APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2004, p. 7251–7259
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 12
In Situ Expression of nifD in Geobacteraceae in Subsurface Sediments
Dawn E. Holmes,* Kelly P. Nevin, and Derek R. Lovley
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts
Received 11 May 2004/Accepted 13 July 2004
In order to determine whether the metabolic state of Geobacteraceae involved in bioremediation of subsurface
sediments might be inferred from levels of mRNA for key genes, in situ expression of nifD, a highly conserved
gene involved in nitrogen fixation, was investigated. When Geobacter sulfurreducens was grown without a source
of fixed nitrogen in chemostats with acetate provided as the limiting electron donor and Fe(III) as the electron
acceptor, levels of nifD transcripts were 4 to 5 orders of magnitude higher than in chemostat cultures provided
with ammonium. In contrast, the number of transcripts of recA and the 16S rRNA gene were slightly lower in
the absence of ammonium. The addition of acetate to organic- and nitrogen-poor subsurface sediments
stimulated the growth of Geobacteraceae and Fe(III) reduction, as well as the expression of nifD in Geobacter-
aceae. Levels of nifD transcripts in Geobacteraceae decreased more than 100-fold within 2 days after the addition
of 100 ?M ammonium, while levels of recA and total bacterial 16S rRNA in Geobacteraceae remained relatively
constant. Ammonium amendments had no effect on rates of Fe(III) reduction in acetate-amended sediments
or toluene degradation in petroleum-contaminated sediments, suggesting that other factors, such as the rate
that Geobacteraceae could access Fe(III) oxides, limited Fe(III) reduction. These results demonstrate that it is
possible to monitor one aspect of the in situ metabolic state of Geobacteraceae species in subsurface sediments
via analysis of mRNA levels, which is the first step toward a more global analysis of in situ gene expression
related to nutrient status and stress response during bioremediation by Geobacteraceae.
The addition of nutrients to stimulate microbial metabolism
is a common practice in the bioremediation of subsurface en-
vironments (20, 26). However, these additions are typically
done in an empirical manner with little or no information
about the actual nutritional requirements of the subsurface
microbial community (36). A more rational approach might be
to evaluate the metabolic state of the community prior to
making amendments by documenting the expression of genes
that respond to the presence or absence of a specific nutrient.
This strategy may be most feasible in environments in which
one group of microorganisms predominates during the biore-
mediation process, because metabolic traits unique to this
group of organisms can be monitored. One example of a group
of microorganisms that frequently dominate microbial commu-
nities that have been associated with the effective bioremedia-
tion of organic and metal contaminants in subsurface environ-
ments is the Geobacteraceae. For example, Geobacteraceae
were the predominant organisms associated with the anaerobic
degradation of petroleum (4, 55, 59) and landfill leachate (54)
contaminants coupled with the reduction of Fe(III) in subsur-
face environments. The addition of acetate to stimulate dis-
similatory metal reduction by the Geobacteraceae was also an
effective strategy for promoting the reductive precipitation of
uranium from contaminated groundwater (5, 19), and active
U(VI) reduction was associated with an enrichment of 16S
rRNA gene sequences of Geobacteraceae that accounted for 40
to 90% of the microbial community (5, 27).
It was hypothesized that fixed nitrogen might be one of the
nutrients limiting the activity of Geobacteraceae during biore-
mediation (8). Petroleum contamination provides significant
quantities of organic carbon but little fixed nitrogen. Further-
more, when U(VI) bioremediation and dissimilatory Fe(III)
reduction were stimulated in field trials and laboratory incu-
bations with the addition of acetate, fixed nitrogen was not
added to the sediments (5, 19). Evaluation of 30 species of
Geobacteraceae demonstrated that they all contain nifD, the
gene that encodes the alpha subunit of the dinitrogenase pro-
tein (28). Previous physiological studies have also demon-
strated that several species of Geobacteraceae are able to fix
nitrogen (8, 14). In contrast, the genomes of other well-studied
metal-reducing microorganisms, such as Shewanella oneidensis
(24), Desulfovibrio vulgari (25), and Geothrix fermentans (www
.jgi.doe.gov) do not contain nifD, suggesting that the ability to
fix nitrogen may be one of the features that permits Geobacter-
aceae to effectively compete in subsurface environments.
Analysis of mRNA levels in subsurface sediments may be
the most direct method for specifically assessing the physiolog-
ical state of microorganisms involved in subsurface bioreme-
diation. Increased levels of mRNA for a particular gene have
been linked to specific metabolic and/or geochemical processes
in pure-culture studies (10, 13, 18, 43, 45, 51, 57, 65, 66, 68) and
in the environment (11, 16, 21, 23, 30, 32, 33, 44, 49–51, 56,
67–70). For example, increased levels of mRNA transcripts
from a gene involved in naphthalene degradation (nahA) were
detected in sediments in which naphthalene was being miner-
alized (21, 67). Expression of tfdA, a gene involved in the
degradation of the herbicide 2,4-dichlorophenoxyacetic acid
was associated with greater herbicide transformation (16), and
higher rates of mercury volatilization were accompanied by
higher levels of merA expression in some environmental sam-
ples (44). However, mRNA analysis does not appear to have
been previously employed to assess the in situ metabolic state
* Corresponding author. Mailing address: Department of Microbi-
ology, 103B Morrill IV North, University of Massachusetts, Amherst,
MA 01003. Phone: (413) 577-0447. Fax: (413) 545-1578. E-mail:
of microorganisms in subsurface environments during biore-
Here we present results from RNA analysis of subsurface
sediments that suggest that Geobacteraceae living in a petro-
leum-contaminated aquifer or in subsurface sediments amended
with acetate in order to stimulate dissimilatory metal reduction
express nifD and that nifD expression is repressed when am-
monium is added to the sediments. These findings represent an
important first step in assessing the metabolic status of the
Geobacteraceae during in situ bioremediation.
MATERIALS AND METHODS
Chemostat culture. Geobacter sulfurreducens (ATCC 51573) was obtained
from our laboratory collection and cultured under anaerobic conditions in ace-
tate-limited chemostats, with acetate (5 mM) provided as the electron donor and
Fe(III) citrate (55 mM) provided as the electron acceptor at 30°C, as described
elsewhere (18a). The dilution rate was 0.05 h?1.
Sediment collection and laboratory incubations. Sediments were collected
from the U.S. Geological Survey Groundwater Toxics Site in Bemidji, Minn.
These aquifer sediments have been contaminated with crude oil for 18 years as
a result of a break in an oil pipeline (7, 15, 29, 37). This site contains extensive
zones of Fe(III) reduction (3, 4, 55), and studies have suggested that microor-
ganisms within the family Geobacteraceae are involved in anaerobic degradation
of toluene and benzene at this site (4, 55, 59). Sediments were collected from the
Fe(III) reduction zone of the contaminant plume and a nearby pristine site, just
outside the contaminant plume in 1999 and in 2003 as previously described (4).
The samples collected from the Fe(III) reduction zone of the contaminant plume
were used to determine whether Geobacteraceae express nitrogen fixation genes
in the environment.
For sediment incubations, 40 g of sediment was added to 60-ml serum bottles
in an anaerobic chamber under a N2atmosphere and processed as previously
described (48). For the ammonium addition experiment, 5 mM acetate and 0, 50,
100, 250, or 500 ?M NH4Cl were added from sterile anaerobic stocks, and the
remainder of the liquid was added as anaerobic groundwater (3.l ml), for a total
of 4 ml. Fe(II) and total HCl-extractable iron were monitored as previously
described (4). In order to monitor toluene degradation, [U-14C]toluene stocks
were prepared as previously described (4). [U-14C]toluene (1 ?Ci) was added to
each sediment bottle. Each bottle was also amended with unlabeled toluene from
a stock prepared with anaerobic groundwater in order to provide a final con-
centration of ca. 50 ?M. NH4Cl (0, 100 or 250 ?M) was added from sterile
anaerobic stocks. Total14CO2was determined from14CO2in the headspace and
the partitioning of H14CO3?added to sediment as previously described (39).
Extraction of mRNA from chemostat cultures and sediments. All sediment
and chemostat incubations were conducted in triplicate. Solutions used during
the RNA extraction process were made with diethyl pyrocarbonate (DEPC)-
treated water (Ambion). Chemostat cultures (200 ml) at steady state were trans-
ferred to prechilled 50-ml conical tubes and centrifuged at 3,150 ? g for 15 min
at 4°C. The supernatant was discarded, and pellets were flash frozen in an
ethanol-dry ice bath and stored at ?80°C. Prior to RNA extraction, the pellet
was resuspended in 1.5 ml of TPE buffer (100 mM Tris-HCl, 100 mM KH2PO4,
10 mM EDTA; pH 8.0) and aliquoted into eight separate 2-ml screw cap tubes.
RNA was extracted from sediment incubations when 60 to 75% of the HCl-
extractable iron in the sediments was reduced. Five grams of sediment was
divided into 10 separate aliquots of 0.5 g each and dispensed into 2-ml screw cap
tubes, and 500 ?l of TPE buffer was added to each tube.
Sediments from the field that were used for in situ RNA expression analysis
were collected as follows: ca. 5 g of sediment was aliquoted into a 50-ml conical
tube containing 10 ml of cold (4°C) TPE buffer and 30 ml of cold (4°C) RNA
Protect solution (QIAGEN). Tubes were mixed manually 20 times, frozen on dry
ice, and immediately transported to the laboratory, where they were stored at
?80°C. Samples were then thawed on ice and pelleted at 4,900 ? g for 15 min.
The pellet was then dispensed into 10 separate 0.5-g aliquots, and 500 ?l of TPE
buffer was added.
The same RNA extraction protocol was followed for all of the sediment and
chemostat culture samples once they were suspended in TPE buffer. First, 100 ?l
of Plant RNA Isolation Aid (Ambion) and 1 ml of cold acetone (stored at
?20°C) were added to the cell or sediment suspensions. Tubes were then mixed
manually ?20 times and centrifuged at 16,100 ? g for 5 min. The supernatant
was discarded, and 2 ?l of Superase-In (Ambion) was added to the pellet, which
was then resuspended in 1 ml of sterile DEPC-treated water (Ambion). Ten
microliters of lysozyme (50 mg/ml), 3 ?l of proteinase K (20 mg/ml), and 30 ?l
of 10% sodium dodecyl sulfate solution were added and this mixture was incu-
bated at 37°C for 10 min. Samples were then centrifuged at 16,100 ? g for 15 min,
and the supernatant was transferred to new 2-ml screw cap tubes. Fifty micro-
liters of Plant RNA Isolation Aid, 10 ?l of yeast tRNA (10 mg/ml; Ambion), 600
?l of hot acidic (70°C; pH 4.5) phenol (Ambion), and 400 ?l of chloroform-
isoamyl alcohol (24:1; Sigma) were added to the supernatant. These tubes were
then mixed on a Labquake rotator (Barnestead/Thermolyne, Dubuque, Iowa) for
10 min and centrifuged at 16,100 ? g for 5 min. The aqueous layer was removed
and transferred to new 2-ml screw cap tubes, and 600 ?l of hot acidic (70°C; pH
4.5) phenol (Ambion) and 400 ?l of chloroform-isoamyl alcohol (24:1; Sigma)
were added. Tubes were mixed on a rotator for 5 min and centrifuged at
16,100 ? g for 5 min. The aqueous layer was removed again and transferred to
a new tube, and 100 ?l of 5 M ammonium acetate (Ambion), 4 ?l of linear
acrylamide (5 mg/ml; Ambion), and 1 ml of cold (?20°C) isopropanol (Sigma)
Nucleic acids were precipitated at ?20°C for 1 h and pelleted by centrifugation
at 16,100 ? g for 0.5 h. The pellet was then cleaned with cold (?20°C) 70%
ethanol, dried, and resuspended in sterile DEPC-treated water (Ambion). The
resuspended pellets were combined and cleaned with the RNeasy RNA cleanup
kit (QIAGEN) according to the manufacturers instructions. The RNA cleanup
product was then treated with DNA-free DNase (Ambion) according to the
The RNA extraction method described here provided high-quality mRNA
from natural populations in aquifer sediments. All of the mRNA samples had
A260/A280ratios of 1.8 to 2.0, indicating that they were of high purity (6). In order
to confirm the fact that PCR products generated from the cDNA template did
not result from the amplification of contaminating DNA, all PCR analyses
included negative controls with RNA that had not been subjected to reverse
transcriptase PCR (RT-PCR).
Testing and design of primers and probes. Degenerate primers targeting nifD
and recA in Geobacteraceae were designed from nucleotide sequences from G.
sulfurreducens, Geobacter metallireducens, Desulfuromonas acetoxidans, and Pe-
lobacter carbinolicus. These sequence data were obtained from The Institute for
Genomic Research (TIGR) website (http://www.tigr.org) and the Department of
Energy (DOE) Joint Genome Institute (JGI) website (www.jgi.doe.gov). These
nucleotide sequences were initially aligned in CLUSTALX and imported into
the Genetics Computer Group (Madison, Wis.) sequence editor (Wisconsin
Package, version 1.0). This alignment was then examined, and conserved regions
were targeted for primer design.
The following primers were used to amplify a 335-bp fragment of the nifD gene
from Geobacteraceae NIFGEO225F (5? ATC GGT GAC GAT ATC AAC GCC
3?) and NIFGEO560R (5? TAG TTC ATG GAA CGG TAG CAG T 3?).
A 468-bp fragment of the recA gene in Geobacteraceae was amplified with
RECGEO202F (5? ATC TWC GGI CCS GAG TCG TCG GGC AA 3?) and
RECGEO670R (5? CCS TCG CCG TAG WAG ATG TCG AA 3?).
RT-PCR of nifD, recA, and 16S rRNA transcripts. The DuraScript enhanced
avian RT single-strand synthesis kit (Sigma) was used to generate cDNA from
extracted nifD, recA, and 16S rRNA transcripts in two steps. The first reaction
mixture had a total volume of 10 ?l and consisted of template RNA (0.5 ?g), 40
pmol of the appropriate reverse primer, 2 ?l of deoxynucleoside triphosphate
solution (2.5 mM), and DEPC-treated water. This mixture was incubated at 70°C
for 10 min. Ten microliters of a second solution containing 2 ?l of 10? RT
buffer, 1 U of avian RT, 1 U of RNase inhibitor, and 6 ?l of DEPC-treated water
was then added to the first solution and allowed to incubate at 50°C for 50 min.
Amplification of nifD, recA, and 16S RT-PCR products and construction of
cDNA libraries. Optimal amplification conditions for NIFGEO225F/560R and
RECGEO202F/670R were determined in a gradient thermal cycler (MJ Re-
search Inc., Waltham, Mass.). The amplification parameters included an initial
denaturation step of 95°C for 5 min, followed by 35 cycles of 95°C (30 s), 60°C
(45 s), and 72°C (45 s), with a final extension at 72°C for 10 min.
In an attempt to reduce PCR bias and provide the most reliable evaluation of
microbial community dynamics, more than one bacterial primer set targeting
different regions of 16S rRNA was used. The primer sets included 8F (17) with
519R (34) and 338F (1) with 907R (34). Once the appropriate 16S cDNA
fragments were generated by RT-PCR, the following PCR parameters were
used: an initial denaturation step at 95°C for 5 min, followed by 35 cycles of 95°C
(45 s), 50°C (1 min), and 72°C (1 min), with a final extension step at 72°C for 7
min. To ensure sterility, the PCR mixtures were exposed to UV radiation for 8
min prior to the addition of the cDNA template and Taq polymerase.
16S rRNA, recA, and nifD PCR products were purified with a gel extraction kit
(QIAGEN), and clone libraries were constructed with a TOPO TA cloning kit,
version M (Invitrogen, Carlsbad, Calif.), according to the manufacturers’ instruc-
7252 HOLMES ET AL.APPL. ENVIRON. MICROBIOL.
tions. One hundred plasmid inserts from each cDNA clone library were then
sequenced with the M13F primer at the University of Massachusetts Sequencing
Quantification of nifD and recA expression with TaqMan PCR. Optimal Taq-
Man PCR conditions were determined by using the manufacturer’s guidelines.
Each PCR mixture consisted of a total volume of 50 ?l and contained 5 ?l of the
appropriate primers (5 ?M) (NIFGEO225F/560R or RECGEO202F/670R), 5 ?l
of the RT-PCR product, 5 ?l of 10? SYBR green PCR buffer (PE Biosystems,
Foster City, Calif.), 6 ?l of MgCl2solution (25 mM; PE Biosystems), 4 ?l of the
deoxynucleoside triphosphate mix (2.5 mM; PE Biosystems), 10 ?l of buffer Q
solution (QIAGEN), 2 ?l of bovine serum albumin (10 mg/ml; New England
Biolabs, Beverly, Mass.), 0.5 U of AmpErase uracil-N-glycosylase (PE Biosys-
tems), and 0.5 U of Taq polymerase (QIAGEN).
The RT-PCR products used to construct the TaqMan standard curve were
purified by the protocol for phenol extraction and ethanol precipitation of nu-
cleic acids outlined in Current Protocols in Molecular Biology (6). Standard curves
were constructed with serial dilutions of known amounts of purified cDNA
quantified with a Shimadzu (Baltimore, Md.) UV2401-PC dual-beam spectro-
photometer at an absorbance of 260 nm. Serial dilutions covered a range of ?8
orders of magnitude. PCR amplification and detection were performed with the
GeneAmp 5700 sequence detection system (PE Biosystems). To verify amplifi-
cation and correct amplicon size, aliquots from real-time PCR were examined on
an ethidium bromide-stained 1% agarose gel.
Quantification of nifD, recA, and 16S rRNA expression by slot blot hybridiza-
tion analysis. cDNA generated from nifD and recA and from bacterial 16S rRNA
transcripts in Geobacteraceae were also analyzed by slot blot hybridization. Stan-
dard curves were constructed with serial dilutions of known amounts of purified
cDNA that covered a range of ?6 orders of magnitude. In order to denature
cDNA, samples were incubated at 65°C for 5 min in 3 volumes of a solution
containing 500 ?l of formamide, 162 ?l of formaldehyde (37% solution), and 100
?l of 10? TBE buffer (0.9 M Tris-borate, 0.02 M EDTA). The cDNA was then
chilled on ice, and 1 volume of cold (4°C) 20? SSC (1? SSC is 0.15 M NaCl plus
0.015 M sodium citrate) buffer was added.
Once cDNA was denatured, all samples were immobilized on the Zeta-Probe
GT membrane (Bio-Rad, Hercules, Calif.) in the same manner. The membrane
was soaked in 10? SSC buffer for 1 h prior to placement in the slot blotting
manifold (Bio-Rad). Equal quantities of samples were applied to the slot blotting
apparatus according to the manufacturer’s instructions. The membrane was then
immersed in denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 5 min, followed
by 1 min in neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl [pH 7.2], 0.001 M
EDTA), and the nucleic acids were fixed to the membrane in a XL-1500 UV
cross-linker (Spectronics Corporation, Westbury, N.Y.) according to the manu-
Probes targeting nifD and recA and bacterial 16S rRNA genes in Geobacter-
aceae were constructed using gene products from the following primer pairs:
NIFGEO225F/560R, RECGEO202F/670R, and 8F/519R. Amplified fragments
from G. sulfurreducens nifD, recA, and 16S rRNA genes were first gel purified
with the QIAGEN gel extraction kit (QIAGEN) according to the manufacturer’s
instructions. Twenty-five nanograms of these amplicons was used as the template
for construction of [32P]dCTP-labeled probes with a NEBlot kit (New England
Biolabs, Inc.), and probe hybridizations were performed with the NorthernMax
Gly kit (Ambion) according to the manufacturer’s instructions. Hybridization
products were visualized on a Typhoon 9210 variable-mode imager (Amersham
Biosciences, Piscataway, N.J.), and spot intensities were quantified and com-
pared with ImageQuant software (Amersham Biosciences).
Phylogenetic analysis. 16S rRNA, nifD, and recA gene sequences were com-
pared to sequences in the GenBank nucleotide and protein databases using
BLASTN and BLASTX algorithms. Nucleotide and amino acid sequences for
each gene were initially aligned in CLUSTAL X (63) and imported into the
Genetics Computer Group sequence editor (Wisconsin Package version 10),
where alignments were checked and hypervariable regions were masked. These
alignments were then imported into CLUSTAL W (64) and Mview (9), where
similarity and identity matrices were generated.
Aligned sequences were imported into PAUP 4.0b10 (62), where phylogenetic
trees were inferred. Distances and branching order were determined and com-
pared using character-based (maximum parsimony and maximum likelihood)
and distance-based (HKY85 and Jukes-Cantor) algorithms. Bootstrap values
were obtained from 100 replicates. Preliminary sequence data from G. sulfurre-
ducens, G. metallireducens, D. acetoxidans, P. carbinolicus, Pelobacter propionicus,
and Geothrix fermentans were obtained from the TIGR website (http://www.tigr
.org) and the DOE JGI website (www.jgi.doe.gov).
RESULTS AND DISCUSSION
nifD, recA, and 16S rRNA gene expression by G. sulfurredu-
cens in chemostat cultures. There have been no previous re-
ports on the regulation of nitrogen fixation genes in the
Geobacteraceae. In order to determine whether transcription
of nitrogen fixation genes was repressed in the presence of
ammonium, pure-culture studies were first conducted with G.
sulfurreducens. G. sulfurreducens was grown in chemostats un-
der the electron donor-limiting conditions that might typically
be found in subsurface environments, with acetate as the lim-
iting electron donor and an excess of Fe(III) as the electron
acceptor. The cultures were either provided with ammonium
or no source of fixed nitrogen. TaqMan and slot blot hybrid-
ization analyses of RNA extracted from these cultures indi-
cated that the number of nifD mRNA transcripts was 4 to 5
orders of magnitude higher in the cultures that were required
to fix nitrogen and that levels of recA, a gene expected to be
constitutively expressed (31), and 16S rRNA transcripts were
slightly lower in the absence of ammonium. TaqMan PCR
analyses indicated that 4.93 ? 106? 2.34 ? 106(three repli-
cates) nifD and 1.26 ? 105? 3.0 ?104recA transcripts were
expressed under nitrogen-fixing conditions compared to 58.5 ?
49.2 nifD and 3.86 ? 106? 8.85 ? 105recA transcripts in
ammonium-rich medium. Slot blot cDNA hybridization anal-
yses gave similar results (Table 1). These results demonstrated
that the up-regulation of nitrogenase genes in G. sulfurredu-
TABLE 1. Concentrations of Geobacteraceae nifD and recA mRNA and total bacterial 16S rRNA gene expression as determined from slot
blot hybridization analysis of cDNA
nifD cDNA recA cDNA 16S cDNA
G. sulfurreducens in ammonium-free medium
G. sulfurreducens in ammonium-amended medium
Acetate-amended sediments (Fig. 2) sampled day 60
Control sediments without acetate (Fig. 2) sampled day 60
Acetate-amended sediments (Fig. 3) day 6
Control sediments without acetate (Fig. 3) day 6
Acetate-amended sediments (Fig. 3) day 8 (2 days after
Control sediments without acetate (Fig. 3) day 8 (2 days
after ammonium added)
1.3 ? 107(?3.2 ? 106)
1.2 ? 103(?3.3 ? 102)
2.2 ? 107(?2.4 ? 106)
5.3 ? 103(?4.4 ? 103)
4.2 ? 106(?1.7 ? 106)
3.2 ? 104(?2.1 ? 104)
4.4 ? 106(?7.8 ? 105)
1.6 ? 107(3.4 ? 106)
9.2 ? 107(?4.5 ? 106)
4.7 ? 105(?1.1 ? 105)
5.9 ? 106(?4.4 ? 106)
1.4 ? 104(?9.2 ? 103)
1.2 ? 106(5.4 ? 105)
4.4 ? 109(?1.1 ? 109)
6.7 ? 1010(?1.7 ? 1010)
8.8 ? 109(?5.7 ? 108)
6.3 ? 107(?1.1 ? 107)
3.7 ? 109(?3.4 ? 108)
1.6 ? 107(?3.2 ? 106)
4.5 ? 109(?2.7 ? 108)
710.2 (?320.2) 9.9 ? 103(?6.6 ? 102)
6.9 ? 107(?4.4 ? 107)
aUnits are numbers of mRNA molecules per microgram of total RNA. All values are the means ? the standard deviations of results of triplicate hybridizations.
VOL. 70, 2004IN SITU EXPRESSION OF nifD IN GEOBACTERACEAE7253
cens, and presumably other closely related organisms, could
readily be detected by monitoring nifD transcripts.
nifD expression in acetate-amended background sediments
and petroleum-contaminated sediments. As previously ob-
served (46), the addition of acetate to pristine, organic-poor
subsurface sediments from the background site stimulated
Fe(III) reduction in anaerobic incubations. After 60 days of
incubation, ca. 75% of the Fe(III) in the acetate-amended
sediment had been reduced, whereas only ca. 10% had been
reduced in sediments incubated without added acetate (data
not shown). As expected from previous results (59), there was
a significant enrichment of Geobacteraceae in the sediments in
which Fe(III) reduction had been stimulated. Seventy percent
of the 16S rRNA gene sequences recovered in a clone library
from the acetate-amended sediments were most closely related
to organisms in the Geobacteraceae, whereas less than 2% of
the sequences could be assigned to the Geobacteraceae in the
sediments not amended with acetate. Associated with this en-
richment in the proportion of Geobacteraceae in the acetate-
amended sediments was a 100-fold increase in the level of
bacterial 16S rRNA transcripts (Table 1). Furthermore, levels
of mRNA for recA recovered with primers or probes specific
for recA in Geobacteraceae were also ca. 2 orders of magnitude
higher in the acetate-amended sediments. Quantitative analy-
ses with TaqMan PCR indicated that 2.73 ? 106? 8.78 ? 105
recA transcripts were detected in Geobacteraceae in acetate-
amended sediments compared to 4.87 ? 104? 2.84 ? 104recA
transcripts expressed in Geobacteraceae in nonamended sedi-
ments. Similar results were obtained from slot blot hybridiza-
tion analyses of these samples (Table 1). These results dem-
onstrate that there was significant growth of Geobacteraceae in
response to the added acetate and the higher rates of Fe(III)
Analysis of levels of nifD mRNA in Geobacteraceae with
both the TaqMan and slot blot hybridization approaches dem-
onstrated that nifD expression in Geobacteraceae was 4 to 5
orders of magnitude greater in acetate-amended sediments
than in nonamended controls (Table 1). TaqMan analyses in-
dicated that 1.60 ? 107? 9.25 ? 106nifD transcripts were
expressed in Geobacteraceae in acetate-amended sediments
compared to only 77.5 ? 20.8 nifD transcripts in nonamended
sediments. Similar results were obtained from slot blot analy-
ses (Table 1). These results suggested that the actively metab-
olizing Geobacteraceae in the sediments highly expressed nifD
in response to the addition of a readily utilizable electron
donor and carbon source, when no fixed nitrogen was added.
In a similar manner, nifD transcripts were detected in
Geobacteraceae in petroleum-contaminated sediments col-
lected from the Fe(III) reduction zone of the aquifer (see Fig.
5), suggesting that the Geobacteraceae were also limited for
fixed nitrogen when petroleum constituents were the electron
donor for Fe(III) reduction.
Effect of added ammonium on nifD expression in Fe(III)-
reducing sediments. In order to further evaluate the metabo-
lism of the Geobacteraceae in the acetate-amended sediments,
it was necessary to provide more sediment Fe(III), which was
nearly depleted. Therefore, fresh sediment was mixed with
some of the acetate-amended sediment (49:1, vol/vol). Addi-
tional acetate (5 mM) was added to ensure that the electron
donor supply would be adequate for continued Fe(III) reduc-
tion. The sediments that had not been amended with acetate
were mixed with fresh sediment in the same manner. Fe(III)
reduction continued without a lag in the acetate-amended sed-
iments (Fig. 1A). On day 6, when ca. 60% of the sediment
Fe(III) had been reduced in the acetate-amended sediments,
mRNA levels for nifD in Geobacteraceae were high (Fig. 1B
and Table 1), suggesting that the Geobacteraceae continued to
highly express nitrogen fixation genes.
Ammonium was then added to the sediments on day 6.
While the addition of ammonium did not appear to effect
FIG. 1. (A) Fe(II) production before and after the addition of
ammonium (100 ?M) in acetate-amended and control sediments.
Each point is the average of results from triplicate samples from
triplicate incubations of each sediment type. (B) recA and nifD con-
centrations determined by TaqMan analysis over time. Each point is
the average of results of five replicates from the triplicate incubations
of the sediments. Error bars represent one standard deviation.
7254 HOLMES ET AL.APPL. ENVIRON. MICROBIOL.
Fe(III) reduction (Fig. 1A), a dramatic decrease in nifD ex-
pression by Geobacteraceae was observed over the next 2 days
(Fig. 1B and Table 1). In contrast, only slight changes were
observed in the number of total bacterial 16S rRNA and recA
mRNA transcripts in Geobacteraceae (Fig. 1 and Table 1).
These results suggest that the addition of ammonium to ace-
tate-amended Fe(III)-reducing sediments resulted in a de-
crease in nifD expression by the Geobacteraceae. The addition
of ammonium to the control sediments that had not been
amended with acetate had little detectable impact on the low
levels of nifD and recA in Geobacteraceae, reflecting the low
activity of Geobacteraceae in these sediments (Fig. 1 and Table
Effect of ammonium on microbial activity. The apparent
lack of stimulation of Fe(III) reduction following the addition
of ammonium in the above studies was further investigated in
studies in which ammonium was added at the start of the
incubation when Fe(III) reduction was first stimulated with the
addition of acetate. The addition of a range of ammonium
concentrations did not have a significant effect on Fe(III) re-
duction (Fig. 2A). This was despite the fact that slot blot
hybridization analysis of sediments collected on day 24, when
ca. 50% of the HCl-extractable iron had been reduced, indicat-
ed that the Geobacteraceae in the sediment were highly ex-
pressing nifD in the sediments not amended with ammonium
but that the expression of nifD was repressed in Geobacter-
aceae in the ammonium-amended sediments (Fig. 2B). In a
similar manner, the addition of 100 or 250 ?M ammonium had
no effect on the anaerobic degradation of toluene in the Fe(III)-
reducing, petroleum-contaminated sediments (Fig. 3).
These results suggest that even though the Geobacteraceae
were limited for fixed nitrogen in both the acetate-amended
sediments and the petroleum-contaminated sediments, this
was not the ultimate factor limiting the rate of Fe(III) reduc-
tion and toluene degradation. For example, the activity of
Fe(III)-reducing microorganisms may be severely limited by
their ability to access insoluble Fe(III) oxides (38, 41, 42, 48).
The addition of chelators that solubilize Fe(III) (41, 42) or
electron shuttles that alleviate the need for direct contact be-
tween Fe(III)-reducing organisms and Fe(III) oxides (38, 40)
can greatly accelerate both Fe(III) reduction and the degrada-
tion of aromatic hydrocarbons in aquifer sediments. Thus, the
need for Geobacteraceae to continually establish direct contact
with fresh Fe(III) oxides over time (12, 47) can kinetically
constrain rates of Fe(III) reduction and may be an overriding
factor limiting the rate of Fe(III) reduction and contaminant
oxidation coupled to Fe(III) reduction.
Effect of added ammonium on community structure. After
24 days of incubation in the presence of added ammonium, the
levels of recA transcripts in Geobacteraceae appeared to be
lower than in sediments not amended with ammonium (Fig.
2B). Given the fact that the pure-culture and sediment studies
with Geobacteraceae summarized above suggested that the ab-
sence of ammonium did not substantially influence recA ex-
pression, these results suggested that long-term exposure to
added ammonium might have resulted in a decrease in
Geobacteraceae in the sediments. Analysis of 16S RNA se-
quences in the RNA that was extracted on day 24 indicated
that Geobacteraceae accounted for ca. 70% of the 16S rRNA
sequences recovered from sediments amended with acetate,
but not those amended with ammonium. In sediments amend-
ed with acetate and ammonium, Geobacteraceae accounted for
only ca. 40% of the sequences (Fig. 4). This decline in the
relative number of sequences of Geobacteraceae was accompa-
nied by an increase in the percentage of sequences most closely
related to organisms in the ?-Proteobacteria, most notably Azo-
arcus species (Fig. 4). The relative number of Azoarcus se-
quences increased from ca. 15% in sediments not amended
with ammonium to ca. 35% of the sequences in clone libraries
from the ammonium-amended sediments.
This enrichment of microorganisms with 16S rRNA gene
sequences closely related to known Azoarcus species was not
expected. Azoarcus are capable of growing anaerobically with
nitrate as the electron acceptor (2, 22, 52, 53, 58, 60, 61, 71),
but previous studies have shown that nitrate is not available
(i.e., there is ?1 ?M) in these sediments (3). To our knowl-
edge, Azoarcus species are not known to use Fe(III) as an
electron acceptor. The increases in Azoarcus species were sim-
ilar whether 100 or 250 ?M ammonium was added, suggesting
FIG. 2. (A) Fe(II) production in acetate-amended sediments sup-
plemented with various concentrations of ammonium. The results are
means of triplicate incubations for each treatment. (B) Slot blot hy-
bridization of the RT-PCR product for nifD, recA, and bacterial 16S
rRNA in Geobacteraceae in sediments collected on day 24, when ca.
50% of the HCl-extractable iron had been reduced.
VOL. 70, 2004IN SITU EXPRESSION OF nifD IN GEOBACTERACEAE 7255
that the ammonium served as a nutrient rather than as a source
of nitrate originating from some form of anaerobic ammonium
oxidation. Further evaluation of this unexpected change in
community structure was beyond the scope of these studies,
but these results emphasize the fact that manipulating envi-
ronmental conditions in subsurface sediments may not only
alter the metabolism of the microorganisms that were predom-
inant prior to the environmental manipulation, but also bring
about significant changes in the structure of the microbial
community. Thus, it is important to study the effect of nutrient
amendments on natural communities rather than single organ-
isms inoculated into sediments as has previously been done
(10, 43, 65). There is also a clear need to properly design
primers and/or probes if the goal is to monitor the physiolog-
ical response of a specific population.
Phylogenetic analysis of 16S, recA, and nifD cDNA clone
libraries. Analysis of cDNA clone libraries constructed from
mRNA extracted from sediments indicated that the nifD and
recA primer sets used in this study preferentially amplified nifD
and recA mRNA being expressed by species of Geobacteraceae.
For example, all of the nifD clones examined showed at least
83.9% amino acid sequence identity to the G. sulfurreducens
nifD sequence (105 amino acids considered). In addition, all of
the nifD amino acid sequences were between 85.6 and 100%
identical to each other. Analysis of recA cDNA clones showed
that all of the recA amino acid sequences were at least 80.1%
identical to the G. sulfurreducens recA sequence (120 amino
FIG. 3. [U-14C]toluene mineralization in Fe(III)-reducing petro-
leum-contaminated subsurface sediments amended with various con-
centration of ammonium. The results are means of results from trip-
licate incubations for each treatment. Error bars represent one
FIG. 4. Relative proportions of sequences in cDNA clone libraries of 16S rRNA gene sequences assembled from acetate-amended sediments
collected on day 24, when ca. 50% of the HCl-extractable iron had been reduced.
7256HOLMES ET AL.APPL. ENVIRON. MICROBIOL.
acids considered). The sequence identity of the recA gene
fragments also ranged from 76.5 to 100%, and the majority
(80.1%) of recA amino acid sequences were ca. 98% identical.
The 16S rRNA sequences expressed in Geobacteraceae in
the acetate-amended background sediments or the petroleum-
contaminated sediments all clustered within the freshwater
Geobacter clade of the family (Fig. 5). The sequences were 81.9
to 92.5% similar to the 16S rRNA sequence of “Geobacter
bemidjiensis,” a pure-culture isolate from this site, sharing 88.4
to 100% sequence identity. All of the nifD and recA sequences
from Geobacteraceae also clustered with freshwater Geobacter
species (Fig. 5). These results further suggest that, as has been
noted elsewhere (E. Shelobolina, H. Vroinis, and D. R. Lovley,
manuscript in preparation), the Geobacteraceae that predom-
FIG. 5. Phylogenetic analysis of 16S rRNA, nifD mRNA, and recA mRNA sequences amplified from Geobacteraceae in acetate-amended
Fe(III)-reducing background sediments and the Fe(III)-reduction zone of a petroleum-contaminated aquifer. (A)16S rRNA nucleotide sequence
comparisons made by the Jukes-Cantor algorithm with Thermotoga maritima used as the outgroup; (B) nifD amino acid sequence comparisons
made by maximum parsimony analysis with Chlorobium tepidum used as the outgroup; (C) recA amino acid sequence comparisons made by
maximum parsimony analysis with C. tepidum used as the outgroup.
VOL. 70, 2004IN SITU EXPRESSION OF nifD IN GEOBACTERACEAE 7257
inate in subsurface environments are related to those that can
be recovered in pure culture.
Implications. The results suggest that it is possible to assess
the in situ metabolic state of Geobacteraceae in the subsurface
by quantifying levels of mRNA specific to Geobacteraceae in
the sediments. nifD expression in Geobacteraceae was high in
sediments amended only with acetate, whereas nifD mRNA
levels dropped significantly when ammonium was also added.
In situ gene expression analyses also indicated that nifD was
being expressed in the Fe(III) reduction zone of the petro-
leum-contaminated aquifer. The fact that similar results were
obtained from analysis of mRNA with PCR-based and slot blot
hybridization techniques provided added assurance of the re-
liability of these results. Most importantly, rather than studying
mRNA extracted from sediments inoculated with pure cul-
tures, these studies focused on gene expression patterns in
Geobacteraceae within mixed natural microbial communities in
The finding that supplying ammonium was not sufficient to
stimulate Fe(III) reduction or toluene degradation emphasizes
the fact that a multitude of factors are likely to be important in
controlling the metabolism and growth of Geobacteraceae in
the subsurface. Thus, a more global analysis of the in situ
metabolic state of the predominant Geobacteraceae, encom-
passing potential stress responses and overall nutrient status,
rather than focusing on a single nutrient, is desirable. The
ability to effectively extract the high-quality mRNA described
here and the increasing availability of genome sequence data
from Geobacteraceae that predominate in environments of in-
terest are providing the tools that will be required for such a
global analysis of gene expression in Geobacteraceae (35, 36).
We thank Tod Anderson for collecting the sediment samples used in
this study. We also thank the DOE JGI for providing us with prelim-
inary sequence data from G. metallireducens, D. acetoxidans, P. carbin-
olicus, P. propionicus, and Geothrix fermentans.
This research was supported by the Office of Science (BER), U.S.
Department of Energy, with funds from the Natural and Accelerated
Bioremediation Research (NABIR) program (grant DE-FG02-
97ER62475), and the Genomes to Life Program (cooperative agree-
ment no. DE-FC02-02ER63446).
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