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Applied and Environmental Microbiology, July 2001, p. 3180-3187, Vol. 67, No. 7
Department of Microbiology, University of
Massachusetts at Amherst, Amherst, Massachusetts 01003
Received 2 February 2001/Accepted 27 April 2001
Members of the genus Geobacter are the dominant
metal-reducing microorganisms in a variety of anaerobic subsurface
environments and have been shown to be involved in the bioremediation
of both organic and metal contaminants. To facilitate the study of the physiology of these organisms, a genetic system was developed for
Geobacter sulfurreducens. The antibiotic sensitivity of
this organism was characterized, and optimal conditions for plating it
at high efficiency were established. A protocol for the introduction of
foreign DNA into G. sulfurreducens by electroporation
was also developed. Two classes of broad-host-range vectors, IncQ and
pBBR1, were found to be capable of replication in G.
sulfurreducens. In particular, the IncQ plasmid pCD342 was
found to be a suitable expression vector for this organism. When the
information and novel methods described above were utilized, the
nifD gene of G. sulfurreducens was
disrupted by the single-step gene replacement method. Insertional
mutagenesis of this key gene in the nitrogen fixation pathway impaired
the ability of G. sulfurreducens to grow in medium
lacking a source of fixed nitrogen. Expression of the
nifD gene in trans complemented this
phenotype. This paper constitutes the first report of genetic
manipulation of a member of the Geobacter genus.
Dissimilatory metal-reducing
microorganisms play an important role in the natural cycling of organic
matter and minerals in aquatic sediments, submerged soils, and
subsurface environments and can be important agents for the
bioremediation of both organic and metal contamination
(13-15). Molecular analyses (16S ribosomal DNA) have
revealed that dissimilatory metal-reducing microorganisms in the
genus Geobacter are prominent members of the microbial community in a diversity of environments in which dissimilatory metal
reduction is either naturally occurring or artificially stimulated
(22, 28). Geobacter species are obligate
anaerobes belonging to the delta subdivision of the
Proteobacteria. These organisms have the ability to
completely oxidize organic compounds to carbon dioxide with either
humic substances or Fe(III) as the sole electron acceptor
(15). Other metals which can serve as electron acceptors
for Geobacter species include Mn(IV), U(IV), Co(III), and
Tc(VII). Several Geobacter species can also reduce nitrate
and fumarate. The organic compounds oxidized by Geobacter species invariably include acetate and other short-chain fatty acids.
In addition, some Geobacter species are capable of
completely oxidizing monoaromatic compounds such as benzoate, phenol,
p-cresol, and toluene to carbon dioxide with Fe(III) as the
electron acceptor.
Little is known about the biochemical pathways that couple the
oxidation of organic compounds to the reduction of metals in Geobacter species or about the regulation of these
processes. Because many electron carriers will nonspecifically reduce
metals and humic substances in vitro, it has been difficult to use
biochemical studies to determine which of the numerous redox active
proteins present in Geobacter species are actually involved
in the reduction of metals and humic substances in vivo. It may
therefore be easier to deduce the physiological roles of redox active
proteins and enzyme complexes via a genetic approach Here we report the development of a genetic system for Geobacter
sulfurreducens. G. sulfurreducens, which was isolated from hydrocarbon-contaminated soil (7), has all of the
important metabolic features of Geobacter species, including
the ability to oxidize monoaromatic compounds (15).
Furthermore, G. sulfurreducens also has the capacity to grow
with fumarate serving as the sole electron acceptor, a property which
is essential for the generation of mutants that are defective in the
transfer of electrons to metals and humic substances.
Preliminary studies have suggested that G. sulfurreducens
might have genes for nitrogen fixation (4). The ability to
fix nitrogen may be required for Geobacter to compete
successfully in petroleum-contaminated subsurface environments which
are carbon rich but contain little fixed nitrogen (4).
Methods for genetically manipulating G. sulfurreducens were
developed as part of a study assessing the capacity of this organism to
fix nitrogen. In this study, the targeted disruption of a G. sulfurreducens homolog of the nifD gene, a gene
required for nitrogen fixation by other microorganisms
(9), was found to eliminate the ability of G. sulfurreducens to grow in a medium devoid of fixed nitrogen. The ability of G. sulfurreducens to grow in this medium was
restored when a functional copy of the gene was reintroduced in
trans. These results indicate that G. sulfurreducens fixes nitrogen in a manner similar to that of other
nitrogen-fixing microorganisms. The genetic techniques described herein
should be applicable to the study of other aspects of G. sulfurreducens physiology and should make it possible to take full
advantage of the information present in the forthcoming sequence of the
genome of this environmentally significant organism.
Bacterial strains and plasmids.
The plasmids and bacterial
strains, G. sulfurreducens (ATCC 51573) and
Escherichia coli, that were used in this study are listed
and described in Table 1.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3180-3187.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Development of a Genetic System for
Geobacter sulfurreducens
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
gene disruption
followed by phenotypic analysis. Until now, the lack of a genetic
system for Geobacter species has prevented the application
of this type of approach to the study of the physiology of these organisms.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
TABLE 1.
Strains and plasmids
DNA manipulations and plasmid construction. Total G. sulfurreducens genomic DNA was prepared using the Genome DNA Kit (Bio 101, Inc., Carlsbad, Calif.). Primers for amplification of DNA fragments from the G. sulfurreducens chromosome were designed using sequence data obtained from The Institute for Genomic Research website at http://www.tigr.org. PCR amplification was performed using AmpliTaq DNA polymerase (Roche Molecular Systems, Branchburg, N.J.) according to the manufacturer's instructions. Plasmid DNA was purified from E. coli and G. sulfurreducens strains using the Qiagen midi- or miniplasmid purification kits (Qiagen Inc., Valencia, Calif.). DNA samples for electrotransformation were subjected to a second ethanol precipitation and 70% ethanol wash followed by resuspension in 0.5× TE buffer (0.5 mM Tris HCl [pH 8.0], 0.5 mM EDTA) in order to reduce their salt concentration and prevent arcing.
The suicide vector, pBRnif::kan, was constructed for the targeted disruption of the G. sulfurreducens nifD gene. A 1.1-kb fragment consisting of the first ~0.9 kb of the nifD coding region and ~0.2 kb of upstream sequence was amplified from the G. sulfurreducens chromosome using the primers prDL1 (CCCCGCTGGAGATGGAAGAGC) and prDL2 (GGCGGATCCAGCCAGGGGATGCC). In primer sequences, restriction sites are italicized and annealing nucleotides are indicated in boldface. This fragment was digested with EcoRI (cleaves 18 bp downstream of prDL1) and BamHI (cleaves prDL2) and was inserted into the corresponding sites of pBR322 (6), resulting in the intermediate construct pBRnif. A kanamycin resistance cassette was amplified from pBBR1MCS-2 (12) with the primers prDL3 (CCCGGTAACCGGATGAATGTCAGC) and prDL4 (CCCGATATCGCGGTGGAATCG). The final suicide vector, pBRnif::kan (Fig. 2B), was constructed by inserting this cassette into a unique EcoRV site within pBRnif (at position 373 of the nifD coding sequence). The nifD expression vector pCDSnifD was constructed in order to complement the nifD1::kan phenotype. The nifD coding sequence was amplified from G. sulfurreducens genomic DNA with the primers prDL5 (CCCGGTACCTGACAGGAGAATAC) and prDL6 (CCCAAGCTTAAAAGCGGACTCCG) and was inserted into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). It was subsequently excised from this plasmid with EcoRI and HindIII and was inserted into the corresponding sites of the IncQ expression vector pCD342 (10). Because the resulting vector pCDnifD conferred resistance to kanamycin, it was not suitable for carrying out complementation studies in our nifD1::kan mutants. Therefore, the kanamycin resistance cassette of pCDnifD was excised with BglII and BstBI and replaced with a streptomycin resistance cassette that had been amplified from pJRD215 (8) with the primers prDL7 (CCCAGATCTTTCTCATGTTTGACAGC) and prDL8 (CCCTTCGAATGAGATTGATGTGTTCC).Culturing conditions and growth media. The two E. coli strains listed in Table 1 were propagated according to established methods (26).
G. sulfurreducens strains were cultured at 30°C under strict anaerobic conditions as previously described (19). Plating and incubations on solid media were performed inside an anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, Mich.) containing a 7% H2-10%CO2-83% N2 atmosphere heated to 30°C. G. sulfurreducens strains were propagated in two types of media (see compositions below): NBAF supplemented with 0.1% yeast extract and 1 mM cysteine (NBAFYE) and FWAFC. NBAFYE was used for general propagation and plating and for the preparation of electrocompetent cells, whereas FWAFC was used for the analysis of nitrogen fixation phenotypes. Antibiotics were added to cultures or plates as needed. During extended incubations of liquid cultures, fresh antibiotics were added every 48 h. All media were dispensed into anaerobic pressure tubes or bottles with butyl rubber stoppers and, unless otherwise indicated, bubbled with an 80% N2-20% CO2 gas mixture to remove dissolved oxygen and obtain a final pH of ~6.7. NBAF, a modified form of the medium described by Lovley et al. (16), contains 15 mM acetate as the electron donor and 40 mM fumarate as the electron acceptor. Its composition per liter of deionized water is 0.42 g of KH2PO4, 0.22 g of K2HPO4, 0.2 g of NH4Cl, 0.38 g of KCl, 0.36 g of NaCl, 0.04 g of CaCl2 · 2H2O, 0.1 g of MgSO4 · 7H2O, 1.8 g of NaHCO3, 0.5 g of Na2CO3, 2.04 g of NaC2H3O2 · 3H2O, 6.4 g of Na2C4H4O4, 0.5 ml of 0.1% resazurin, 1.0 ml of 100 mM Na2SeO4, 10.0 ml of a vitamin solution (17), and 10.0 ml of NB trace mineral solution. The composition of the NB trace mineral solution per liter of deionized water is 2.14 g of nitriloacetic acid, 0.1 g of MnCl2 · 4H2O, 0.3 g of FeSO4 · 7H2O, 0.17 g of CoCl2 · 6H2O, 0.2 g of ZnSO4 · 7H2O, 0.3 g of CuCl2 · 2H2O, 0.005 g of AlK(SO4)2 · 12H2O, 0.005 g of H3BO3, 0.09 g of Na2MoO4, 0.11 g of NiSO4 · 6H2O, and 0.2 g of Na2WO4 · 2H2O. Liquid NBAFYE medium was prepared by adding appropriate volumes of sterile anoxic stock solutions of yeast extract and cysteine to NBAF medium to achieve final concentrations of 0.1% (wt/vol) and 1 mM, respectively. If NBAFYE medium was to be used for manipulations within the anaerobic chamber, the final cysteine concentration was increased to 5 mM. Solid NBAFYE was prepared by amending NBAF medium lacking CaCl2 and MgSO4 with 15 g of purified agar/liter. Following sterilization, the molten medium was cooled to 50°C and appropriate volumes of anaerobic stock solutions of CaCl2, MgSO4, yeast extract, and cysteine were added to yield final concentrations of 0.004%, 0.01%, 0.1%, and 5 mM, respectively. If necessary, antibiotics (concentrations listed below) were also added at this time. FWAFC is a modification of FWA-Fe(III) citrate medium (19), a freshwater minimal medium containing 20 mM acetate as the electron donor and 55.9 mM Fe(III) citrate as the electron acceptor. Its composition per liter of deionized water is 13.7 g of FeC6O7, 2.5 g of NaHCO3, 0.25 g of NH4Cl, 0.6 g of NaH3PO4 · H2O, 0.1 g of KCl, 1.0 ml of 100 mM Na2SeO4, 10.0 ml of a vitamin solution (17), and 10.0 ml of a trace mineral solution (17). Ammonium- and nitrogen-free FWAFC medium was prepared by sparging and overlaying ammonium-free FWAFC medium with an 80% Ar-20% CO2 gas mixture. Antibiotics (200 µg of kanamycin, 400 µg of streptomycin, or 10 µg of chloramphenicol per ml) were added to liquid and solid medium as needed. All antibiotic stock solutions were sterile and anoxic (sparged and overlaid with an N2 atmosphere).Determination of plating efficiency. The cell density (cells/milliliter) of prestationary-phase liquid cultures was determined by staining cells with acridine orange and utilizing epifluorescence microscopy as previously described (19). The density of CFU in these cultures was determined by plating serial dilutions.
Preparation of electrocompetent cells. Electrocompetent E. coli was prepared according to Sambrook et al. (26). Electrocompetent G. sulfurreducens was prepared from cultures maintained in NBAFYE medium as described below.
All manipulations were carried out on ice in an anaerobic chamber, and any buffers used were ice-cold and anoxic. Four hundred milliliters of mid-log-phase cultures (optical density at 600 nm = 0.2 to 0.35; 9 × 107 to 1.8 × 108 cells/ml) was harvested by centrifugation at 4°C for 8 min at 4,300 × g. The cells were washed twice with 400 ml of electroporation buffer (1 mM HEPES [pH 7.0], 1 mM MgCl2, and 175 mM sucrose) and resuspended in the same buffer at a final concentration of ~1011 cells/ml. Because G. sulfurreducens was found to be particularly susceptible to shearing, pipetting of cell suspensions was minimized and, when necessary, performed with large-bore pipette tips. An appropriate volume of a 60% dimethyl sulfoxide (DMSO)-40% electroporation buffer solution was added to the final cell suspension to achieve a final DMSO concentration of 10%. The resulting electrocompetent cells were either electroporated immediately or stored at
70°C for future use.
Electrotransformation procedures. All electrotransformations were performed in 0.15-cm-gap microelectroporation chambers using a Cell-Porator equipped with a Voltage Booster (Life Technologies Inc., Gaithersburg, Md.). Electrocompetent E. coli cells were transformed according to the manufacturer's instructions.
Electrocompetent G. sulfurreducens cells (25 µl), either freshly prepared or thawed on ice, were pulsed at 14.7 kV/cm for ~6 ms (resistance = 4 k
; capacitance = 25 µF). Immediately
following electroporation, cells were washed into the bottom of the
microelectroporation chamber with 1 ml of room temperature
phosphate-buffered NBAF (NBAF medium containing 50 mM potassium
phosphate instead of bicarbonate). The cells were then transferred to a
prewarmed anaerobic pressure tube containing 9 ml of NBAFYE. The
electroporated cells were allowed to recover for 5 h at 30°C,
prior to plating onto the appropriate solid growth medium.
Assessment of plasmid stability under nonselective conditions. G. sulfurreducens was electrotransformed with either pBBR1MCS-2 or pJRD215 (Table 1) and plated onto selective medium (NBAFYE containing 200 µg of kanamycin/ml). The resulting kanamycin-resistant colonies were transferred to liquid selective medium until the cultures became turbid (3 to 6 days). Selective pressure was maintained throughout this incubation by adding fresh kanamycin to the cultures every 48 h. Twenty-four hours prior to the beginning of the experiment, cultures were diluted 10-fold into fresh selective medium. At time zero, the cultures were serially diluted into nonselective medium (NBAFYE) and were then plated onto both selective and nonselective media. The remainder of the various serial dilutions was incubated at 30°C. Every 48 h, a log-phase culture was selected from among the various dilutions for analysis and the process was repeated. The total number of CFU in the various cultures was determined on nonselective medium, and the percentage of kanamycin-resistant CFU was ascertained by comparison. The number of generations between time points was determined from the total CFU/milliliter using the following formula: log (final CFU per milliliter/initial CFU per milliliter)/log2.
Southern blotting.
Following digestion of genomic DNA with
the restriction enzyme EcoRV, Southern blot analysis was
performed according to Sambrook et al. (26). Probes were
labeled with [
-32P]dCTP using the Multiprime
DNA labeling system (Amersham Pharmacia Biotech, Piscataway, N.J.).
[-32P]dCTP was obtained from New England
Nuclear (Boston, Mass.).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Growth on solid medium and characterization of antibiotic sensitivity. In order to be able to rapidly isolate clonal populations of G. sulfurreducens, conditions for high-efficiency plating onto solid growth medium were established. Simply solidifying the two liquid media for G. sulfurreducens, NBAF and FWAFC (see Materials and Methods), was not sufficient to achieve the rapid growth of G. sulfurreducens on plates. After considerable trial and error, it was found that supplementation of the solid form of NBAF medium with 0.1% yeast extract and a high concentration (5 mM) of the reductant cysteine yielded a solid medium, NBAFYE, on which both the growth rate and the plating efficiency of G. sulfurreducens were reproducibly high. Colonies were visible on solid NBAFYE medium after only 5 days of incubation at 30°C. In addition, when pre-stationary-phase liquid cultures with a density of (3.25 ± 0.54) × 108 cells/ml were plated onto this medium, (2.85 ± 0.48) × 108 CFU/ml were recovered (mean ± standard deviation; n = 3), demonstrating a high plating efficiency.
The growth of G. sulfurreducens on solid medium could be inhibited by a variety of commonly used antibiotics. Growth of 108 cells on solid medium could be inhibited by choramphenicol (10 µg/ml), nalidixic acid (10 µg/ml), tetracycline (10 µg/ml), kanamycin (200 µg/ml), spectinomycin (50 µg/ml), streptomycin (400 µg/ml), and ampicillin (400 µg/ml). The sensitivity of G. sulfurreducens to these antibiotics in liquid culture was similar (data not shown). These results indicated that it should be possible to select for the acquisition of multiple antibiotic resistance markers by G. sulfurreducens, a requirement for many genetic manipulations.Development of an electrotransformation procedure.
A protocol
for the electrotransformation of G. sulfurreducens was
developed by modifying existing protocols (21) to account for the unique properties of G. sulfurreducens. The
preparation of electrocompetent cells typically involves extensive
washing of cells derived from mid-log-phase cultures in a suitable
low-ionic-strength electroporation buffer, followed by resuspension of
the cells at high density (109 to 1011
cells/ml) in this buffer (21). In order to attain
sufficient G. sulfurreducens biomass at mid-log phase, it
was necessary to supplement the NBAF medium routinely used to culture
the organism with 0.1% (wt/vol) yeast extract. When grown on this rich
medium at 30°C, G. sulfurreducens had a generation time of
~4.5 h and reached a cell density of ~108
cells/ml at mid-log phase (data not shown). The composition of the
electroporation buffer was found to be critical for maintaining the viability of G. sulfurreducens throughout the washing
and electrotransformation procedures. The optimal composition of the electroporation buffer was found to be 1 mM HEPES (pH 7.0), 1 mM
MgCl2, and 175 mM sucrose. The
addition of MgCl2 to the electroporation buffer
was absolutely required if G. sulfurreducens was to remain viable during washing and storage. In the absence of
MgCl2, 90% of the cells lysed prior to
electroporation (data not shown). MgCl2 is
thought to prevent bacterial lysis by stabilizing the outer membrane
(27). The concentration of sucrose was another crucial
variable in the composition of the electroporation buffer. Electrotransformed G. sulfurreducens could not tolerate
large changes in osmolarity upon dilution back into nonselective growth medium. To avoid such changes in osmotic pressure, 175 mM sucrose was
added to the electroporation buffer such that its osmolarity was
equivalent to that of NBAFYE medium. The addition of 10% DMSO to the
final cell suspension permitted storage of electrocompetent G. sulfurreducens at 70°C with little or no loss in
transformation efficiency. Glycerol was not a suitable cryopreservant
due to the sensitivity of G. sulfurreducens to rapid changes
in osmolarity.
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Identification of potential "suicide" and expression vectors. A variety of plasmids were tested for the ability to replicate in and confer antibiotic resistance upon G. sulfurreducens (Table 2). In addition to pJRD215, two other IncQ plasmids, pJRC2 and pCD342 (Table 1), were evaluated. The plasmid pJRC2 is identical to pJRD215 except that it confers resistance to chloramphenicol. pCD342 is an expression vector containing a polylinker which is preceded by the hybrid tac-lac promoter (tac and lacUV5 promoters in tandem) and followed by the rrnB transcriptional terminator (1, 20). Electroporation of G. sulfurreducens with all three IncQ plasmids resulted in the growth of numerous antibiotic-resistant transformants (Table 2), indicating that all of these plasmids were capable of replication in G. sulfurreducens. In addition, all three of the IncQ plasmids could be purified from cultures of G. sulfurreducens in sufficient quantity to be identified by digestion with restriction enzymes (Fig. 1A), suggesting that IncQ plasmids are stable in this organism. In fact, pJRD215 has a half-life of ~60 generations in G. sulfurreducens in the absence of antibiotic selection (Table 2; Fig. 1B). Similarly, we were able to purify pCD342 from G. sulfurreducens (Fig. 1A, lane 3) after culturing a transformant for approximately 30 generations in the absence of antibiotic selection.
To determine whether pCD342 could serve as an expression vector for G. sulfurreducens, cells were transformed with pCD354 (10), a green fluorescent protein expression vector derived from pCD342. The green fluorescent protein was detected in the resulting transformants (Fig. 1C), indicating that pCD342 is indeed a suitable expression vector for G. sulfurreducens. Due to their large size (
10 kb) and low copy number, IncQ vectors
are difficult to manipulate (8, 10). The pBBR1MCS series
of broad-host-range expression vectors (12), in contrast, are fairly small (~5.3 kb) and have a higher copy number. In
addition, they contain a variety of antibiotic resistance cassettes and possess an extensive multicloning site located within the
lacZ alpha peptide (12). Kanamycin-resistant
transformants were isolated following electroporation of G. sulfurreducens with pBBR1MCS-2 (Table 2), indicating that this
class of plasmids is capable of replication in G. sulfurreducens. However, pBBR1MCS-2 was found to be unstable in
G. sulfurreducens (Fig. 1B). Despite the rigorous maintenance of selective pressure during propagation of pBBR1MCS-2 in
liquid culture, only 15% of the CFU in these cultures were able to
grow on solid medium containing kanamycin. Furthermore, in the absence
of continued selection, kanamycin resistance was lost within 12 generations. Thus, despite the fact that pBBR1MCS vectors can replicate
in G. sulfurreducens, their instability renders them
unsuitable for use as expression vectors.
In addition to the two types of broad-host-range plasmids mentioned
above, one plasmid with limited host specificity, pBMK7 (23), was evaluated for the ability to replicate in
G. sulfurreducens. The plasmid pBMK7 is a shuttle vector
that contains two replicons: the pBM1 replicon, which functions in
E. coli, and the pBG1 replicon, which functions in
Desulfovibrio species. Members of the genus Desulfovibrio, like those of the Geobacter genus,
are obligate anaerobes belonging to the delta subdivision of the
Proteobacteria. However, electrotransformation of G. sulfurreducens with pBMK7 repeatedly failed to yield
kanamycin-resistant transformants (Table 2). Thus, even though
Geobacter and Desulfovibrio species are closely
related, the pBG1 replicon did not appear to function in G. sulfurreducens. Furthermore, these results indicated that plasmids containing the pBG1 or pBM1 replicons acted as suicide vectors
in G. sulfurreducens and could, therefore, be used to introduce mutations into its chromosome.
Mutagenesis of the G. sulfurreducens chromosome by gene replacement. The next step in the development of a genetic system for G. sulfurreducens was to determine whether the function of a specific gene could be eliminated by placing a mutation in the G. sulfurreducens chromosome. A gene suspected of being involved in nitrogen fixation by G. sulfurreducens was selected as the first target, because, as noted above, it is important to conclusively demonstrate that G. sulfurreducens has the capacity to fix nitrogen. In other microorganisms, nitrogen reduction is catalyzed by the nitrogenase enzyme complex, which is encoded by the highly conserved nifH, nifD, and nifK genes (9, 30). Because the nitrogenase complex functions specifically in the nitrogen fixation pathway, inactivation of the genes for any of its components yields a predictable and easily characterized phenotype, an inability to grow in medium lacking fixed nitrogen (9). By using the nifD genes of other organisms to probe the partial sequence of the G. sulfurreducens genome, a potential G. sulfurreducens nifHDK operon was identified. Studies were initiated to determine whether disruption of the putative nifD gene of G. sulfurreducens by the single-step gene replacement method (25) would affect the ability of G. sulfurreducens to grow in a medium devoid of fixed nitrogen.
In order to disrupt the putative G. sulfurreducens nifD gene, we constructed the suicide vector pBRnif::kan (Table 1; Fig. 2B), which contained a fragment of the nifD coding sequence disrupted by a kanamycin resistance cassette. To ensure that incorporation of the disrupted nifD coding sequence into the chromosome occurred via a double recombination event, the suicide vector was linearized with NruI and ScaI prior to electroporation. The recovery period following electroporation was extended to ~18 h to allow ample time for recombination and expression of kanamycin resistance. This procedure resulted in the growth of 2.77 × 105 kanamycin-resistant colonies per µg of linear DNA. Eight of these kanamycin-resistant colonies were screened for the presence of the disrupted nifD (nifD1::kan) coding sequence by PCR, and all eight were found to contain the nifD1::kan insertion mutation (data not shown). These results indicated that recombination between linear DNA fragments and the G. sulfurreducens chromosome was a highly efficient process.
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Complementation of the nifD1::kan
phenotype by in trans expression of the
nifD gene.
In order to demonstrate that the
phenotype of mutant DL2D was in fact due to disruption of the
nifD gene and not to an unexpected secondary mutation or to
an unanticipated effect on downstream gene expression, the
nifD expression vector pCDSnifD was introduced into DL2D by electroporation. To determine if expression of the nifD gene in trans complemented the
nifD1::kan phenotype, the ability of
DL2D/pCDSnifD to grow in the absence of fixed nitrogen was
evaluated (Fig. 4). The extent of cell
growth and iron reduction by both DL2D/pCDSnifD and the
wild-type strain (DL1) was similar under all three conditions tested
(Fig. 4): (i) in the presence of both gaseous and fixed nitrogen, (ii)
in the presence of gaseous nitrogen alone, and (iii) in the absence of
both gaseous and fixed nitrogen. Thus, expression of the
nifD gene in trans restored the ability of the
nifD1::kan mutant DL2D to grow in the absence of
exogenous fixed nitrogen. In fact, DL2D/pCDSnifD can be
routinely cultured in ammonium-free FWAFC medium (data not shown).
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Concluding remarks. In summary, this study has demonstrated that G. sulfurreducens is a genetically tractable microorganism. It is now possible to introduce foreign DNA into G. sulfurreducens by electroporation, to place mutations at specific locations within its chromosome, and to express proteins from extrachromosomal elements. These novel genetic techniques coupled with the availability of the sequence of the G. sulfurreducens genome will enable the rapid study of the physiological function of multiple G. sulfurreducens genes. The finding that the physiology of G. sulfurreducens can be studied by genetic techniques is significant, because microorganisms closely related to G. sulfurreducens are prominent members of a variety of Fe(III)-reducing microbial communities (22, 28). Thus, studies on the physiology of G. sulfurreducens, using the techniques outlined here, are likely to provide insights into microbial processes occurring in the Fe(III) reduction zone of subsurface environments. Studies similar to those described here but focusing on mechanisms for the reduction of Fe(III) and contaminant metals by G. sulfurreducens are currently in progress.
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ACKNOWLEDGMENTS |
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This work was supported by the U.S. Department of Energy (grants DE-FG02-98ER20306 and DE-FG02-97ER-62475-A005).
All G. sulfurreducens sequence data used in this report were obtained from The Institute for Genomic Research website at http://www.tigr.org. We thank Judy Wall, Christoph Dehio, and Michael Kovach for sending us plasmids. We also acknowledge Betsy Harris for her guidance and assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, 203 Morrill Science Center IVN, University of Massachusetts at Amherst, Amherst, MA 01003. Phone: (413) 545-9651. Fax: (413) 545-1578. E-mail: dlovley{at}microbio.umass.edu.
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Applied and Environmental Microbiology, July 2001, p. 3180-3187, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3180-3187.2001
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