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Applied and Environmental Microbiology, May 2000, p. 1980-1986, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Horizontal Heterogeneity of Denitrifying Bacterial
Communities in Marine Sediments by Terminal Restriction Fragment
Length Polymorphism Analysis
David J.
Scala1 and
Lee J.
Kerkhof2,*
Department of Chemical and Biochemical
Engineering, Rutgers University, Piscataway, New Jersey
08854,1 and Institute of Marine and
Coastal Sciences, Cook College, Rutgers University, New Brunswick,
New Jersey 08901-85212
Received 9 December 1999/Accepted 15 February 2000
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ABSTRACT |
Although it is widely believed that horizontal patchiness exists in
microbial sediment communities, determining the extent of variability
or the particular members of the bacterial community which account for
the observed differences among sites at various scales has not been
routinely demonstrated. In this study, horizontal heterogeneity was
examined in time and space for denitrifying bacteria in continental
shelf sediments off Tuckerton, N.J., at the Rutgers University
Long-Term Ecosystem Observatory (LEO-15). Characterization of the
denitrifying community was done using PCR amplification of the nitrous
oxide reductase (nosZ) gene combined with terminal
restriction fragment length polymorphism analysis. Spatial scales from
centimeters to kilometers were examined, while temporal variation was
assayed over the course of 1995 to 1996. Sorenson's indices (pairwise
similarity values) were calculated to permit comparison between
samples. The similarities of benthic denitrifiers ranged from 0.80 to
0.85 for centimeter scale comparisons, from 0.52 to 0.79 for meter
level comparisons, and from 0.23 to 0.53 for kilometer scale
comparisons. Sorenson's indices for temporal comparisons varied from
0.12 to 0.74. A cluster analysis of the similarity values indicated
that the composition of the denitrifier assemblages varied most
significantly at the kilometer scale and between seasons at individual
stations. Specific nosZ genes were identified which varied
at centimeter, meter, or kilometer scales and may be associated with
variability in meio- or macrofaunal abundance (centimeter scale),
bottom topography (meter scale), or sediment characteristics (kilometer scale).
| |
INTRODUCTION |
Measurements of spatial and temporal
heterogeneity in microbial communities are of considerable interest to
microbial ecologists. Knowledge of the spatial patchiness of bacteria
is important for determining the appropriate sampling scales and for
addressing basic ecological questions such as which factors may control
microbial communities (e.g., top-down control by predators). For
instance, marine microbial abundance in seawater is fairly uniform at
ca. 109 cells/liter (2, 7). However, when small
volumes of water are analyzed (<1 ml), high-density clusters can be
revealed (6, 10) and nanoscale patches of bacteria have been
induced by the addition of organic substrates (11). So
routine marine sampling, which generally involves sample sizes greater
than 1 liter, homogenizes bacterial populations, destroying any spatial
patchiness information (6). As another example, several
studies have shown the process of soil denitrification exhibits both
temporal and spatial heterogeneity (8, 15, 16), with one
study reporting that 25 to 85% of denitrification occurs in microsites
comprising <1% of the soil mass (15). Given that the
denitrification process itself exhibits temporal and spatial
patchiness, it seems likely that the microbes responsible for the
process are also nonrandomly distributed.
In this study, we monitored the temporal and spatial heterogeneity of
denitrifying bacteria in a continental shelf environment using the
nitrous oxide reductase (nosZ) gene (18, 19). The study was conducted at a Long-Term Ecosystem Observatory (LEO-15) centered on a sand ridge in 15 m of water offshore from the
Rutgers University Marine Field Station (22). In order to
rapidly characterize the nosZ target genes from the samples,
we used terminal restriction fragment length polymorphism (T-RFLP)
analysis (1). T-RFLP analysis is a powerful tool that has
been used to compare microbial community structure and diversity in a
variety of different laboratory and natural settings (4, 9, 12,
17, 21). Our analysis demonstrated variability in denitrifier
community structure on horizontal scales ranging from centimeters to
kilometers. Additionally, changes at single sites resulting from
seasonal differences were observed. This information is critical for
understanding the microbial dynamics of denitrifying bacteria in the
marine environment and may help elucidate how the oceanic microbial
ecosystem is structured and maintained.
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MATERIALS AND METHODS |
Environmental site.
Continental shelf sediment samples were
collected on 6 November 1995 (11/6/95), 12/5/95, 5/15/96, 6/25/96,
8/9/96, and 10/13/96 from a long-term ecosystem observatory site
(LEO-15) described in detail elsewhere (22). Four stations
(stations 9, 32, C, and C-2) were occupied over this time period at the
LEO-15 site. Latitude and longitude for the LEO-15 stations are as
follows (see Fig. 3): 74°15.73'W, 39°27.68'N (station 9),
74°15.23'W, 39°27.85'N (station C), 74°12.88'W, 39°23.08'N
(station C-2), and 74°14.50'W, 39°29.38'N (station 32).
Some general characteristics of the LEO-15 stations are as follows.
Station 9 contains medium to coarse sand, with a mean sediment grain
size of 1.0 phi unit, and is usually populated by Spisula
solidissima (surfclams). [Phi units are calculated as follows:
= 
log2(S); where S is the grain size in
millimeters.] Furthermore, station 9 consists of approximately 99.5%
sand, with 0.5% silt-clay (20). Station C contains medium
to fine sand, with a mean sediment grain size of 1.8 phi units.
Sediments at stations 32 and C-2 are predominantly very fine sands,
with a mean grain size of 3.7 phi units (5). S. solidissima is rare at these locations; however, polychetes are
found in abundance (20). Carbon and nitrogen analyses were
performed by the Rutgers University Stable Isotope Facility on a
ANCA-GSL elemental analyzer coupled to a Europa Scientific 20/20
isotope mass spectrometer. This analysis indicated that C/N ratios
varied from 6 to 28.7 for the different sediments (Fig.
1).
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FIG. 1.
Graph of carbon-to-nitrogen ratio and bottom water
temperature (in degrees centigrade) during sampling for the various
samples used in this study.
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Sample collection.
For the 11/6/95, 12/5/95, 5/15/96, and
6/25/96 sampling dates, single sediment cores (size: 30-cm length,
10-cm internal diameter) were hand retrieved by scuba divers.
Triplicate subsamples were obtained by collecting the top 2-cm portion
of the sediment cores with a sterile, trimmed 3-ml syringe. For the
8/9/96 and 10/13/96 sampling dates, divers retrieved duplicate
surficial sediment samples (top 0 to 3 cm) by scraping sterile 50-ml
polypropylene centrifuge tubes (Phenix Research Products, Hayward,
Calif.) across the sediment surface. The bottom water temperature at
the time of collection was recorded for most of the samples (Fig. 1).
Despite differences in collection methods, all of the samples used in these studies represent the upper 0 to 3 cm of sediments at the various
stations and were stored frozen at 20°C until analysis.
Fingerprinting by T-RFLP analysis.
Total genomic DNA was
extracted from approximately 100 mg (wet weight) of sediment, as
described previously (18). The Nos661F (5'-CGGCTGGGGGCTGACCAA; labeled at the 5' end with
6-carboxyfluorescein [6-FAM; Perkin-Elmer]) and Nos1773R
(5'-ATRTCGATCARCTGBTCGTT) primers were used to amplify
~1,100 bp of the nosZ gene. The PCR reactions contained 20 ng of template DNA and 200 pmol of primer, and the amplification
conditions were 1 cycle at 94°C for 5 min, followed by 35 cycles of
95°C for 0.5 min, 55°C for 0.5 min, and 72°C for 1.5 min, with a
final extension step at 72°C for 10 min (18). After
amplification, 15 µl of the PCR products was digested with
HinPI restriction enzyme (New England Biolabs) at 37°C for 6 h.
The digested DNA was precipitated with a 0.1 volume of 3 M sodium
acetate and 2.0 volumes 95% ethanol, followed by spinning
at
16,000 ×
g in an Eppendorf microcentrifuge for 15 min.
The
DNA pellet was washed with 70% ethanol, dried, and resuspended
in
a mixture of 12 µl deionized formamide and 0.5 µl of DNA fragment
length internal standard (TAMRA 500; Perkin-Elmer).
Pairwise similarity calculations and cluster analysis.
The
fluorescent moiety on the end of the digested PCR product was detected
using the PE/ABI GeneScan Software which displays the various
T-RFLPs as a series of peaks. The presence or absence of the T-RFLP
peaks allows for two samples to be compared using Sorenson's index:
Cs = 2Nab/(Na + Nb), where Nab is the
number of shared peaks between two samples, and
Na and Nb are the number of peaks for samples A and B, respectively (13, 14).
Phenograms of denitrifier similarity indices were constructed using
unweighted-pair-group mean-average (UPGMA) analysis. All indices and
clusters were calculated using the COMbinatorial Polythetic
Agglomerative Hierarchical clustering package (COMPAH96;
http://www.es.umb.edu/edgwebp.htm).
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RESULTS |
In this study, T-RFLP technology was used to characterize the
target nosZ genes from the samples. In order to accomplish
this, it was necessary to select restriction enzyme(s) capable of
resolving as many nosZ target genes as possible. We found
that HinPI could produce the largest number of diagnostic
terminal restriction fragments (Table 1).
For example, 15 of the 47 known nosZ sequences could be
unambiguously identified, and five TRFLPs contained only 2 nosZ sequences. Only TRFLPs 151, 191, 194, and 248 contained three or more different nosZ genes. Finally, three
nosZ genes were found to not cut with this particular
restriction enzyme and would remain undetectable.
To assess the level of variation inherent in our T-RFLP technique,
duplicate samples from station 9 (12/5/95) and a freshwater marsh were
fingerprinted. These duplicate samples were extracted, amplified, and
digested separately. The fingerprints are found to be nearly identical
(Fig. 2). Analysis of the duplicate
samples indicated a similarity value (Cs) of 0.99 between
duplicate samples from the LEO-15 site and a comparable similarity
value of Cs of 0.95 from the freshwater marsh samples.
These duplicate fingerprints demonstrating high reproducibility in both
banding pattern and peak height suggest that the appearance or
disappearance of T-RFLP peaks is not a methodological artifact.
However, this analysis did not directly test the extraction efficiency
or the potential artifacts from PCR and does not preclude the existence
of nosZ genes (denitrifiers) which may remain undetectable
using current methodologies.
Heterogeneity of nosZ genes on different spatial
scales.
Variability in denitrifier assemblages was measured at
LEO-15 over the three horizontal spatial scales indicated in Fig.
3. Centimeter scale variability in the
nosZ gene was examined between two subsamples from three
cores collected on 11/6/95, 5/15/96, and 6/25/96 at station 32. Meter
scale variability was investigated at station C2 on 10/13/96 using four
surficial sediment samples (top 0 to 3 cm) taken a few meters apart,
and all possible pairwise comparisons were made. The kilometer scale
variability of denitrifying bacterial populations was assessed on two
occasions, on 5/15/96 and 6/25/96, for stations 9, C, and 32.
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FIG. 3.
Schematic showing the LEO-15 sampling site and the three
spatial scales over which denitrifying bacteria were monitored.
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The individual C
s values for the centimeter scale study
were 0.82, 0.80, and 0.85 (Fig.
4). The
meter scale values ranged
between 0.52 and 0.79, exhibiting greater
variability than the
centimeter scale values. Finally, the kilometer
scale comparisons
approach the lower end of the range for
variability at the meter
level, i.e., the similarity values ranged from
0.23 to 0.53. In
addition, similarity values were highest for
comparisons made
between stations 32 and C (C
s = 0.52 to 0.53), while the lowest
values were calculated for comparisons
between station 9 and either
station 32 or station C (e.g.,
C
s(station 9 versus station 32 [5/96]) = 0.23).
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FIG. 4.
Cluster diagram of similarity values for LEO-15 samples.
Similarity levels are indicated above the diagram. Similarity values
were calculated using Sorenson's index, and clustering was done by the
UPGMA method. Stars indicate samples included in the 6/25/96 kilometer
level comparison. Phenograms of denitrifier similarity indices were
constructed using UPGMA analysis. Samples are indicated using the
following code: station identifier (9, 32, C, and C2), sampling date
(month and year), and subsample identifier (A, B, etc.). Therefore,
321195A indicates a subsample of the 11/95 station 32 sample.
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Temporal variability was examined at all LEO-15 stations for 12 months.
The C
s value for all temporal assays ranged between
0.12 and 0.74, which nearly encompasses the range of values seen
in the
spatial study. In particular, the spread in the temporal
values is
twice as large as that seen for the meter or kilometer
scale
comparisons. Station C showed significant variation between
sampling
dates, but there was no systematic pattern of change
between May 1996 (5/96) and 8/96. Station 32 showed a more distinct
pattern, with the
greatest differences occurring between 11/95
and 5/96
(C
s = 0.49), while the 5/96 and 6/96 samples displayed
much greater similarity (C
s = 0.69). The most dramatic
seasonal
difference occurred at station 9, where the similarity between
the 12/95 and 5/96 samples was only 0.12, while between 5/96 and
6/96
the C
s value was 0.74.
Cluster analysis of similarity values.
The COMPAH96 software
program was used to perform cluster analysis of the similarity values
generated in this study. A phenogram of the output of this analysis was
reconstructed using an UPGMA algorithm (Fig. 4). The cluster diagram
depicts the very close grouping of the individual centimeter scale
comparisons. In addition, the meter level samples are grouped together
with samples C2-4, C2-5, and C2-6, forming a tight cluster, and C2-2,
splitting off separately. The kilometer scale samples for the 6/25/96
sampling date are indicated by a star. As Fig. 4 shows, these kilometer level samples are grouped at a very low similarity level (~0.40).
The cluster diagram also illustrates some of the temporal impact on
denitrifying bacteria at the various LEO-15 stations.
In general,
samples tend to cluster according to the time of year.
For example, the
11/6/95 station 32 and 10/13/96 station C samples
cluster together, as
do the station 32 and C samples from 5/15/96
and 6/25/96. The most
dramatic temporal changes are seen between
the late fall and the
5/15/96 samples at stations 9 and
32.
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DISCUSSION |
This study demonstrated that the composition of denitrifier
assemblages at the various LEO-15 stations change both spatially and
temporally, with the greatest dissimilarity occurring over the
kilometer and seasonal scales. Our results are consistent with those of
other reports. For example, phenotypic variation of 6% in
phenanthrene-degrading bacterial populations in intertidal sediments
has been noted over spatial scales of centimeters, while temporal
effects accounted for 21% of the variation (3).
Additionally, a study in the coastal waters of Antarctica on the
variability of Archaeal assemblages (14) also yielded a
summer-winter comparison (Cs = 0.29), very similar
to the fall-late spring results obtained at LEO-15, where
Cs(station 9) = 0.12 and
Cs(station 32) = 0.49.
Our analysis of T-RFLP number (denitrifier diversity) and the
environmental parameters of C/N ratio or bottom water temperature was
inconclusive (0.01 
r2 0.1). This
suggests that it is not a simple matter of a single global regulator
controlling all denitrifier populations at this site but is most likely
a myriad of regulators affecting individual members of the denitrifying
community. Although the variability we describe in denitrifier
communities can provide clues as to when and where sampling should
occur in the coastal ocean, there is another aspect to the data that is
equally important. In particular, the T-RFLP technique can lead
directly to testable hypotheses about the ecology of particular
denitrifiers in the continental shelf sediment environment.
For example, a testable hypothesis would be that some of the
denitrifiers demonstrating variability at the centimeter scale are
associated with abiotic or biotic processes which also vary on the
centimeter scale at the LEO-15 site. Specifically, the denitrifiers
that differed between subsamples of the station 32 samples (e.g.,
terminal restriction fragments [T-RFs] 112, 154, and 184; station 32, 11/95, A [Table 2]) may be explained by the presence or absence of Ampharetid worm tubes at the site. Figure 4
also illustrates the temporal similarity seen in denitrifiers between
the fall and spring samples taken at stations 9 and 32 at LEO-15.
Temperature variations between the sampling dates were approximately
5°C and may be responsible for the variation in denitrifiers at these
sites and times. For instance, the unique denitrifiers present at
station 9 during 12/95 with HinPI T-RFs of 122, 124, 131, or
133 (station 9, 12/95 [Table 3]) may only be found in samples with
lower temperatures.
The analysis of meter scale variation at station C (10/13/96) provides
an interesting example of the possible effects of microsites on the
indigenous denitrifying bacteria. Of the four meter level samples
examined, three clustered at the 0.75 similarity level or higher.
However, C2-2 only grouped with these samples at the 0.52 similarity
level. This could indicate that even though the spacing between all
four samples was roughly identical, sample C2-2 encompassed some unique
feature. The seafloor at station C2 is characterized by sand ridges and
troughs, which vary on the scale of meters (5). It is
possible that the three similar samples were taken across the crest of
a ridge, while the fourth was taken in a trough (or vice versa). The
key point is that while station C is a medium to fine sandy
environment, troughs adjacent to sand ridges are often characterized by
high concentrations of organic "fluff" (5, 20) which
could alter the species composition of the denitrifiers present.
Therefore, a testable hypothesis to explain meter scale variation in
denitrifiers is that denitrifier species vary across a sand ridge, from
trough to crest. For example, denitrifiers with T-RFs of 94, 108, or 206 (columns C2-4 and C2-5 in Table 3)
might only occur on the crest or troughs of sand ridges.
The kilometer level comparisons also produced results that may reveal
the importance of environmental factors on denitrifying bacteria. For
the two sampling dates for which kilometer scale comparisons were made
(5/15/96 and 6/25/96), comparisons between stations C and 32 displayed
much higher similarity values than did comparisons between either
stations 9 and 32 or stations 9 and C (Fig. 4). In addition, Table 3
demonstrates that stations 9 and C both contained unique T-RFs for the
June samples. Diver observations indicated clear benthic community
differences in the three stations for the 5/15/96 sampling date (data
not shown). Worm tubes and an organic fluff layer were present at
station 32, moon snails and megaripple topography were observed at
station 9, and many surfclams were found in the sediments at station C. A testable hypothesis to explain kilometer level variability in denitrifiers is that there are specific suites of denitrifiers associated with the various environments present at LEO-15. For example, denitrifiers with T-RFs of 44 would only be located at station 9.
Finally, Fig. 5 shows a frequency
distribution of the T-RFs generated in this study. It is apparent from
this diagram that one denitrifier was present in all 16 samples
examined and that a dozen were present in 10 or more samples. Several
of these abundant T-RFs align with known nosZ genes (Table
1), while other common T-RFs (e.g., T-RF of 97 bp) are not currently in
the nosZ database. This indicates that there is still much
work to be done in order to characterize the denitrifying bacteria at
LEO-15. The majority of T-RFs are present in low numbers (i.e., 4
samples). This could indicate that there is a core suite of
denitrifiers common to the shelf environment, with ancillary species
appearing and disappearing over space and time. However, T-RFLP
analyses will tend to underestimate the species diversity of a
microbial community since species present in low abundance may be below
the detection limit or may share a common terminal restriction site,
thereby yielding peaks of an identical size.
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FIG. 5.
Frequency plot of the individual T-RFs generated in this
study. Only the 186-bp T-RF appeared in all 16 samples. Sizes are
indicated above those T-RFs appearing in 12 or more samples (75% or
higher). Note that the horizontal scale is not linear due to the
omission of T-RF sizes with zero occurrences.
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In conclusion, denitrifying bacteria exhibit seasonal and spatial
variability at the LEO-15 site. Seasonal changes and kilometer scale
variations seem to have the greatest effect on denitrifying assemblages, while the smallest differences are observed at the centimeter spatial scale. Although significant temporal variability in
denitrifier communities is seen at LEO-15, care must be taken to avoid
spatial confounding. It is clear from the data that non-negligible variations occur in as little as a few centimeters, therefore sampling
over time must occur at the same location. Otherwise, it may be
impossible to fully separate effects of time versus simple variation in
sampling location. For example, the variability between the 5/15/96 and
6/25/96 samples does fall within that observed for meter level
comparisons, so changes in denitrifying bacteria at these stations
cannot unambiguously be ascribed to temporal effects. With care, the
T-RFLP methodology has proven to be a capable tool that can be used to
address fundamental questions in microbial ecology.
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ACKNOWLEDGMENTS |
This work was supported in part from NOAA grant NA46RU0149 to
Sybil P. Seitzinger and L.J.K., a New York Sea Grant/Hudson River
National Estuarine Research Reserve Fellowship to D.J.S., and NSF grant
OCE9872024 to L.J.K.
We thank Rose Petrecca, Robert DeKorsey, Andy Laursen, and the divers
of the Institute of Marine and Coastal Sciences for their assistance in
obtaining sediment samples.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Marine and Coastal Sciences, Cook Campus, Rutgers University, 71 Dudley Rd., New Brunswick, NJ 08901-8521. Phone: (732) 932-6555, x335. Fax:
(732) 932-6520. E-mail: kerkhof{at}ahab.rutgers.edu.
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Applied and Environmental Microbiology, May 2000, p. 1980-1986, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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