre-NotI (sense) and
nre-EcoRI (antisense) (Table 1) was used to change the NotI
site with the sequence GCGGCCGC into GCGGCAGC.
This resulted in two PCR fragments that were approximately 1.6 and 1.1 kb long, respectively. Subsequently, these fragments were mixed
and joined by using a PCR-based ligation strategy and were amplified
with primers nre-PstI (sense) and nre-EcoRI
(antisense). This resulted in a 2.7-kb PstI-EcoRI
fragment with the mutated nre operon. The mutation did not
affect the amino acid sequence of the NreB protein, since GCC and GCA
both encode alanine. The 2.7-kb PstI-EcoRI
fragment was subsequently cloned into pUC18/NotI, resulting in plasmid pMOL1525 (E. coli CM2438). Plasmid
pMOL1525 was digested with NotI, and the
nre-containing NotI fragment was cloned in the
unique NotI site of pUTmini-Tn5-Km1. This
resulted in plasmid
pUTminiTn5-Km1/nre(NotI)
(pMOL1527 in E. coli CM2442).
The region with the mutated
nreB gene was also amplified as
a 2.7-kb
PstI fragment by using primers
nre-PstI
(sense) and
nre-PstI
(antisense). Subsequently, this
fragment was cloned in the unique
PstI site of pMOL1522.
This resulted in a 10.8-kb
ncc-nre fragment
flanked by
two
NotI sites (plasmid pMOL1548 in
E. coli
CM2500).
This fragment was subsequently cloned in the unique
NotI site
of pUTmini-Tn
5-Km1, resulting in
plasmid pUTminiTn
5-Km1/
ncc-nre (NotI)
(pMOL1554 in
E. coli CM2520).
To inactivate the
SfiI site in
nreA, we used a
strategy similar to that used for mutation of the
NotI site,
except that primers
nre-PstI (sense) and
nre-SfiI
(antisense) and primers
nre-SfiI
(sense) and
nre-EcoRI (antisense) were used. The mutations did
not
affect the amino acid sequence of the NreA protein. A 2.7-kb
PstI-
EcoRI fragment with the mutated
nre operon (
SfiI site) was
subsequently cloned in
pUC18/
SfiI (
8), resulting in plasmid
pMOL1526
(
E. coli CM2440). Plasmid pMOL1526 was digested with
SfiI, and the
nre-containing
SfiI
fragment was cloned in
SfiI-digested
pUTmini-Tn
5-Km1. This resulted in plasmids
pUTminiTn
5-Km1/
nre(SfiI)
(pMOL1529 in
E. coli CM2446) and pUTminiTn
5-
nre(SfiI)
(pMOL1528
in
E. coli CM2444), in which the kanamycin
resistance gene was
replaced by
nre. The restriction maps of
the mini-Tn
5 Ni resistance
transposons are presented in Fig.
1.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 1.
Schematic representation of different
mini-Tn5 Ni resistance transposons. The positions of the
kanamycin resistance marker (Km), the Ni resistance determinants
ncc and nre, the inverted repeats at the
extremities of the minitransposons, and important restriction sites are
indicated. The sizes of the minitransposons are given in parentheses.
|
|
Range of expression of Ni resistance.
The range of expression
of Ni resistance was examined for all mini-Tn5 Ni resistance
transposons. To do this, the pUT-based constructs were introduced into
E. coli S17-1 (
pir) (5) and subsequently transferred by conjugation into the nickel-sensitive strains R. eutropha AE104 (12), E. coli DH10B, Burkholderia cepacia W1.2 (isolated from
wheat) and LS2.4 (isolated from lupine shoots) (a gift from K. Ophel-Keller), Herbaspirillum seropedicae LMG2284 (associated with rye grass) (1), Pseudomonas
stutzeri A15 (associated with rice roots) (13, 25),
Azospirillum irakense KBC1 (a rice endophyte)
(9), and Pseudomonas putida VMO433. The last
strain was isolated as an endophytic bacterium after surface
sterilization of Brassica napus plants (Lodewyckx and van
der Lelie, unpublished data). Transfer frequencies, as well as the
appearance of nickel- and kanamycin-resistant mutants, were examined.
The results are presented in Table 2.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Mutation and transconjugation frequencies of the strains
used to test transfer and heterologous expression of ncc,
nre, and ncc plus nrea
|
|
Comparing the efficiencies of transfer of the minitransposons, we
observed that
R. eutropha AE104,
E. coli DH10B,
and
P. putida VM0433 showed the highest transfer
frequencies. The lowest transfer
frequencies were observed for
B. cepacia W1.2 and
P. stutzeri A15; this might have been
due to the presence of efficient restriction-modification
systems
present in these two
strains.
No spontaneous Ni-resistant mutants were found for the strains used in
the experiments; this is in contrast to the kanamycin-resistant
mutants
that were observed at low frequencies (~10
8) for most
of the strains tested. This indicates that nickel resistance
is a more
reliable marker for selecting transconjugants than
kanamycin.
Transconjugants were selected for kanamycin or nickel resistance (Table
2). The stabilities of the transconjugants were confirmed
by growing
them for more than 100 generations under nonselective
conditions.
Subsequently, the Ni resistance of these organisms
was compared to that
of the wild-type strains. As expected, both
ncc- and
nre-containing mini-Tn
5 transposons gave Ni
resistance
in
R. eutropha AE104, and the MICs on 284 gluconate medium (Lodewyckx
et al., submitted) were 3 and 40 mM Ni for
nre and
ncc, respectively
(Table
3).
For all of the other strains tested except
B. cepacia W1.2,
Ni resistance was observed when the
nre determinant was
present.
For these strains
miniTn
5-Km1/
ncc-containing transconjugants had
to
be selected for kanamycin resistance. The presence of
nre
resulted
in MICs of Ni for Ni resistance on 284 minimal medium with an
appropriate C source that varied from 2 to 3 mM depending on the
bacterial species (Table
3). In all cases the presence of
nre was confirmed by PCR (results not shown). No Ni
resistance was
observed for transconjugants containing
ncc,
and the presence
of both
ncc and
nre in general
did not increase the MIC for Ni
resistance, as determined for
nre. However, two exceptions were
found: in
P. stutzeri A15 the presence of
ncc resulted in an
increase
in Ni resistance (MIC) from 0.6 to 1.0 mM, while
B. cepacia W1.2
transconjugants showed Ni resistance only when both
ncc and
nre were present. The latter phenomenon
might imply that both
ncc and
nre contribute to
Ni resistance, but it is not clear in what
way. Therefore, it can be
concluded that in general broad-host-range
Ni resistance is encoded by
nre and that the
ncc determinant is
expressed
only in
R. eutropha-like strains. This implies that
only the
nre-based nickel resistance minitransposons, such as
miniTn
5-nre(
SfiI), are suitable as
broad-host-range selection
markers for construction of antibiotic
resistance-free but selectable
strains belonging to the families of the
,
, and
subclasses
of the class
Proteobacteria.
| |
ACKNOWLEDGMENTS |
This work was financially supported by the European Commission and
OVAM as an EFRO project.
We are grateful to K. Ophel-Keller and J. Balandreau for providing the
B. cepacia strains used in this study and to J. Vanderleyden and M. Gillis for providing the H. seropedicae strain. We
also thank T. Engelen and A. Bossus for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Vlaamse
Instelling voor Technologisch Onderzoek (Vito), Environmental
Technology Centre, Boeretang 200, 2400 Mol, Belgium. Phone:
32-14-33.51.66. Fax: 32-14-58.05.23. E-mail:
niels.vanderlelie{at}vito.be.
| |
REFERENCES |
| 1.
|
Baldani, J. I.,
V. D. L. Baldani,
F. L. Olivares,
G. Kirchof,
A. Hartmann,
B. Pot,
B. Hoste,
E. Falsen,
K. Kersters,
M. Gillis, and J. Döbereiner.
1996.
Emended description of Herbaspirillum; inclusion of (Pseudomonas) rubrisubalbicans, a milk plant pathogen, as Herbaspirillum rubrisubalbicans comb. nov.; and classification of a group of clinical isolates (EF group 1) as Herbaspirillum species.
Int. J. Syst. Bacteriol.
46:802-810[Abstract/Free Full Text].
|
| 2.
|
Brim, H.,
M. Heyndrickx,
P. De Vos,
A. Wilmotte,
D. Springael,
H. G. Schlegel, and M. Mergeay.
1999.
Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs.
Syst. Appl. Microbiol.
22:258-268[Medline].
|
| 3.
|
Collard, J.-M.,
A. Provoost,
S. Taghavi, and M. Mergeay.
1993.
A new type of Alcaligenes eutrophus CH34 zinc resistance generated by mutations affecting regulation of the cnr cobalt-nickel resistance system.
J. Bacteriol.
175:779-784[Abstract/Free Full Text].
|
| 4.
|
de Lorenzo, V.,
M. Herrero,
U. Jakubzik, and K. Timmis.
1990.
Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria.
J. Bacteriol.
172:6568-6572[Abstract/Free Full Text].
|
| 5.
|
de Lorenzo, V., and K. N. Timmis.
1994.
Analysis and construction of stable phenotypes in Gram-negative bacteria with Tn5 and Tn10-derived mini-transposons.
Methods Enzymol.
235:386-405[Medline].
|
| 6.
|
Diels, L.,
Q. Dong,
D. van der Lelie,
W. Baeyens, and M. Mergeay.
1995.
The czc operon of Alcaligenes eutrophus CH34: from resistance mechanism to the removal of heavy metals.
J. Ind. Microbiol.
14:142-153[CrossRef][Medline].
|
| 7.
|
Dong, Q.,
D. Springael,
J. Schoeters,
G. Nuyts,
M. Mergeay, and L. Diels.
1998.
Horizontal transfer of bacterial heavy metal resistance genes and its applications in activated sludge systems.
Water Sci. Technol.
37:465-468[CrossRef].
|
| 8.
|
Herrero, M.,
V. de Lorenzo, and K. Timmis.
1990.
Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.
J. Bacteriol.
172:6557-6567[Abstract/Free Full Text].
|
| 9.
|
Khammas, K. M.,
E. Ageron,
P. A. D. Grimont, and P. Kaiser.
1989.
Azospirillum irakense sp. nov., nitrogen fixing bacterium associated with rice roots and rhizosphere soil.
Res. Microbiol.
140:679-693[Medline].
|
| 10.
|
Lemke, K.
1994.
Schwermetallresistenz in den zwei Alcaligenes-Stämmen A. eutrophus CH34 und A. xylosoxydans 31A: Transposonmutagenese, Klonierung und Sequenzierung. Ph.D. thesis.
Universität Göttingen. Cuvillier Verlag Göttingen, Göttingen, Germany.
|
| 11.
|
Liesegang, H.,
K. Lemke,
R. A. Siddiqui, and H. G. Schlegel.
1993.
Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34.
J. Bacteriol.
175:767-778[Abstract/Free Full Text].
|
| 12.
|
Mergeay, M.,
D. Nies,
H. G. Schlegel,
J. Gerits,
P. Charles, and F. Van Gijsegem.
1985.
Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals.
J. Bacteriol.
162:328-334[Abstract/Free Full Text].
|
| 13.
|
Qiu, Y. S.,
S. P. Zhou,
X. Z. Mo,
D. Wang, and J. H. Hong.
1981.
Study of nitrogen fixing bacteria associated with rice root.
Acta Microbiol. Sin.
21:468-472. (In Chinese.)
|
| 14.
|
Sanchez-Romero, J. M.,
R. Diaz-Orejas, and V. de Lorenzo.
1998.
Resistance to tellurite as a selection marker for genetic manipulations of Pseudomonas strains.
Appl. Environ. Microbiol.
64:4040-4046[Abstract/Free Full Text].
|
| 15.
|
Schmidt, T.,
R.-D. Stoppel, and H. G. Schlegel.
1991.
High-level nickel resistance in Alcaligenes xylosoxidans 31A and Alcaligenes eutrophus KTO2.
Appl. Environ. Microbiol.
57:3301-3309[Abstract/Free Full Text].
|
| 16.
|
Schmidt, T., and H. G. Schlegel.
1994.
Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A.
J. Bacteriol.
176:7045-7054[Abstract/Free Full Text].
|
| 17.
|
Sensfuss, C., and H. G. Schlegel.
1988.
Plasmid pMOL28-encoded resistance to nickel is due to specific efflux.
FEMS Microbiol. Lett.
55:295-298[CrossRef].
|
| 18.
|
Siddiqui, R. A.,
K. Benthin, and H. G. Schlegel.
1989.
Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp.
J. Bacteriol.
171:5071-5078[Abstract/Free Full Text].
|
| 19.
|
Stoppel, R. D.,
M. Meyer, and H. G. Schlegel.
1995.
The nickel resistance determinant cloned from the enterobacterium Klebsiella oxytoca: conjugational transfer, expression, regulation and DNA homologies to various nickel-resistant bacteria.
Biometals
8:70-79[Medline].
|
| 20.
|
Stoppel, R.-D., and H. G. Schlegel.
1995.
Nickel-resistant bacteria from anthropogenically nickel-polluted and naturally nickel-percolated ecosystems.
Appl. Environ. Microbiol.
61:2276-2285[Abstract].
|
| 21.
|
Taghavi, S.,
M. Mergeay, and D. van der Lelie.
1997.
Genetic and physical maps of the Alcaligenes eutrophus CH34 megaplasmid pMOL28 and its derivative pMOL150 obtained after temperature induced mutagenesis and mortality (TIMM).
Plasmid
37:22-34[CrossRef][Medline].
|
| 22.
|
Tibazarwa, C.,
S. Wuertz,
M. Mergeay,
L. Wyns, and D. van der Lelie.
2000.
Regulation of the cnr cobalt and nickel resistance determinant of Ralstonia eutropha (Alcaligenes eutrophus) CH34.
J. Bacteriol.
182:1399-1409[Abstract/Free Full Text].
|
| 23.
|
Varma, A. K.,
C. Sensfuss, and H. G. Schlegel.
1990.
Inhibitor effects on the accumulation and efflux of nickel ions in plasmid pMOL28-harboring strains of Alcaligenes eutrophus.
Arch. Microbiol.
154:42-49[CrossRef].
|
| 24.
|
Yabuuchi, E.,
Y. Kosako,
I. Yano,
H. Hotta, and Y. Nishiuchi.
1995.
Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb. nov. and Ralstonia eutropha (Davis 1969) comb. nov.
Microbiol. Immunol.
39:897-904[Medline].
|
| 25.
|
You, C. B.,
X. Li,
H. X. Wang,
Y. S. Qiu,
X. Z. Mo, and Y. L. Zhang.
1983.
Associative nitrogen fixation of Alcaligenes faecalis with rice plant. Biol. N2
Fixation Newsl. Sydney
11:92-103.
|
Applied and Environmental Microbiology, February 2001, p. 1015-1019, Vol. 67, No. 2
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.2.1015-1019.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Taghavi, S., Garafola, C., Monchy, S., Newman, L., Hoffman, A., Weyens, N., Barac, T., Vangronsveld, J., van der Lelie, D.
(2009). Genome Survey and Characterization of Endophytic Bacteria Exhibiting a Beneficial Effect on Growth and Development of Poplar Trees. Appl. Environ. Microbiol.
75: 748-757
[Abstract]
[Full Text]
-
Chaintreuil, C., Rigault, F., Moulin, L., Jaffre, T., Fardoux, J., Giraud, E., Dreyfus, B., Bailly, X.
(2007). Nickel Resistance Determinants in Bradyrhizobium Strains from Nodules of the Endemic New Caledonia Legume Serianthes calycina. Appl. Environ. Microbiol.
73: 8018-8022
[Abstract]
[Full Text]
-
Taghavi, S., Barac, T., Greenberg, B., Borremans, B., Vangronsveld, J., van der Lelie, D.
(2005). Horizontal Gene Transfer to Endogenous Endophytic Bacteria from Poplar Improves Phytoremediation of Toluene. Appl. Environ. Microbiol.
71: 8500-8505
[Abstract]
[Full Text]
Top
Abstract
Text
