Previous Article | Next Article 
Appl Environ Microbiol, April 1998, p. 1210-1219, Vol. 64, No. 4
Research Institute for Plant Protection
(IPO-DLO), 6700 GW Wageningen, The Netherlands
Received 27 February 1997/Accepted 2 January 1998
A set of mercury resistance plasmids was obtained from wheat
rhizosphere soil amended or not amended with mercuric chloride via
exogenous plasmid isolation by using Pseudomonas
fluorescens R2f, Pseudomonas putida UWC1, and
Enterobacter cloacae BE1 as recipient strains. The
isolation frequencies were highest from soil amended with high levels
of mercury, and the isolation frequencies from unamended soil were low.
With P. putida UWC1 as the recipient, the isolation
frequency was significantly enhanced in wheat rhizosphere compared to
bulk soil. Twenty transconjugants were analyzed per recipient strain.
All of the transconjugants contained plasmids which were between 40 and
50 kb long. Eight selected plasmids were distributed among five groups,
as shown by restriction digestion coupled with a similarity matrix
analysis. However, all of the plasmids formed a tight group,
as judged by hybridization with two whole-plasmid probes and
comparisons with other plasmids in dot blot hybridization analyses. The
results of replicon typing and broad-host-range incompatibility
(Inc) group-specific PCR suggested that the plasmid isolates were not
related to any previously described Inc group. Although resistance
to copper, resistance to streptomycin, and/or resistance to
chloramphenicol was found in several plasmids, catabolic sequences were
generally not identified. One plasmid, pEC10, transferred into a
variety of bacteria belonging to the The potential use of genetically
modified bacteria in agriculture has raised questions pertaining to the
spread of introduced recombinant DNA through soil bacterial
communities. Gene transfer in soil via conjugation has received much
attention, and the focus of most studies has been the transfer and fate
of introduced plasmids (6, 22, 27-29, 39). Under favorable
conditions, in specific soil microhabitats, or under selection
conditions, both self-transmissible and mobilizable plasmids present in
introduced hosts can be transferred to introduced recipients, as well
as to a variety of indigenous bacteria (15, 20, 27, 28, 33).
In particular, rhizospheres of crop plants, such as wheat and sugar
beet, provide conditions conducive to conjugal plasmid transfer between
bacterial inhabitants (15, 36). When genetically modified
bacteria are developed as inoculants for the rhizosphere, insertion of
heterologous DNA into non-self-transmissible plasmids or the chromosome
might restrict conjugal transfer of this DNA to members of the
indigenous bacterial community. However, mobilizing or retromobilizing
(33) plasmids present in indigenous soil bacteria could
potentially still effect the transfer of the less mobile heterologous
DNA via chromosome or plasmid mobilization, which may involve
cointegration (9, 19, 31). Such plasmids might thus be
responsible for the escape of heterologous DNA from genetically
modified bacteria introduced into soil.
There is a paucity of knowledge concerning the incidence of plasmids
with mobilizing capacity in soils and rhizospheres, as well as
concerning the effects of soil factors, such as stresses resulting from
pollution or from natural causes (e.g., rhizosphere acidity), on
plasmid prevalence and transfer (e.g., reference 38). Whereas it has been suggested that chemical
stress often does not enhance plasmid incidence in selected soil
bacterial populations (40), pollution in river water or
mines (in particular mercury pollution) has been found to exert a
selective (enhancing) effect (4, 13).
Plasmids of environmental bacteria have classically been obtained by
endogenous isolation procedures (20). Endogenous isolation implies that putative plasmid hosts with the phenotype of interest are
isolated from soil, after which plasmids are extracted from pure
cultures of these strains. On the other hand, pioneering studies
performed with river stone epilithon (9) and later extended
to soil and sediment (32) have shown that plasmids can be
obtained directly from indigenous bacterial communities in new hosts by
exogenous isolation. In this approach, plasmids are captured in
selectable recipient strains by using mating between these strains and
the total bacterial community obtained from an environmental sample.
Following incubation, the mating mixture is plated with selection for
the recipient and an additional marker gene presumedly located on a
plasmid present in the indigenous bacteria (6). The
advantage of the exogenous isolation procedure is that no culturing
step is required in the mating, which thus allows isolation of plasmids
from nonculturable hosts. Furthermore, plasmids are directly selected
for their transfer capacity, in addition to the presence of a specific
selectable marker.
In this study, exogenous plasmid isolation was employed to obtain
transferable plasmids from soil bacteria by using mercury resistance as
the selectable marker. The objective of this work was to gain insight
into the potential present in soil bacterial populations to
(retro)mobilize genes out of introduced bacteria into members of the
soil bacterial community. Since the incidence of plasmids in soil
bacteria is likely influenced by soil ecological factors and selection
pressure, the presence of wheat roots and selection by mercury
(25) were studied as experimental variables.
Strains and plasmids used.
The strains and plasmids used in
this study are listed in Table 1. The new
plasmids exogenously isolated from soil, as summarized in Table
2, are listed in Table
3. All strains were routinely grown in
Luria-Bertani (LB) broth (10 g of tryptone, 5 g of yeast extract,
5 g of NaCl, 1 liter of H2O; pH 7.2) to which
appropriate antibiotics (each at a concentration of 50 µg
ml
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Self-Transmissible Mercury Resistance Plasmids with
Gene-Mobilizing Capacity in Soil Bacterial Populations: Influence
of Wheat Roots and Mercury Addition
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and 
subdivisions of the
class Proteobacteria and mobilized as well as
retromobilized the IncQ plasmid pSUP104. A PCR method for detection of
pEC10-like replicons was used, in conjunction with other methods, to
monitor pEC10-homologous sequences in mercury-polluted and unpolluted
soils. The presence of mercury enhanced the prevalence of pEC10-like
replicons in soil and rhizosphere bacterial populations.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1) were added (Table 1). Strains were maintained in
20% glycerol at 
80°C.
TABLE 1.
Strains and plasmids used and host range of mercury
resistance plasmid pEC10
TABLE 2.
Exogenous isolation of mercury resistance plasmids in
three recipient strains from bulk soil and wheat rhizosphere soil
portions 10 days after different amounts of HgCl2 had
been added
TABLE 3.
Grouping of eight mercury resistance plasmids based on
replicon, broad-host-range Inc group PCR, and
restriction-hybridization typing data
Soil-plant microcosms and sampling. Flevo silt loam (FSL) freshly collected from a field microplot at the Instituut voor Plantenziektekundig Onderzoek, Directie Landbouwkundig Onderzoek (IPO-DLO; Wageningen, The Netherlands), was used in all microcosm experiments. The FSL soil had a low organic matter content (about 2%), and its pH was approximately neutral (22). In order to establish selective pressure for resistance to mercury, different concentrations of mercury (4, 28, and 55 µg of HgCl2 per g of soil) were added to separate soil portions. Control soil portions received equivalent amounts of water. To prepare soil microcosms, 60-g portions of soil with a moisture content of 35% (about 60% of the moisture-holding capacity, corresponding to pF 2) were, after treatment with the different concentrations of mercuric chloride, placed in polypropylene containers (diameter, 62 mm; height, 65 mm). One day after HgCl2 was added, a subset of microcosms was planted with pregerminated seeds of wheat (Triticum aestivum cv. Sicco) (three seeds per microcosm). The microcosms containing wheat were incubated for different periods of time with a cycle consisting of 16 h of light at 20°C and 8 h of darkness at 16°C per day. Soil microcosms not containing wheat seedlings were kept in the dark. The planted microcosms were weighed daily and watered until they were at their original weight when needed in order to keep the soil moisture content stable throughout the experiments.
Following incubation, samples were removed from duplicate or triplicate microcosms at regular times after the start of the experiment (at zero time and after 3 and 8 h and 1, 2, 3, 5, 14, and 28 days). Bulk soil samples were obtained from the unplanted soil microcosms. Rhizosphere samples were obtained by carefully removing plants from planted soil microcosms. Plants were shaken to remove loosely adhering soil particles, and roots were separated from shoots. Soil closely associated with the roots was considered rhizosphere soil. For enumeration by selective plating, 5- to 10-g portions of bulk soil or rhizosphere soil (roots included) were shaken in 250-ml Erlenmeyer flasks containing 95 ml of sterile 0.1% sodium pyrophosphate and 10 g of gravel (diameter, 2 to 4 mm) for 10 min at 280 rpm. Appropriate dilutions of the resulting suspensions were plated onto 10% tryptic soy broth agar (0.1× TSA) (Oxoid) supplemented with 100 µg of cycloheximide per ml to estimate the total culturable bacterial populations and on 0.1× TSA supplemented with 20 µg of HgCl2 per ml and 100 µg of cycloheximide per ml to determine the mercury-resistant bacterial populations (12). The colonies appearing on plates were counted after 2 and 5 days of incubation at 27°C, and the average number of log CFU per gram of dry soil was inferred from the total counts obtained for (at least) duplicate experimental units. For exogenous plasmid isolations, 5-g samples of bulk soil or rhizosphere soil were suspended in 95 ml of sterile 0.85% NaCl. The flasks were shaken on a gyratory shaker at 280 rpm for 30 min, and the bacterial suspensions were used as described below.Exogenous isolation of mercury resistance plasmids from soil and rhizosphere. On three occasions, samples of maize and wheat plants with adhering soil were obtained from agricultural fields around Wageningen, The Netherlands. These samples were immediately taken to the laboratory, where rhizospheres (root parts plus adhering soil) were separated, and rhizosphere soil suspensions were prepared as described above. In addition, suspensions were obtained in the same way from the rhizospheres of young (7-, 10-, or 14-day-old) wheat plants grown in microcosms.
Following shaking of the rhizosphere soil suspensions, large soil particles were allowed to settle by leaving the flasks untouched for 10 min. Thirty milliliters of each supernatant was filtered with filter paper (mittelschnell; Schleicher & Schuell, Dassel, Germany), and the filtrate was passed over a sterile membrane filter (pore size, 0.22 µm) by using a vacuum filtration unit (Millipore Corp., Bedford, Mass.). Washed overnight cultures (1 ml) of one of the recipient strains (Pseudomonas fluorescens R2f Rpr, Pseudomonas fluorescens R2f [chr::Tn5] [used only for exogenous isolation of added plasmid pEC10], Pseudomonas putida UWC1 Rpr, or Enterobacter cloacae BE1 Rpr) were then added to the filters. Control filters contained either only the soil bacterial fractions or one of the recipient strains. The filters with the mating mixtures, as well as the controls, were placed on LB agar plates containing 1.5% agar and incubated overnight at 27°C. Following incubation, the filters were each shaken in 5 ml of 0.85% NaCl to dislodge bacterial cells, and serial 10-fold dilutions were plated onto 0.1× TSA plates supplemented with rifampin (50 µg ml1) and cycloheximide (100 µg
ml1) to enumerate the rifampin-resistant recipient CFU
and onto 0.1× TSA plates supplemented with 20 µg of
HgCl2 per ml, 50 µg of rifampin per ml, and 100 µg of
cycloheximide per ml to select for transconjugants. For the
Pseudomonas fluorescens R2f
(chr::Tn5) recipient strain, King's
medium B (35) agar supplemented with kanamycin (50 µg ml1), streptomycin (50 µg ml1),
HgCl2 (20 µg ml1), and cycloheximide (100 µg ml1) was used. The plates were incubated at
27°C for 2 to 5 days, after which they were used for CFU analyses,
for colony hybridization experiments and PCR analysis, and for
isolation procedures. Mutants resistant to mercury were not obtained
with all recipient strains. Moreover, the indigenous bacterial
populations sampled generally did not give rise to any colonies on the
transconjugant-selective plates.
Plasmid extraction.
Plasmids were extracted by a
modification of the method of Kado and Liu (11), as
developed by Bailey and Lilley (1). Briefly, 1.5 ml of an
overnight culture of each putative plasmid host was centrifuged (1 min,
14,000 rpm in an Eppendorf centrifuge), suspended in 200 µl of a
solution containing 50 mM Tris, 3% sodium dodecyl sulfate, and 120 mM
NaOH (pH 12.6), and kept at 57°C for 70 min. The DNA was extracted
with 200 µl of phenol-chloroform (24). The aqueous phase
was diluted with 200 µl of distilled H2O, and the DNA was
precipitated by adding 50 µl of 3 M sodium acetate (pH 5.2) and 1 ml
of absolute ethanol and incubating the preparation at 20°C. The
pellet containing the plasmid DNA was taken up in 25 µl of distilled
water, and 5 to 8 µl was analyzed by agarose gel electrophoresis by
using 0.7% agarose gels electrophoresed in TBE buffer (24).
Determination of the host ranges of selected plasmids.
To
determine the plasmid host ranges, filter matings were performed with
selected rifampin-, tetracycline-, or erythromycin-resistant recipient
strains in order to include a wide range of members of the 
, ,
and subdivisions of the class Proteobacteria, as well as
gram-positive bacteria (Table 1). Pseudomonas fluorescens (R2f chr::Tn5) containing the
plasmids was used as the donor. Millipore membrane filters onto which
100-µl aliquots of washed cultures of both donor and recipient
strains had been pipetted were placed on LB agar plates and incubated
overnight at 27°C. After incubation, the filters were each shaken in
5 ml of 0.85% NaCl, and appropriate dilutions were spread plated onto
transconjugant-selective plates (LB agar supplemented with the
appropriate antibiotic [rifampin, tetracycline, or erythromycin] at a
concentration of 50 µg ml1 and 20 µg of
HgCl2 ml1) and onto plates for enumeration of
donor CFU (LB agar supplemented with 20 µg of HgCl2 per
ml) and recipient CFU (LB agar supplemented with 50 µg of rifampin
per ml, 50 µg of tetracycline per ml, or 50 µg of erythromycin per
ml). Suspensions of donor and recipient bacteria were also incubated
separately on filters and subsequently plated onto
transconjugant-selective plates to check for the occurrence of
spontaneous antibiotic- or mercury-resistant mutants.
Determination of plasmid phenotypes.
The presence of
plasmid-encoded antibiotic or heavy-metal resistance genes was assessed
by streaking both plasmid-containing and corresponding plasmidless
strains onto 0.1× TSA plates supplemented with various concentrations
of different antibiotics and heavy metals and examining the plates for
the development of single colonies. Presumptively resistant clones,
which were identified by enhanced resistance compared to the
corresponding plasmidless strains, were rechecked by using the same
concentration of inhibitory agent. The following inhibitory agents and
concentration ranges were used: Ag2SO4, 0.025 to 5.0 mM; Na2HAsO4 · 7H2O,
0.025 to 5.0 mM; CdNO3, 0.25 to 2.5 mM;
CoCl2 · 2H2O, 0.25 to 5.0 mM;
K2Cr2O7, 0.025 to 5.0 mM;
CuSO4 · 5H2O, 0.15 to 1.6 mM;
HgCl2, 0.015 to 1 mM; NiSO4 · 6H2O, 0.25 to 2.5 mM; ZnCl2, 2 to 12 mM; sodium ampicillin, 10 to 500 µg ml1; chloramphenicol, 10 to
150 µg ml1; erythromycin, 5 to 100 µg
ml1; gentamicin sulfate, 10 to 200 µg
ml1; kanamycin sulfate, 10 to 200 µg ml1;
lincomycin, 7.5 to 50 µg ml1; nalidixic acid, 10 to 200 µg ml1; streptomycin sulfate, 10 to 200 µg
ml1; tetracycline hydrochloride, 5 to 100 µg
ml1; and trimethoprim, 10 to 100 µg ml1.
Hybridization studies. To screen for homology between the exogenously isolated plasmids and selected DNA sequences, colony hybridization, dot blot hybridization, and Southern blot hybridization were used. To prepare probes for (reverse) hybridization, preparations of selected plasmids were digested with HindIII, and the DNA was labeled with digoxigenin-11-dUTP by using the megaprime labeling system (Boehringer, Mannheim, Germany). The filters used for hybridization (24) contained dot blots of a range of novel and reference plasmids, as well as colony material from a plasmid host (Escherichia coli) with a set of 26 plasmids belonging to various incompatibility (Inc) groups (kindly provided by Helmut Tschäpe, Bundes Gesundheits Amt, Wernigerode, Germany). Other filters contained restriction enzyme-digested DNAs of selected novel plasmid isolates, of replicon typing probes (based on genes involved in plasmid replication of about 20 Inc groups [5]), and of 17 genes selected for their involvement in xenobiotic compound-catabolic processes (kindly donated by Dirk Springael, Vlaams Instituut vor Technologische Onderzoek, Mol, Belgium). These genes were xylDLEGFJIHS, catA, benABC, catBCDE, pcaACBDFE, bphC, todC123BADE, alkST, alkBAC, tcbCD, bphABCD, dmpABCD, nahR/nahG, nahAa, Ab, tbuA1, tbmB, and tbmC.
Hybridizations were carried out under high-stringency conditions by using standard procedures (24). Blots were washed under high-stringency conditions before nonradioactive detection was performed with the chemiluminescent substrate disodium 3-(4-methoxyspiro{1,2-dioxetane-3,21-(51-chloro)tricyclo [3.3.1.13.7]decan}-4-yl)phenyl phosphate(CSPD) combined with the digoxigenin-11-dUTP labeling-enzymatic detection system (Boehringer).Soil DNA extraction.
DNA was extracted directly from FSL
bulk and rhizosphere soil samples by the method of Smalla et al.
(26), as modified by van Elsas and Smalla (35). A
good yield of pure DNA (25 µg g of soil1; average size,
>20 kb) was obtained in all cases in which a subsequent PCR analysis
was performed.
PCR analysis. PCR were performed with pure plasmid or chromosomal DNA, with colonies, and with soil DNA. A suite of primer systems specific for selected plasmid Inc groups (7, 16, 17), for mer genes (3), and for pEC10-like plasmids was used. Unless noted otherwise, the PCR mixtures and temperature cycling regimens used were the same as those described previously (7, 16, 37). PCR products were analyzed by electrophoresis in appropriate 1.4% agarose gels (24). The nature of the PCR products was determined by blotting gels onto nylon filters and hybridizing these filters with the appropriate amplicon-specific probes.
Two primers were used to detect pEC10-like replicons. These primers were primers pEC10-f (5'-GCA CCC TGC CAT TTG CAGG-3') and pEC10-r (5'-GGC TTT TGC CCT TCT GGTG-3'). A touchdown temperature cycling scheme (1 min at 94°C; 1.5 min at [sequentially, two cycles for each temperature] 65, 63, 61, 59, and 57°C; 2 min at 72°C) was used, followed by 40 similar cycles consisting of annealing at 55°C and, finally, extension for 10 min at 72°C. The PCR mixture was the mixture described previously for Taq polymerase (Stoffel fragment) (37). For PCR with colony DNA as well as soil DNA as the target, 0.1% skim milk was added since it was found to enhance specific target amplification. This system yielded a product that was about 250 bp long with pEC10 DNA, pEC10-containing colonies, and pEC10-supplemented soil DNA. The product generated on pEC10 was used as a probe for pEC10 sequences with blots of PCR products, of DNA, and of colonies. The quantitative PCR for pEC10-like sequences in soil DNA, which included blotting and hybridization analysis, was based on a triplicate, threefold-dilution most-probable-number (MPN) scheme as described previously (23). The efficiency of PCR amplification in the soil DNA background was monitored by using a eubacterial 16S rRNA sequence-based PCR system, which yielded a product that was about 450 bp long.Prevalence of pEC10 and homologous plasmids in soil bacterial
populations subjected to mercury stress.
The effect of mercury in
soil on the prevalence of pEC10 type plasmids was assessed in
microcosms containing FSL not treated with HgCl2 (control)
or treated with 28 µg of HgCl2 g1. A subset
of mercury-treated microcosms received about 105 CFU of
Enterobacter cloacae BE1 Rpr(pEC10)
per g of soil, which served as a control for the selective effect of
mercury. Microcosms were incubated at 20°C by using a daily regimen
consisting of 16 h of light and 8 h of darkness. Three days
after incubation, replicate microcosms receiving each treatment were
planted with young wheat seedlings.
1). In addition, dilutions
of soil portions that had received Enterobacter cloacae BE1
Rpr(pEC10) were also plated onto 0.1× TSA supplemented
with 50 µg of rifampin per ml. The colonies appearing on these media
were counted after incubation at 27°C for 2 to 5 days. Selected
plates, as well as randomly picked colonies, were used for colony lifts to obtain filters for colony hybridization and in PCR studies targeting
pEC10-like plasmids. Hybridizations with total pEC10 DNA, as well as
with the pEC10-specific probe generated by PCR, were performed to
assess the occurrence of pEC10-homologous DNA in selected colonies from
soil. PCR with pEC10-specific primers was then performed with a subset
of the probe-positive colonies to confirm the presence of pEC10-like
sequences. The numbers of colonies that reacted with the probe and the
specific PCR system were determined, and the numbers of CFU that
presumably contained pEC10-like plasmids were calculated. In addition,
soil suspensions were used to assess the exogenous isolation
frequencies of mercury resistance plasmids in matings between the
bacterial suspensions and either Enterobacter cloacae BE1
Rpr, Pseudomonas fluorescens R2f
Rpr, or Pseudomonas fluorescens R2f
(chr::Tn5) [for the soil portions supplemented with Enterobacter cloacae BE1
Rpr(pEC10)]. The presence of pEC10-like sequences in
transconjugant colonies was assessed by performing colony hybridization
with the pEC10 probe, as well as by pEC10-specific PCR. Finally, the numbers of pEC10 targets were determined in soil DNA extracts as
described above.
Statistics. All experiments were performed in duplicate or triplicate. Means of bacterial counts and plasmid isolation frequencies, as well as standard errors, were calculated. Analysis of variance was used to assess the significance (at a P of <0.05) of the differences between means due to treatments or experimental factors. The pEC10 target numbers were estimated by MPN PCR in a triplicate, threefold-dilution set-up, with confidence levels of MPN/2.8 and MPN × 2.8 (23).
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Effect of mercury on indigenous soil bacterial populations.
The addition of different concentrations of HgCl2 to FSL
soil dramatically affected the initial total bacterial counts (Fig. 1A). Whereas without added mercury the
total counts initially decreased slightly but then remained roughly
constant at levels of 4 × 107 to 6.3 × 107 CFU g of dry soil1, the counts after
mercuric chloride was added decreased rapidly to 1 × 107 CFU g1 (4 µg of HgCl2 g of
dry soil1) or even to 1 × 105 CFU
g1 (28 or 55 µg of HgCl2 g of dry
soil1). Following these initial decreases, the bacterial
counts increased again. After a few days, they were in all cases back
to approximately the initial levels. For the two highest mercury
concentrations (28 and 55 µg of dry soil1), the total
counts even increased to significantly higher levels (around
1.6 × 108 CFU g of dry soil1) than the
counts in the control.
|
, no HgCl2; 
, 4 µg
of HgCl2 g of soil
, 28 µg of
HgCl2 g of soil
, 55 µg of
HgCl2 g of soil1, and they
remained at this level in the control and low-mercury treatments
throughout the experiment (Fig. 1B). However, in the soil portions that
received the two highest mercury concentrations, the numbers of
mercury-resistant CFU initially decreased, after which the values
increased to levels significantly above those in the no-mercury and
low-mercury treatments. The levels reached were up to 1 order of
magnitude below those of the total counts (i.e., 1 × 107 to 1.6 × 107 CFU g of dry
soil1) after 14 days and remained stable thereafter.
Exogenous isolation of mercury resistance plasmids from bulk and
rhizosphere soil.
When the exogenous plasmid isolation procedure
was used with Pseudomonas fluorescens R2f Rpr,
Pseudomonas putida UWC1 Rpr, or
Enterobacter cloacae BE1 Rpr as the recipient
strain, transconjugants were not obtained on three occasions with
bacterial populations from the rhizospheres of mature maize or wheat
plants growing in the field (data not shown). The total bacterial
counts ranged from 1.5 × 108 to 4 × 108 CFU g of dry soil1, whereas the counts
for the mercury-resistant populations were around 2 × 104 CFU g of dry soil1. The estimated
limits of detection, expressed as exogenous isolation frequencies
(number of transconjugants per recipient), were about 1010 to 1011 in both cases.
Molecular characterization of selected mercury resistance plasmids. Eight plasmids of different sizes (two or three plasmids per recipient strain) were characterized further (Table 3). All of the plasmids transferred mercury resistance to a Pseudomonas fluorescens R2f (chr::Tn5) recipient strain, suggesting that their resistance determinant was readily expressed in Pseudomonas fluorescens.
The restriction patterns of the eight plasmids generated with EcoRV, PstI, and XbaI revealed differences among the plasmids, as well as similarities. Digestion with XbaI resulted in up to 4 fragments, digestion with EcoRV resulted in 5 to 8 fragments, and digestion with PstI resulted in 9 to 13 fragments. A similarity matrix constructed with the Dice coefficient of similarity was based on the PstI digests, which were the most discriminating digests. The corresponding dendrogram (Fig. 2) showed that the plasmids fell into five groups that differed by a genetic distance of more than 20%. The largest group, designated group II, consisted of three plasmids (pEC8, pP4, and pF7), and another group (group III) consisted of two indistinguishable plasmids (pP2 and pF17) (Table 3). Several bands of all plasmids hybridized strongly with pEC10 and pP11 when they were used as probes, and the hybridization patterns of group II plasmids, as well as the hybridization patterns of group III plasmids, were internally consistent, whereas the hybridization patterns of the other groups were different. Hence, all of the plasmids were related to pEC10 at the sequence level, but their restriction and hybridization patterns clearly separated them.
|
P primers and probe
(3). Hence, these plasmids contained sequences of prototypic
mercury resistance transposon Tn501.
Screening for marker genes in selected plasmids.
All eight
plasmids were subjected to a heavy-metal resistance and antibiotic
resistance analysis, and one plasmid from each group (groups I through
V) was also screened for the presence of catabolic genes (Table 3). All
of the plasmids conferred resistance to 0.3 to 0.6 mM Cu to their
hosts, and one plasmid, pP11, also showed As resistance (Table
3). Furthermore, five plasmids (pEC10, pEC8, pP2, pF17, and pP11)
encoded resistance to 25 to 50 µg of streptomycin per ml, whereas
resistance to chloramphenicol (100 to 200 µg ml1) was
found in six plasmids (pEC10, pEC8, pF7, pF17, pP11, and pF10). The
latter resistance was difficult to detect in Pseudomonas putida UWC1 plasmids pP2 and pP4 due to the high intrinsic
resistance of the host (Table 3). Plasmids pEC10 and pEC8 expressed
resistance to chloramphenicol in both Pseudomonas
fluorescens R2f and Enterobacter cloacae BE1. In the
latter strain (but not in Pseudomonas fluorescens), they also expressed streptomycin resistance.
Host ranges of selected plasmids and (retro)mobilization by plasmid
pEC10.
All eight plasmids were transferred between their original
hosts and Pseudomonas fluorescens R2f
(chr::Tn5). Several also were
transferred to two members of the subdivision of the
Proteobacteria, Alcaligenes eutrophus AE815 and
Burkholderia cepacia P2. None of the plasmids could be
transferred to Agrobacterium tumefaciens Gmi9023 (a member
of the subdivision of the Proteobacteria), to
Escherichia coli CV601 or Sm10
(chr::Tn5::luxAB-tet),
or to the gram-positive bacteria Bacillus subtilis SEm-2 and
Paenibacillus azotofixans P3L5. These limited data suggested
that all of the plasmids had a preference for hosts belonging to the
and subdivisions of the Proteobacteria.
and subdivisions of the Proteobacteria (Table 1). Plasmid pEC10 was
transferred to and maintained in all fluorescent pseudomonads tested,
as well as Pseudomonas stutzeri, Enterobacter
cloacae, Alcaligenes eutrophus, and Burkholderia
cepacia. The Acinetobacter, Agrobacterium,
Rhizobium, Bacillus, Paenibacillus,
Escherichia coli, and soil isolate F4 strains tested did not
support transfer or maintenance of pEC10.
In a mating between Enterobacter cloacae BE1(pEC10) and
Pseudomonas fluorescens R2f(pSUP104), direct
transconjugants (Pseudomonas fluorescens cells
containing pEC10 in addition to pSUP104) were found at a
frequency of 1.5 × 102 transconjugants
per recipient, whereas retrotransconjugants (Enterobacter cloacae BE1 with pEC10 and pSUP104) appeared at a frequency of 7 × 107 transconjugants per recipient. In a
subsequent mating between Pseudomonas fluorescens R2f(pEC10,
pSUP104) and Pseudomonas fluorescens R2f
(chr::Tn5), pSUP104 was mobilized
by pEC10 at a frequency of 2.2 × 103
transconjugants per recipient. Both plasmids were found in
several selected transconjugants, suggesting that
transfer of pEC10 and mobilization of pSUP104 took place
at similar rates. These data showed that pEC10 was capable of
mobilizing as well as retromobilizing IncQ plasmid pSUP104. Plasmids
belonging to group III (e.g., pF17) were also capable of mobilizing
pSUP104 (data not shown).
Detection of pEC10-like plasmids. A strategy for molecular detection of pEC10 was based on the observation that the IncQ-oriV-specific PCR system generated an amplification product of about 300 bp with pEC10 as the target (Table 3). This amplicon did not produce a hybridization signal with the IncQ-oriV-specific probe generated by PCR on IncQ plasmid RSF1010. The product was sequenced; alignment of the sequence with the sequences in the EMBL database (both total sequences and bacterial sequences) showed that the only similarities to the database sequences were in short (20-bp or smaller) regions of the amplicon. Primers for selected inner regions of the amplicon were designed, and the PCR system obtained was tested with 37 different plasmids (including the eight new plasmids and three IncQ plasmids, pSKTG, pSUP104, and pIE723), as well as with nine chromosomal DNAs obtained from seven different bacterial species (Escherichia coli, Enterobacter cloacae, Pseudomonas fluorescens, Pseudomonas putida, Acinetobacter calcoaceticus, Bacillus polymyxa, and Mycobacterium chlorophenolicum). The results revealed that neither the chromosomal DNAs of the nine strains nor 30 different plasmid DNAs produced a PCR product. On the other hand, all of the eight novel plasmids except pF7 were PCR positive (Table 3), and the products obtained hybridized with the probe generated by PCR with pEC10. Hence, we considered the PCR primers, as well as the probe generated on plasmid pEC10, specific for pEC10 and "like" replicons in the soil environment.
Effect of mercury addition to soil on the prevalence of mercury
resistance and pEC10-like plasmids.
The effect of mercury as a
selective agent on the occurrence of pEC10-like plasmids in soil and
wheat rhizosphere bacterial populations was assessed in a microcosm
study. At 3, 7, and 15 days after the start of the experiment, the
mercury added had no effect on the total bacterial counts on 0.1× TSA
(Fig. 3A and B). After 7 and 15 days, the
counts on Gould's S1 agar were equivalent to 0.1 to 0.5% of the total
counts on 0.1× TSA, and effects of mercury were also not observed. In
accordance with the experiment shown in Fig. 1, the numbers of CFU on
0.1× TSA amended with mercury increased progressively in
mercury-amended soil, whereas the counts were roughly stable (at the
initial level, around 104 CFU g of dry soil1)
in soil without added mercury. From day 3, the numbers of
mercury-resistant CFU were significantly higher in the mercury-amended
soil than in the unamended soil (Fig. 3A and B). The presence of wheat
roots in all cases enhanced the total and mercury-resistant counts
after 7 and 15 days, albeit not always significantly (Fig. 3A and B). On days 7 and 15 the proportions of mercury-resistant CFU in the total
counts on Gould's S1 agar obtained with unamended soil were 1 to 4%;
however, the proportions in mercury-amended soil samples were 57%
(bulk soil) and 80% (rhizosphere soil).
|
1 at the
beginning of the experiment, decreased to 2.6 × 103
CFU g of dry soil1 shortly after the organism was added
to the mercury-amended soil. There was a small but significant increase
in the number of organisms in the wheat rhizosphere after 15 days (Fig.
3C). Colony filter hybridization and a PCR analysis of randomly
selected colonies indicated that pEC10 was present in all colonies
grown on the selective plates.
The exogenous plasmid isolation frequencies obtained in matings
performed with Enterobacter cloacae BE1 Rpr,
Pseudomonas fluorescens R2f Rpr, and the
bacterial flora from unamended soil were at or below the limit
of detection throughout the experiment. With Pseudomonas fluorescens R2f Rpr, the levels of exogenous
transconjugants increased to detectable values in the rhizosphere of
wheat after 7 and 15 days. In matings performed with the bacterial
flora from mercury-treated soil portions, the frequencies were
initially (at zero time and on day 3) also below the limit of
detection, whereas they had increased dramatically and significantly
after 7 and 15 days (Fig. 3B). The presence of wheat roots did not
affect these frequencies to a great extent after 7 and 15 days (Fig.
3B). A high proportion (52 to 62%) of the transconjugant colonies
obtained contained pEC10-like sequences, as determined by colony PCR.
Plasmid pEC10 was exogenously isolated in Pseudomonas
fluorescens R2f (chr::Tn5) from
soil portions that had received Enterobacter cloacae BE1
Rpr(pEC10) at frequencies that differed significantly, from
1.3 × 109 transconjugants per recipient (day 3, bulk soil) to 4.8 × 106 transconjugants per
recipient (day 15, rhizosphere soil). On day 15, the frequency of
isolation was significantly greater with rhizosphere bacterial
populations than with populations from corresponding bulk soil (Fig.
3C). The majority (82 to 85%) of the putative exogenous
transconjugants from day-7 and -15 samples carried pEC10-like plasmids.
Furthermore, a significant (17-fold) increase in the number of colonies
with homology to pEC10 in mercury-amended bulk soil samples was
observed; the level increased from 2.3 × 103 CFU g of
dry soil1 initially to 4.16 × 104 CFU g
of dry soil1 after 15 days (day-3 and -7 data could not
be assessed). Moreover, on days 7 and 15, with bulk and rhizosphere
samples the number of colonies containing pEC10-homologous sequences on
mercury-containing Gould's S1 agar was approximately 104
CFU g of dry soil1. In unamended soil portions, such
increases were not noticeable, and the number of pEC10-homologous
colonies on both 0.1× TSA and Gould's S1 agar remained below the
limit of detection (3 × 103 to 5 × 103 CFU g of dry soil1).
The prevalence of pEC10-like sequences in bulk soil samples increased
as a result of the addition of mercury (Fig. 3B). Whereas the initial
pEC10 copy number was estimated to be 2.45 × 103
copies g of dry soil1, the copy numbers increased to
3.98 × 104 and 4.2 × 104 copies g
of dry soil1 in mercury-amended soil on days 7 and 15, respectively. On the other hand, the levels of pEC10-like sequences in
the unamended soil portions remained stable at about the initial level
(Fig. 3A). The soil portions containing added Enterobacter
cloacae BE1 Rpr(pEC10) at similar low levels also
showed positive amplification and were used to monitor the PCR
amplification efficiency.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
In this study, we first assessed the responses of culturable
bacterial populations in soil to different levels of mercury, and
subsequently we assessed the possible involvement of plasmids in
conferring mercury resistance, as well as gene-mobilizing capacity, to
selected bacterial populations. We chose this combination since mercury
resistance is a ubiquitous marker in bacterial populations in the
environment which is often associated with mobile genetic elements
(1a, 10, 12, 21, 30). Both the total culturable bacterial
populations (Fig. 1) and the mercury-resistant culturable bacterial
populations (Fig. 1 and 3) reacted quickly to high levels of added
mercury, whereas a strong response was not noticeable at low mercury
levels (Fig. 1). The highest mercury concentration used in this study
(55 µg g of soil1) was still about 10-fold below the
concentration which has been found to inhibit soil microbial activity
(34), but it was in the range which can elicit a response by
bacteria in soil (12). The presence of mercury in soil
imposes in situ stress on cells that are affected by sufficiently high
local levels of mercury. In general, bacterial populations in soil can
respond to mercury stress by a variety of mechanisms, including (i)
inhibition of cellular metabolism, resulting in growth inhibition or
death; (ii) induction of existing mercury resistance operons, possibly followed by outgrowth of resistant forms; and (iii) acquisition of
plasmids with functional mercury resistance genes via conjugation. The
population response can thus be observed as an increase in the
frequency with which mercury-resistant bacteria are isolated (18). Cell death, induction of mer operons, gene
transfer of mer determinants, and clonal selection of
mer operon-containing organisms very likely all played a
role in the response measured by CFU counting in our study.
The exogenous plasmid isolations were performed primarily to assess the prevalence and nature of transferable plasmids with mercury resistance determinants in soil bacterial populations. Exogenous isolation in a preselected host is a good method to obtain and assess plasmids with self-transfer and possibly gene-mobilizing capacity. It abolishes the need to culture the plasmid hosts and therefore even permits isolation of plasmids from nonculturable bacteria (32). However, a lack of knowledge about the natural plasmid host obviously leaves doubts concerning the role of the isolated plasmid in its natural host.
The preferential isolation of mercury resistance plasmids from young wheat roots suggests that the dynamics of the plasmids and their hosts are related to temporal and spatial aspects of plant growth, inasmuch as these may reach a peak in abundance and/or activity at a specific time and in specific sites during root development. Although the original plasmid hosts are not known, they might be found in copiotrophic early-root-colonizing groups, such as the fluorescent pseudomonads or enteric bacteria. This hypothesis is also consistent with the host range data obtained with pEC10. Lilley et al. (14) also found that mercury resistance plasmids of similar types were prevalent only at specific times of plant development in three consecutive years in the phyllospheres and rhizospheres of sugar beet; this fluctuating prevalence very likely reflected the fluctuating abundance of the presumed plasmid hosts.
In the soil microcosms, the exogenous isolation frequencies of the mercury resistance plasmids increased as higher concentrations of mercury were added to soil. The wheat rhizosphere enhanced these frequencies only for one recipient strain. Top et al. (32) suggested that exogenous isolation frequencies could be used in a quantitative way to estimate the abundance of the plasmid types isolated. In our view, the enhanced isolation frequencies that resulted from mercury addition indicated that the mercury pressure increased either the prevalence of mercury resistance determinants located on mobile genetic elements or the activity of the determinants via induction of mercury resistance genes. Selection by mercury may also have resulted in clonal selection of cells containing plasmids with mercury resistance determinants, as well as plasmid transfer to new hosts at a stage when the densities of potential plasmid donor bacteria were increased.
Despite the proposed grouping of the eight selected plasmids in five
groups based on restriction patterns and plasmid sizes (Fig. 2 and
Table 3), all of these plasmids are probably related. This was evident
from the clustering of the plasmids in the dot blot hybridization
assay, from the cross-hybridization results, from the similar (albeit
not identical) phenotypes of the plasmids, and from the reactions
of most of the plasmids with the pEC10-specific detection systems, as
well as the merRTP detection system. The (weak)
reactions with the PCR system specific for the IncA/C rep gene and the absence of hybridization under high-stringency conditions suggested that these plasmids were related at lower levels to a
rep gene of the broad-host-range IncA/C group, a synonym of the IncP3 group of Pseudomonas plasmids (2, 16,
17). Interestingly, the 88-kb prototype IncP3 plasmid, pBS73 (of
Pseudomonas aeruginosa), also carries
resistance to mercury, resistance to streptomycin, and resistance to
chloramphenicol. It is possible that the bacterial populations from
which the plasmids were obtained naturally contain a myriad of closely
related plasmids, from which a closely knit yet diverse set of plasmids
was obtained in the three gram-negative hosts. It is unclear whether
each representative of this plasmid pool is restricted to a different
specific host (which would result in mainly vertical inheritance) or
whether ample gene transfer and recombination among the different
(gram-negative) hosts can take place. The primary isolation of the
group II plasmids pEC8, pP4, and pF7 in three different gram-negative
hosts was interesting and may support the latter possibility. Moreover,
as the group II plasmids, as well as pEC10 and pF10, reacted with a
0.7-kb tniA probe generated by PCR from novel plasmid pMOL96
(41), a transposon with the Tn5090
(Tn402) transposase of IncP plasmid R751 may be present.
These findings support a myriad of different internal relationships
among our plasmids, as well as the possible presence of a
tniA gene.
The selected plasmids obtained from the rhizosphere of wheat could be
transferred to a range of members of the and subdivisions of
the Proteobacteria at moderate to high frequencies. As
exemplified by pEC10, they also mobilized and retromobilized IncQ
plasmid pSUP104. Hence, these plasmids form a pool of elements which
may play a role in conjugal gene transfer events in the initial stages of plant root development (i.e., at times when their hosts are prevalent). Given the presumably low levels of antibiotics
(streptomycin, chloramphenicol) and heavy metals (Hg, Cu) in the FSL
soil used, it is unclear from the plasmid phenotypes and genotypes what
their actual role in the ecophysiology of their hosts is, other than conferring gene-mobilizing capacity. It would be interesting to assess
whether these plasmids enhance their mobilizing activity and extend it
to, for instance, the bacterial chromosome under stress conditions in
soil and rhizospheres.
From the final experiment in mercury-treated soil it became clear that
pEC10-like plasmids are indeed selected for or "activated" by
mercury stress. The following three lines of evidence supported this
contention: (i) the number of colonies reacting with the pEC10-specific
probe, as well as the PCR system, increased; (ii) the exogenous plasmid
isolation frequencies increased, and most of the plasmids isolated were
pEC10-like; and (iii) direct PCR performed with soil-extracted DNA
revealed that there was enhanced prevalence of pEC10-like plasmids
under the influence of mercury. The simplest explanation for these
three phenomena is that there was an increase in the abundance of
bacteria carrying pEC10-like plasmids, although an increase in the
plasmid copy number per cell, as well as progressive induction of
repressed mercury resistance genes, may also have played a role, either
by affecting the numbers of targets measured by direct soil DNA
extraction and PCR or by affecting the numbers of probe-positive
colonies and the exogenous isolation frequencies. The similar numbers
of probe- and PCR-positive colonies (around 104 g of dry
soil1) found on 0.1× TSA and Gould's S1 medium might
indicate that fluorescent pseudomonads are prime carriers of these
plasmids in bulk and wheat rhizosphere soils. If this is true, it might also explain the enhancement of the isolation frequency in the rhizosphere, as fluorescent pseudomonads are known to be favored by
young rhizospheres. On the other hand, the introduced pEC10-carrying strain Enterobacter cloacae BE1 did not survive in high
numbers in the mercury-treated soil, and its abundance was enhanced
only to a small extent in the wheat rhizosphere. These observations suggested that Enterobacter cloacae BE1(pEC10) did not have
a strong selective advantage over other competing soil microorganisms in the mercury-stressed soil system.
We suggest that the gene-mobilizing capacity of soil bacterial populations increases as mercury stress is applied, since the prevalence (dominance) of pEC10-like mercury resistance plasmids with gene-mobilizing capacity is enhanced. This observation has an important bearing on the potential for gene spread via conjugation in soil microbial communities under stress conditions. In contrast, Wickham and Atlas (40), in a limited survey, found that mercury selective pressure did not enhance the incidence of plasmids in selected culturable soil bacterial populations. However, the data of these authors were based solely on plasmid incidence as judged by plasmid extraction from soil isolates, which represents a very different level of resolution. In our view, this does not imply that the incidence of transfer-proficient mercury resistance plasmids is unaffected in total or specific soil bacterial fractions. As exemplified by the mercury resistance determinants present on the pEC10-like plasmids, the self-transmissible plasmids could well provide a means of genetic adaptation to changing environmental conditions to their hosts.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by grant BIO2-CT92-0491 from the EU-BIOTECH program.
We are grateful to all partners of the EU-BIOTECH consortium for their help, particularly for sharing dot blot hybridization results obtained with the pEC10-like plasmids. In particular, Helmut Tschäpe, Dirk Springael, and Annick Wilmotte are acknowledged for providing Inc determinants and catabolic genes for the hybridization studies and for sharing results obtained with the tniA probe. Andrew Lilley is thanked for his assistance with the dot blot analysis. Sander Worst, Alexandre Rosado, Gert-Jan ten Thij, and Ludwina Lankwarden are acknowledged for their help with some of the experiments.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: IPO-DLO, P.O. Box 9060, 7600GW Wageningen, The Netherlands. Phone: 31.317.476210. Fax: 31.317.410113. E-mail: j.d.vanelsas{at}ipo.dlo.nl.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. | Bailey, M. J., and A. K. Lilley. 1996. Personal communication. |
| 1a. | Belliveau, B., and J. T. Trevors. 1989. Mercury resistance and detoxification in bacteria. Appl. Organometall. Chem. 3:283-294. |
| 2. | Boronin, A. M. 1992. Diversity and relationships of Pseudomonas plasmids, p. 329-340. In E. Galli, S. Silver, and B. Witholt (ed.), Pseudomonas: molecular biology and biotechnology. American Society for Microbiology, Washington, D.C. |
| 3. |
Bruce, K. D.,
W. D. Hiorns,
J. L. Hobman,
A. M. Osborn,
P. Strike, and D. A. Ritchie.
1992.
Amplification of DNA from native populations of soil bacteria by using the polymerase chain reaction.
Appl. Environ. Microbiol.
58:3413-3416 |
| 4. |
Burton, N. F.,
M. J. Day, and A. T. Bull.
1982.
Distribution of bacterial plasmids in clean and polluted sites in a South Wales river.
Appl. Environ. Microbiol.
44:1026-1029 |
| 5. |
Couturier, M.,
F. Bex,
P. L. Bergquist, and W. K. Maas.
1988.
Identification and classification of bacterial plasmids.
Microbiol. Rev.
52:375-395 |
| 6. | Fry, J. C., and M. J. Day. 1990. Plasmid transfer in the epilithon, p. 55-80. In J. C. Fry, and M. J. Day (ed.), Bacterial genetics in natural environments. Chapman and Hall, London, United Kingdom. |
| 7. | Götz, A., R. Pukall, E. Smit, E. Tietze, R. Prager, H. Tschäpe, J. D. van Elsas, and K. Smalla. 1996. Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Appl. Environ. Microbiol. 62:2621-2628[Abstract]. |
| 8. |
Gould, W. D.,
C. Hagedorn,
T. R. Bardinelli, and R. M. Zablotowicz.
1985.
New selective media for enumeration and recovery of fluorescent pseudomonads from various habitats.
Appl. Environ. Microbiol.
49:28-32 |
| 9. |
Hill, K. E.,
A. J. Weightman, and J. C. Fry.
1992.
Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.
Appl. Environ. Microbiol.
58:1292-1300 |
| 10. | Jobling, M. G., S. E. Peters, and D. A. Ritchie. 1988. Plasmid-borne mercury resistance in aquatic bacteria. FEMS Microbiol. Lett. 49:31-37. |
| 11. |
Kado, C. I., and S. T. Liu.
1981.
Rapid procedure for detection and isolation of large and small plasmids.
J. Bacteriol.
145:1365-1373 |
| 12. | Kelly, W. J., and D. C. Reanney. 1984. Mercury resistance among soil bacteria: ecology and transferability of gene encoding resistance. Soil. Biol. Biochem. 16:1-8. |
| 13. | Khesin, R., and E. V. Karasyova. 1984. Mercury-resistance plasmids in bacteria from a mercury and antimony deposit area. Mol. Gen. Genet. 197:280-285[Medline]. |
| 13a. | Lilley, A. K. 1997. Unpublished results. |
| 14. | Lilley, A. K., M. J. Bailey, M. J. Day, and J. C. Fry. 1996. Diversity of mercury resistance plasmids obtained by exogenous isolation from the bacteria of sugar beet in three successive years. FEMS Microbiol. Ecol. 20:211-227. |
| 15. | Lilley, A. K., J. C. Fry, M. J. Day, and M. J. Bailey. 1994. In situ transfer of an exogenously isolated plasmid between Pseudomonas spp. in sugar beet rhizosphere. Microbiology 140:27-33. |
| 16. | Llanes, C., P. Gabant, M. Couturier, L. Bayer, and P. Plesiat. 1996. Molecular analysis of the replication elements of the broad-host-range repA/C replicon. Plasmid 36:26-35[Medline]. |
| 17. |
Llanes, C.,
P. Gabant,
M. Couturier, and Y. Michel-Briand.
1994.
Cloning and characterization of the IncA/C plasmid RA1 replicon.
J. Bacteriol.
176:3403-3407 |
| 18. |
Osborn, A. M.,
K. D. Bruce,
P. Strike, and D. A. Ritchie.
1993.
Polymerase chain reaction-restriction fragment length polymorphism analysis shows divergence among mer determinants from gram-negative soil bacteria indistinguishable by DNA-DNA hybridization.
Appl. Environ. Microbiol.
59:4024-4030 |
| 19. | Powell, B. J., K. J. Purdy, I. P. Thompson, and M. J. Bailey. 1993. Demonstration of tra+ plasmid activity in bacteria indigenous to the phyllosphere of sugarbeet; gene transfer to a recombinant pseudomonad. FEMS Microbiol. Ecol. 12:195-206. |
| 20. | Pukall, R., H. Tschäpe, and K. Smalla. 1996. Monitoring the spread of broad host and narrow host range plasmids in soil microcosms. FEMS Microbiol. Ecol. 20:53-66. |
| 21. |
Radford, A. J.,
J. Oliver,
W. J. Kelly, and D. C. Reanney.
1981.
Translocatable resistance to mercuric and phenylmercuric ions in soil bacteria.
J. Bacteriol.
147:1110-1112 |
| 22. | Richaume, A., E. Smit, G. Fauri, and J. D. van Elsas. 1992. Influence of soil type on the transfer of RP4p from Pseudomonas fluorescens to indigenous bacteria. FEMS Microbiol. Ecol. 101:263-292. |
| 23. | Rosado, A., L. Seldin, A. C. Wolters, and J. D. van Elsas. 1996. Quantitative 16S rDNA-targeted polymerase chain reaction and oligonucleotide hybridization for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere. FEMS Microbiol. Ecol. 19:153-164. |
| 24. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. . Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 25. | Silver, S., and T. K. Misra. 1988. Plasmid-mediated heavy metal resistances. Annu. Rev. Microbiol. 42:717-743[Medline]. |
| 26. | Smalla, K., N. Cresswell, L. C. Mendonca-Hagler, A. Wolters, and J. D. van Elsas. 1993. Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification. J. Appl. Bacteriol. 74:78-85. |
| 27. |
Smit, E.,
D. Venne, and J. D. van Elsas.
1993.
Mobilization of a recombinant IncQ plasmid between bacteria on agar and in soil via cotransfer or retrotransfer.
Appl. Environ. Microbiol.
59:2257-2263 |
| 28. |
Smit, E.,
J. D. van Elsas,
J. A. van Veen, and W. M. de Vos.
1991.
Detection of plasmid transfer from Pseudomonas fluorescens to indigenous bacteria in soil using phage phiR2f for donor counterselection.
Appl. Environ. Microbiol.
57:3482-3488 |
| 29. | Stotzky, G. 1989. Gene transfer among bacteria in soil, p. 165-222. In S. B. Levy, and R. V. Miller (ed.), Gene transfer in the environment. McGraw-Hill, New York, N.Y. |
| 30. |
Tanaka, M.,
T. Yamamoto, and T. Sawai.
1983.
Evolution of complex resistance transposons from an ancestral mercury transposon.
J. Bacteriol.
153:1432-1438 |
| 31. | Thomas, C. M. 1989. . Promiscuous plasmids of gram-negative bacteria. Academic Press, London, United Kingdom. |
| 32. |
Top, E.,
I. de Smet,
W. Verstraete,
R. Dijkmans, and M. Mergeay.
1994.
Exogenous isolation of mobilizing plasmids from polluted soils and sludges.
Appl. Environ. Microbiol.
60:831-839 |
| 33. | Top, E. M., H. de Rore, J.-M. Collard, V. Gellens, G. Slobodkina, W. Verstraete, and M. Mergeay. 1995. Retromobilization of heavy metal resistance genes in unpolluted and heavy metal polluted soil. FEMS Microbiol. Ecol. 18:191-203. |
| 34. | Trevors, J. T. 1996. Sterilization and inhibition of microbial activity in soil. J. Microbiol. Methods 26:53-59. |
| 35. | van Elsas, J. D., and K. Smalla. 1995. Extraction of DNA from soil, p. 1-11. In A. D. L. Akkermans, J. D. van Elsas, and F. J. de Bruijn (ed.), Molecular microbial ecology manual, 1.3.3. Kluwer Academic Publishers, Dordrecht, The Netherlands. |
| 36. | van Elsas, J. D., J. T. Trevors, and M.-E. Starodub. 1988. Bacterial conjugation in the rhizosphere of wheat. FEMS Microbiol. Ecol. 53:299-306. |
| 37. | van Elsas, J. D., and A. C. Wolters. 1995. Polymerase chain reaction (PCR) analysis of soil microbial DNA, p. 1-10. In A. D. L. Akkermans, J. D. van Elsas, and F. J. de Bruijn (ed.), Molecular microbial ecology manual, 2.7.2. Kluwer Academic Publishers, Dordrecht, The Netherlands. |
| 38. | Verhagen, C., E. Smit, D. B. Janssen, and J. D. van Elsas. 1995. Bacterial dichloropropene degradation in soil; screening of soils and involvement of plasmids carrying a dhlA-like gene. Soil Biol. Biochem. 27:1547-1557. |
| 39. | Wellington, E. M. H., and J. D. van Elsas. 1992. . Genetic interaction among microorganisms in the natural environment. Pergamon Press, London, United Kingdom. |
| 40. |
Wickham, G. S., and R. M. Atlas.
1988.
Plasmid frequency fluctuations in bacterial populations from chemically stressed soil communities.
Appl. Environ. Microbiol.
54:2192-2196 |
| 41. | Wilmotte, A. 1997. Unpublished results. |
| 42. | Wilmotte, A., A. Hennen, D. van der Lelie, and M. Mergeay. 1996. The broad host range plasmid pMOL96 contains a putative transposon similar to Tn402 (Tn5090) and Tn5053, p. 30. In A. Karagouni, and D. Koraki (ed.), Abstracts of the BAGECO-5 Conference, Nafplion, Greece. University of Athens Press, Athens, Greece. |
Appl Environ Microbiol, April 1998, p. 1210-1219, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Slater, F. R., Bruce, K. D., Ellis, R. J., Lilley, A. K., Turner, S. L.
(2008). Heterogeneous Selection in a Spatially Structured Environment Affects Fitness Tradeoffs of Plasmid Carriage in Pseudomonads. Appl. Environ. Microbiol.
74: 3189-3197
[Abstract] [Full Text] -
El Yacoubi, B., Brunings, A. M., Yuan, Q., Shankar, S., Gabriel, D. W.
(2007). In Planta Horizontal Transfer of a Major Pathogenicity Effector Gene. Appl. Environ. Microbiol.
73: 1612-1621
[Abstract] [Full Text] -
Coombs, J. M., Barkay, T.
(2004). Molecular Evidence for the Evolution of Metal Homeostasis Genes by Lateral Gene Transfer in Bacteria from the Deep Terrestrial Subsurface. Appl. Environ. Microbiol.
70: 1698-1707
[Abstract] [Full Text] -
Schneiker, S., Keller, M., Droge, M., Lanka, E., Puhler, A., Selbitschka, W.
(2001). The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa. Nucleic Acids Res
29: 5169-5181
[Abstract] [Full Text] -
Cook, M. A., Osborn, A. M., Bettandorff, J., Sobecky, P. A.
(2001). Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities. Microbiology
147: 2089-2101
[Abstract] [Full Text] -
Hansen, L. H., Ferrari, B., Sørensen, A. H., Veal, D., Sørensen, S. J.
(2001). Detection of Oxytetracycline Production by Streptomyces rimosus in Soil Microcosms by Combining Whole-Cell Biosensors and Flow Cytometry. Appl. Environ. Microbiol.
67: 239-244
[Abstract] [Full Text] -
Smalla, K., Heuer, H., Götz, A., Niemeyer, D., Krögerrecklenfort, E., Tietze, E.
(2000). Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids. Appl. Environ. Microbiol.
66: 4854-4862
[Abstract] [Full Text] -
Hohnstock, A. M., Stuart-Keil, K. G., Kull, E. E., Madsen, E. L.
(2000). Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids. Appl. Environ. Microbiol.
66: 3088-3092
[Abstract] [Full Text] -
Stuart-Keil, K. G., Hohnstock, A. M., Drees, K. P., Herrick, J. B., Madsen, E. L.
(1998). Plasmids Responsible for Horizontal Transfer of Naphthalene Catabolism Genes between Bacteria at a Coal Tar-Contaminated Site Are Homologous to pDTG1 from Pseudomonas putida NCIB 9816-4. Appl. Environ. Microbiol.
64: 3633-3640
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»