Post on 15-May-2023
Denaturing gradient gel electrophoresis (DGGE) approaches to
study the diversity of ammonia-oxidizing bacteria
Mette Haubjerg Nicolaisen, Niels Birger Ramsing *
Department of Microbial Ecology, Institute of Biological Sciences, University of Aarhus,
Ny Munkegade, Building 540, DK-8000 Aarhus C, Denmark
Received 20 September 2001; received in revised form 5 February 2002; accepted 5 February 2002
Abstract
Denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of the ammonia monooxygenase gene (amoA) was
developed and employed to investigate the diversity of ammonia-oxidizing bacteria (AOB) in four different habitats. The results
were compared to DGGE of PCR-amplified partial 16S rDNA sequences made with primers specific for ammonia-oxidizing
bacteria. Potential problems, such as primer degeneracy and multiple gene copies of the amoA gene, were investigated to
evaluate and minimize their possible impact on the outcome of a DGGE analysis. amoA and 16S rDNA amplicons were cloned,
and a number of clones screened by DGGE to determine the abundance of different motility types in the clone library. The
abundance of clones was compared to the relative intensity of bands emerging in the band pattern produced by direct
amplification of the genes from the environmental sample. Selected clones were sequenced to evaluate the specificity of the
respective primers. The 16S rDNA primer pair, reported to be specific for ammonia-oxidizing bacteria (AOB), generated several
sequences that were not related to the known Nitrosospira–Nitrosomonas group and, thus, not likely to be ammonia oxidizers.
However, no false positives were found among the sequences retrieved with the modified amoA primers. Some phylogenetic
information could be deduced from the position of amoA bands in DGGE gels. The Nitrosomonas-like sequences were found
within a denaturant range from 30% to 46%, whereas the Nitrosospira-like sequences migrated to 50% to 60% denaturant. The
majority of retrieved sequences from all four habitats with high ammonia loads were Nitrosomonas-like and only few
Nitrosospira-like sequences were detected. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Denaturing gradient gel electrophoresis; Diversity; Ammonia-oxidizing bacteria
1. Introduction
Knowledge of microbial diversity is essential to
understand the relationship between environmental
parameters and ecosystem function (Chao and Chao,
1997; Torsvik et al., 1996). For some environmentally
important but very slow growing bacteria, such as the
chemolithoautotrophic ammonia-oxidizing bacteria
(AOB), culture-dependent diversity studies can be
difficult and time consuming (Matulewich et al.,
1975), and the results may be biased due to our in-
ability to cultivate common organisms (MacDonald,
1986). Molecular techniques for identification that do
not require cultivation have been useful in studies of
different groups of microorganisms, including AOB.
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0167 -7012 (02 )00026 -X
* Corresponding author. Tel.: +45-8942-3248; fax: +45-8612-
7191.
E-mail address: niels.ramsing@biology.au.dk (N.B. Ramsing).
www.elsevier.com/locate/jmicmeth
Journal of Microbiological Methods 50 (2002) 189–203
One of these techniques is denaturing gradient gel
electrophoresis (DGGE) of PCR-amplified genes,
which can be used to evaluate the diversity of com-
plex microbial systems (Muyzer et al., 1993).
In DGGE, the separation of equal length DNA
fragments is based on sequence-dependent melting
behavior in a polyacrylamide gel containing a con-
centration gradient of increasing denaturant (Muyzer
et al., 1998). DGGE is a powerful technique whereby
the diversity of PCR-amplified genes from a large
number of samples can be compared in one gel to
reveal changes in community structure over time or
space. The DGGE technique has been applied to
investigate the diversity of AOB and other organisms
in many diverse habitats (Bano and Hollibaugh, 2000;
de Bie et al., 2001; Ivanova et al., 2000; Kowalchuk et
al., 1998, 1999, 2000; Luxmy et al., 2000; McCaig et
al., 1999). The method is much less time consuming
than traditional analysis by cloning and sequencing.
Furthermore, the gel provides direct information about
the abundance of each PCR amplicon, without intro-
ducing any additional bias inherent in a traditional
cloning step. On the other hand, the use of DGGE
primers with a GC-clamp and possibly additional PCR
cycles may likewise bias the outcome. AOB play an
important role in the global cycling of nitrogen
(Prosser, 1989), in wastewater treatment (Wagner et
al., 1995), and in many agricultural systems (McCarty,
1999), as they are responsible for the first and
frequently rate-limiting step in the process of nitrifi-
cation. 16S rDNA studies have revealed that the
autotrophic AOB are related to two distinct phyloge-
netic lineages. Most strains belong to a monophyletic
branch of the h-subclass of the Proteobacteria, whichincludes the genera Nitrosomonas and Nitrosospira
(the latter encompassing the former genera Nitrosovi-
brio and Nitrosolobus). However, a few strains belong
to the g-subclass of the Proteobacteria. This lineage
includes only two known species Nitrosococcus
oceanus and N. halophilus (Head et al., 1998; Purk-
hold et al., 2000; Teske et al., 1994). Studies of AOB
diversity using DGGE have until recently been
restricted to 16S rDNA analysis. However, a major
problem with the 16S rDNA approach is the limited
specificity of commonly used primers (Kowalchuk et
al., 1998; Rotthauwe et al., 1997). A subsequent
hybridization step (de Bie et al., 2001; Hastings et
al., 1998; Stephen et al., 1998) is usually required to
confirm the phylogenetic origin of the bands in a
DGGE analysis. A further problem with the 16S rDNA
approach is the high similarity of rDNA sequences,
which may make it impossible to resolve and identify
closely related AOB species.
An alternative approach is to use a translated gene
as a molecular marker (Sinigalliano et al., 1995; Wag-
ner et al., 1998; Wawer et al., 1997). AOB convert
ammonia to nitrite in a two step process, where the first
step is the conversion of ammonia to hydroxylamine
catalyzed by the enzyme ammonia monooxygenase
(Hooper et al., 1997). Rotthauwe et al. (1997) have
developed a primer pair (AmoA-1F and AmoA-2R),
which amplify a 491 bp fragment of the ammonia mo-
nooxygenase subunit A gene (amoA). amoA is present
in all autotrophic AOB and is believed to contain
enough information to make phylogenetic inferences
based on its sequence (Purkhold et al., 2000; Rot-
thauwe et al., 1997). The primers only amplify amoA
genes from AOB belonging to the h-Proteobacteriallineage; however, they were shown to be very specific
for detecting AOB in complex environments, includ-
ing soils and sediment (Rotthauwe et al., 1997).
Recently, Oved et al. (2001) have applied the amoA
amplicons to DGGE analysis in their analysis of AOB
diversity in soil, but without a thorough evaluation of
this new approach. In this study, we describe optimi-
zation and characterization of DGGE of partial amoA
amplicons. We have evaluated potential problems such
as multiple copies of the amoA gene and primer
degeneracy. Finally, we have compared a DGGE ana-
lysis of amoA amplicons from four environmental
samples with clone libraries from the same samples.
A similar analysis of amplicons obtained with the 16S
rDNA primers for AOB made an overall comparison
of the two approaches possible.
2. Materials and methods
2.1. Environmental samples
Samples were collected at four locations. (1) S:
Activated sludge from the aeration tank of Marselis-
borg sewage treatment plant, Arhus, Denmark, where
aeration cycles promote removal of nitrogen com-
pounds by coupled nitrification–denitrification. (2)
T: Air-exposed water-saturated surface sediment from
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203190
an intertidal mudflat in the Tagus Estuary, Portugal.
The mudflat received discharge from Lisbon and the
average annual load of nitrogen to the estuary was 323
Amol m�2 h�1 (130 kg N ha�1 (4 months)�1) (Ottosen
et al., 2001). The tidal amplitude was 1 to 4 m, and the
salinity varied from 5–30xat low tide to 20–33xat high tide. (3) R: Surface soil from rice fields at
the International Rice Research Institute (IRRI), Los
Banos, Philippines. The fields were supplied with
nitrogen fertilizer (130 kg N ha�1 crop cycle�1). (4)
K: Surface sediment from Kysing Fjord, Denmark.
The Fjord is a shallow estuary with virtually no tidal
flow and a stable salinity of 14x. The nitrogen load
from freshwater input to the Fjord is high and keeps
the nitrogen content in the estuary at a constant high
level (Nielsen et al., 1995). All samples were imme-
diately frozen after sampling until DNA extraction.
2.2. Pure cultures
Cultures of N. europaea NCIMB #11850 and N.
multiformis ATCC #25196 were kindly provided by
Kristian Brandt, KVL, Denmark, and used for GC-
clamp evaluation as well as time travel experiments.
Both strains were grown at 30 jC in liquid media to
stationary phase as described by Donaldson and
Henderson (1989).
2.3. DNA extraction
For pure cultures, 20 ml of log phase cultures were
centrifuged for 30 min at 5000 rpm. The pellet was
dissolved in 2 ml of media. Two hundred microliters
was used for DNA extraction with the Fast DNA MH
kit (Bio 101, Vista, CA). For environmental samples,
direct DNA extractions were performed with the
FastDNA spin kit for soil (Bio 101, Vista, CA). In
both procedures, cell lysis was performed by vigorous
shaking in a bead beater at intensity 5.5 for 2� 15 s
(FastPrep DNA extractor, Bio 101, Vista, CA). The
kits were used according to the manufacturer’s
instructions. The quality of the DNA extracts was
evaluated by electrophoresis on a 1% SeaKem GTG
agarose gel (FMC BioProducts, Rockland, ME) that
was subsequently stained with 100 ng/ml SYBRGold
(Molecular Probes, Leiden, NL) for 20 min. The DNA
was visualized by UV transillumination (Gel Doc
2000, BioRad, Hercules, CA). Digital images of the
gels were acquired with a CCD camera controlled by
the software Quantity One (BioRad). This staining
and imaging protocol was used for all subsequent
gels. DNA extracts were stored at � 20 jC. To obtain
suitable amplicons, 10- to 100-fold dilutions of the
extract were used as template for subsequent PCR
reactions.
2.4. PCR-primers
Primers AmoA-1F (forward) and AmoA-2R (re-
verse), targeting a stretch corresponding to positions
332 to 349 and 802 to 822 of the open reading frame of
the ammonia monooxygenase subunit A gene (amoA)
of N. europaea, were developed and tested by Rot-
thauwe et al. (1997). This primer pair targets the amoA
gene from h-Proteobacteria AOB. For DGGE-PCR,
adding a 33 nt GC-clamp to one of the primers sta-
bilizes melting behavior of the amplified PCR frag-
ments (Kowalchuk et al., 1997; Sheffield et al., 1989).
To evaluate the optimal position of the GC-clamp, both
primers were tested with the GC-clamp. Primer
sequences are shown in Table 1. The AmoA-2R primer
has two degeneracies: a K ( =G or T) and a S ( =G or
C) at seven and nine nucleotides from the 5V-end,respectively. Primers with one or no degeneracy were
also constructed, giving a total of seven different
versions of the reverse primers (Table 1).
An alternative AOB primer pair, CTO189f-GC and
CTO654r, described by Kowalchuk et al. (1997) to
amplify a 465 bp fragment of the 16S rDNA gene
from Proteobacteria h-subgroup AOB, was used for
comparison. The length of both the amoA and the
partial 16S rDNA amplicons are less than 500 bp,
which makes them amenable to DGGE analysis
(Myers et al., 1985).
2.5. PCR
For GC-clamp evaluation, 50 Al PCR reactions
were performed with the two different primer pairs
AmoA-1F-Clamp/AmoA-2R and AmoA-1F/AmoA-
2R-Clamp [35.5 Al dH2O, 5 Al buffer (100 nM
Tris–HCl pH 8.8, 750 mM KCl, 15 mM MgCl2); 5
Al dNTP (1.25 mM of each dNTP); 1 Al forward
primer (50 pmol/Al); 1 Al reverse primer (50 pmol/Al);5U Taq DNA polymerase (Amersham Pharmacia
Biotech, Uppsala, Sweden); and 2 Al template]. The
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203 191
PCR thermocycling regime was: 92 jC for 1 min, 35
cycles of 92 jC for 30 s, 57 jC for 30 s, 72 jC for 45
s + 1 s/cycle; the last cycle had a 5-min final extension
(modified from Kowalchuk et al., 1997). The time
travel experiments (Muyzer et al., 1993) were done
with the same setup and PCR conditions as above, but
with the primer pair AmoA-1F-Clamp/AmoA-2R-T
(Table 1). PCR amplicon aliquots were loaded every
half hour to evaluate the progressive migration of
bands in the gel.
All other PCR were performed as semi-nested
PCR. An initial hotstart PCR using primers without
the GC-clamp [7.5 Al HotstartTaq mastermix (Qiagen,
Hilden, Germany), 5.5 Al primer mix [0.3 Al AmoA-
1F primer, 0.3 Al AmoA-2R-TC primer and 4.9 AldH2O], 2 Al template,] was made with a 15-min pre-
incubation at 95 jC added to the program listed above.
The outcome was evaluated by agarose gel electro-
phoresis (see below). Bands of the expected length
from this PCR were cut out and used as templates for a
second PCR that was run with 16–18 cycles in a 50-Alsetup as described for GC-clamp evaluation. The latter
PCR reaction was made with GC-clamp primers for
DGGE analysis or with non-GC-clamp primers for
cloning. Partial 16S rDNA amplicons were obtained
with the CTO-16S rDNA primer pair using the same
nested protocol and PCR conditions as was used for
the amoA amplification.
When checking the clone insert size, a pipet tip was
used to transfer a small amount (less than 1 Al) of cellsfrom growing colonies directly into the PCR tube. The
AmoA-1F-Clamp/AmoA-2R-TC or the CTO 16S
rDNA primer pair was used in a 50-Al setup as de-
scribed above for the GC-clamp evaluation, but with
an initial step of 8 min at 95 jC to soften the cell walls.
All PCR products were evaluated by electropho-
resis on a 2% Nusieve 3:1 agarose gel (FMC Bio-
Products), and subsequently stained, illuminated and
photographed as described above.
2.6. Cloning
PCR amplicons were purified using the QIAquick
PCR purification kit (Qiagen) prior to cloning. The
amplicons were ligated into the pCR II-TOPO vector
(Invitrogen, Groningen, NL) and were further trans-
formed into competent cells using the TOPO TA
cloning kit (Invitrogen), following the manufacturer’s
protocol. White colonies were randomly picked from
each cloned sample, grown overnight, reamplified and
run on DGGE.
2.7. DGGE
DGGE of PCR products generated by the amoA
and CTO-16S rDNA primers were performed with the
D-Gene System (BioRad). Polyacrylamide gels [8%
of a 37:1 acrylamide–bisacrylamide mixture in
0.5�TAE buffer, 0.75 mm thick, 16� 10 cm], with
a gradient of 30–70% denaturant, were made with a
gradient maker (BioRad) according to the manufac-
turer’s guidelines. 100% denaturing acrylamide was
defined as 7 M Urea and 40% formamide by Muyzer
et al. (1993) so that 30% denaturant corresponds to
2.1 M Urea and 12% formamide. A top gel without
denaturant was cast above the denaturing gel before
the polymerization started. Gels were run for 6 h at
200 Vor 15 h at 100 V in 1�TAE buffer at a constant
Table 1
Primers used for PCR
Primer Sequence
AmoA-1F-Clampa 5V-Clamp-GGG GTT
TCT ACT GGT GGT-3VAmoA-1F 5V-GGG GTT TCT ACT
GGT GGT-3VAmoA-2R-Clampa,b 5V-Clamp-CCC CTC KGS
AAA GCC TTC TTC-3VAmoA-2R-KSb 5V-CCC CTC KGS AAA
GCC TTC TTC-3VAmoA-2R-TSb 5V-CCC CTC TGS AAA
GCC TTC TTC-3VAmoA-2R-GSb 5V-CCC CTC GGS AAA
GCC TTC TTC-3VAmoA-2R-TC 5V-CCC CTC TGC AAA
GCC TTC TTC-3VAmoA-2R-TG 5V-CCC CTC TGG AAA
GCC TTC TTC-3VAmoA-2R-GC 5V-CCC CTC GGC AAA
GCC TTC TTC-3VAmoA-2R-GG 5V-CCC CTC GGG AAA
GCC TTC TTC-3VCTO189f-Clampa,b 5V-Clamp-GGA GRA AAG
YAG GGG ATC G-3VCTO654rb 5V-CTA GCY TTG TAG
TTT CAA ACG C-3Va The GC-clamp added for DGGE-PCR: 5V-CGCCGCGCGGC-
GGGCGGGGCGGGGGC.b Degeneracies are shown in bold.
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203192
temperature of 60 jC. The gels were stained for 20
min in 150 ml 1�TAE buffer containing 100 ng/ml
SYBR Gold (Molecular Probes). Visualization and di-
gital photography was performed as described above
for agarose gels.
The DGGE-ladder consist of the following ampli-
cons (read from low denaturant concentration to high
denaturant concentration): amoA N. europaea, amoA
S clone #14 and #31, amoA T clone #27, CTO-16S
rDNA K clone #29, amoA N. multiformis (producing
four bands; two weak and two strong bands). For all
pictures, the uppermost visible band is N. europaea at
approximately 35% denaturant and the lowest N.
multiformis at about 55% denaturant.
2.8. Sequencing of DGGE bands
DGGE bands were excised using a sterile pipet tip,
transferred to 100 Al DNA buffer and frozen at � 20
jC until reamplification. Reamplification was done as
described for excised agarose bands. The reamplifica-
tion product was evaluated by DGGE to verify purity
and correct mobility of the reamplified product com-
pared to the environmental sample. Excised and ream-
plified bands, which in the DGGE rerun gave one band
at the expected position, were chosen for sequencing.
The PCR product was purified by the QIAquick
PCR purification kit (Qiagen) according to the man-
ufacturer’s instructions. Sequences were obtained
using Cy-5-labelled PCR primers and the Thermose-
quenase fluorescent cycle sequencing kit (Amersham
Pharmacia Biotech) according to manufacturer’s
guidelines. The cycle sequencing reactions were run
as 94 jC for 3 min, then 40 cycles of 92 jC for 30 s
and 57 jC for 30 s; 5-min final extension. Sequencing
products were subsequently loaded on an acrylamide
gel (Reprogelk Long Read, Amersham Pharmacia
Biotech) and run for 15 h at constant temperature (55
jC) and high voltage using an Alfexpressk DNA se-
quencer (Amersham Pharmacia Biotech).
2.9. Sequencing of clones
PCR amplicons of clone inserts were evaluated
with DGGE. Representative clones were picked for
plasmid isolation (Qiagen). The sequencing was done
as described above, but using Cy-5-labelled M13 F or
M13 R vector primers for the sequence reactions
(Invitrogen). Sequences were deposited at Genbank
with the following accession numbers: amoA partial
sequences: K_1: AF489630; K_2: AF489631; K_3:
AF489632; K_4: AF489633; K_5: AF489634; K_6:
AF489635; K_7: AF489636; K_8: AF489637; K_9:
AF489638; K_10: AF489639; K_11: AF489640;
K_12: AF489641; K_13: AF489642; K_14:
AF489643; T_1: AF489644; T_2: AF489645; T_3:
AF489646; T_4: AF489647; T_5: AF489648; T_6:
AF489649; T_7: AF489650; T_8: AF489651; T_9:
AF489652; T_10: AF489653; T_11: AF489654;
T_12: AF489655; T_13: AF489656; T_14:
AF489657; T_15: AF489658; S_1: AF489659; S_2:
AF489660; S_3: AF489661; S_4: AF489662; S_5:
AF489663; S_6: AF489664; S_7: AF489665; S_8:
AF48966; S_9: AF489667; S_10: AF489668; S_11:
AF489669; S_12: AF489670; S_13: AF489671;
S_14: AF489672; S_15: AF489673; R_1:
AF489674; R_2: AF489675; R_3: AF489676; R_4:
AF489677; R_5: AF489678; R_6: AF489679; R_7:
AF489680 16S rDNA partial sequences:K_1:
AF489681; K_2: AF489682; K_3: AF489683; K_4:
AF489684; K_5: AF489685; K_6: AF489686; K_7:
AF489687; K_8: AF489688; K_9: AF489689; R_1:
AF489690; R_2: AF489691; R_3: AF489692; R_4:
AF489693; R_5: AF489694.
2.10. Phylogenetic analysis
amoA sequences were manually aligned in SeqPup
v. 0.6 (http://www.iubio.bio.indiana.edu/soft/molbio/
seqpup/java/seqpup-doc.html) against a pure culture
sequence database kindly provided by Ulrike Purk-
hold, TU, Munich. 16S rDNA sequences were aligned
using the general small-subunit alignment from the
Ribosomal Database Project v. 7.1 (http://www.rdp.
cme.msu.edu/html/analyses.html). Aligned amoA
nucleotide sequences were translated to amino acid
sequences by SeqPup. Only nucleotide or amino acid
positions that were unambiguously aligned were used
in the subsequent phylogenetic analysis. Phylogenetic
analysis was performed by the computer program
PAUP v. 4.0 (Sinauer Associates, Sunderland, MA)
using default settings for the various algorithms. Phy-
logenetic relationships were inferred by distance
matrix, maximum likelihood and maximum parsimony
algorithms, whereas the amino acid sequences were
only evaluated by distance matrix and parsimony
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203 193
algorithms. A bootstrap analysis with 100 resamplings
was made for all datasets to evaluate the stability of the
phylogeny derived from distance matrices.
3. Results
3.1. Pure culture studies
Preliminary investigations revealed that separation
of amoA fragments from different organisms by
DGGE was possible. With the GC-clamp affixed to
the forward primer, amplicons of pure cultures pro-
duced a clear and sharp band pattern after DGGE,
whereas a smear of bands was found when the GC-
clamp was affixed to the reverse primer (data not
shown). The GC-clamp was, therefore, affixed to the
AmoA-1F primer in all following PCR amplifications
of amoA for DGGE. A time travel experiment
(Muyzer et al., 1993), where identical samples were
loaded at half-hour intervals on a DGGE gel, was used
to determine the optimal runtime, which was found to
be 6 h at 200 V (data not shown). DGGE of the pure
culture amplicons revealed four bands for N. multi-
formis (Fig. 1). Excision and reamplification of bands
1 and 2 gave a single band at their respective positions
on a DGGE rerun. However, reamplification of either
of the two uppermost bands, bands 3 and 4, gave rise
to all four bands with similar intensities as in the
original band pattern (Fig. 1). Bands 3 and 4 appeared
to be heteroduplexes of the two lower bands, bands 1
and 2, which was confirmed when a mixture of bands
1 and 2 likewise reproduced the original four-band
pattern. This indicates that N. multiformis has at least
two copies of the amoA gene and that, though similar,
there are differences in the gene sequence of these
copies.
N. europaea only produced one band after the
DGGE (Fig. 1), suggesting that the organism only
carry one copy of the amoA gene or possibly multiple
copies with identical mobility.
Sequencing of the two lower bands from N. multi-
formis and the one band from N. europaea confirmed
the interpretation. The sequence obtained from N.
europaea was identical to the sequence published by
Hommes et al. (1998). The N. multiformis sequences
differed at two base positions; yet both sequences
were virtually identical to the sequence published by
Rotthauwe et al. (1995), with only 3 and 4 nt differ-
ences, respectively.
3.2. Optimization of PCR
Direct use of DGGE primers to amplify environ-
mental DNA, often caused unspecific priming (data
not shown). It was especially pronounced when only
small numbers of AOB were expected in the sample
(e.g. soil from rice paddies without fertilizer amend-
Fig. 1. DGGE of amoA amplicons from Nitrosomonas europaea and
Nitrosospira multiformis. The N. europaea amplicon only contain
one band which was sequenced and found to be identical to the
sequence published by (Hommes et al., 1998). N. multiformis gene-
rate four bands. Bands 1 and 2 were sequenced and found to be two
different copies of the amoA gene. Bands 3 and 4 of N. multiformis
are heteroduplexes of bands 1 and 2 since a reamplification of ex-
cised band 3 or of band 4 recreate the complete band pattern of four
bands.
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203194
ment). However, changing the PCR protocol to a
semi-nested strategy, comprised of an initial PCR
without the GC-clamp using a hotstart polymerase
setup, followed by a second PCR using the GC-clamp
primers and a standard Taq polymerase, solved the
problem. All amplifications of the amoA gene using
this protocol produced a single band of the expected
length on agarose gels and the product was amendable
to DGGE analysis.
3.3. AmoA-2R primer degeneracy
Fig. 2 shows PCR amplicons of amoA genes in a
rice soil sample, R, made using seven different ver-
sions of the AmoA reverse primer (see Table 1 for
primer sequences). The original reverse primer,
AmoA-2R, proposed by Rotthauwe et al. (1997), con-
tains two base degeneracies: a mixture of G and T at
position 7 and a mixture of G and C at position 9. This
primer gave a complex picture with many indistinct
bands and high background intensity, presumably due
to heteroduplex formation. However, the nondegener-
ate primers gave a much clearer band pattern produc-
ing only four bands each. The patterns obtained from
the four nondegenerate primers each consisted of two
distinct bands and a double band. The AmoA-2R-TC
and AmoA-2R-GG, which were believed to be the
most different of the four nondegenerate primers (see
discussion), were evaluated more closely. The two
distinct bands from each primer amplicon were
excised from the DGGE gel and sequenced. The
sequences of the upper bands with either primer were
found to be identical, except for the reverse priming
site. The same was found for the lower band with
either primer. The observed differences in mobility by
DGGE between the two-band patterns were, thus,
ascribed to the primer sequence difference. Hence,
the primers seem to amplify the same target sequences
and the distinct differences in the banding patterns
produced by the nondegenerate primers simply arise
because of differences in the primer sequence. The
apparent complexity of the DGGE pattern produced
by the original primer set AmoA-1F/AmoA-2R is,
thus, a side effect of using a degenerate primer and
does not reflect the original diversity. We, therefore,
chose of the AmoA-2R-TC primer as the reverse
primer in all further studies.
3.4. DGGE
Primer specificity and PCR reproducibility is essen-
tial when working with the PCR-DGGE approach. The
DGGE band pattern of environmental samples showed
a high degree of reproducibility both between PCR
amplifications, when the same DNA extract was am-
plified multiple times, and between DGGE gels, when
the same PCR product was loaded several times. How-
ever, a few samples were encountered in which a direct
PCR protocol with fewer cycles was required to obtain
a reproducible DGGE band pattern.
DGGE was run on both amoA and 16S rDNA
amplicons from the four environments to reveal the
differences in band pattern (Fig. 3). The band pattern
for amoA amplicons were generally more complex
than the band pattern obtained by the 16S rDNA am-
plicons. A notable exception was the R sample. To
evaluate the specificity of the amoA primer pair and
compare it to the CTO-16S rDNA approach, we cloned
and sequenced both types of amplicons from different
environments. Amplified amoA fragments from four
Fig. 2. DGGE of sample R amplified with seven different reverse
primers. The same template is amplified with all primerpairs The
complex pattern generated by degenerate primers is a simple sum of
the band patterns generated by the respective nondegenerate
primers. Subsequent sequencing of bands produced by the two
nondegenerate primers AmoA-2R-GG and AmoA-2R-TC thus gave
identical sequences except for the primer sequences themselves.
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203 195
locations, S, T, R and K and 16S rDNA fragments of
AOB from two locations R and K were investigated.
Randomly picked clones from each site were amplified
and the mobility on a DGGE gel was determined. The
most intense bands from the DGGE analysis of envi-
ronmental samples were also the bands most fre-
quently encountered amongst the examined clones
(Fig. 4). This indicated a semiquantitative performance
of the cloning step, which is supported by the fact that
only a few weak bands present in the environmental
sample were not retrieved among the clones. The slight
discrepancy is likely to be a statistical effect of picking
too few clones. However, one prominent DGGE band
from the sewage sample and another prominent DGGE
band from the Tagus Estuary were never found among
the examined clones. Reamplification and rerun of
these bands did not reproduce their position and they
were interpreted as PCR artifacts such as single
stranded DNA or heteroduplexes. Some of the cloned
bands were not directly visible in the DGGE pattern of
the environmental sample. Possible explanations are
discussed below.
3.5. Phylogeny
The overall branching order for the phylogenetic
trees shown in Figs. 5 and 6 was supported by all the
different algorithms used (i.e. maximum parsimony,
distance matrix and maximum likelihood). The amoA
amino acid-based tree and the amoA nucleotide-based
tree revealed similar branching patterns (Fig. 5a and
b). Even though amino acid-based trees are probably
less biased by multiple substitutions, fine scale reso-
lution of the very closely related amoA sequences is
often better resolved by the nucleotide-based trees
(Rotthauwe et al., 1997).
3.5.1. Activated sludge and Tagus Estuary amoA
sequences
As seen in Fig. 5a, the major part of the retrieved S
and T clones belonged to two distinct phylogenetic
lineages. Group I were related to the N. europaea/N.
eutropha cluster 7 (according to Kowalchuk et al.,
2000) and group II were related to N. oligotropha
cluster 6a.
Fig. 3. DGGE of amplicons from a waste water treatment plant, S;
an estuary, K; a tidal mudflat, T; and rice paddy soil, R after
amplification with the amoA primers and the CTO-16S rDNA
primers, respectively. amoA amplicons always gave clear bandpat-
terns with several distinct bands, whereas the CTO amplicons often
gave a smeary pattern with no distinct bands (e.g. sample S).
However, the number of bands cannot be used as a direct indication
of diversity due to the multiple copies of amoA.
Fig. 4. Relative clone abundance in library vs. the relative intensity
of the corresponding DGGE band from the environmental amplicon.
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203196
Fig. 5. Phylogenetic trees of amoA sequences based on an uncorrected distance matrix. (a) Phylogenetic tree based on amino acid sequences. (b)
Phylogenetic tree based on nucleotide sequences. Branching patterns that were not supported by bootstrap values above 50% have been
collapsed to multifurcations. Closely related sequences originating from the same sample are represented by triangles to give a better overview.
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203 197
3.5.2. Kysing Fjord amoA sequences
Eleven out of 14 sequences from K were related to
N. marina and N. aestuarii (cluster 6). Two sequences
(K1 and K22) were positioned together and possibly
related to the Nitrosospira group, but not closely
related to any pure cultures. One sequence was very
closely related to N. europaea.
3.5.3. Rice field amoA sequences
The retrieved R sequences all fell together in one
group with the closest relative in pure culture being N.
nitrosa.
3.5.4. 16S rDNA sequences
16S rDNA sequences from K fell into two groups,
both closely related to the Nitrosomonas group (Fig.
6). One group was related to N. marina (cluster 6) and
the other one a distinct, but related, sister group. This
was in excellent agreement with the sequences found
for the K sample with the amoA primers, where 11 of
14 sequences were related to N. marina.
The 16S rDNA sequences retrieved with the CTO
primer pair from rice field sample R only revealed one
AOB sequence, which was related to the Nitrosospira
branch of AOB. Four other sequences retrieved with
these primers belonged to a lineage related to Meth-
ylophilus methylotrophus in another branch of the h-Proteobacteria (Fig. 6). It is difficult to compare the
consistency of the phylogeny obtained from R using
the two different genes since there is only a single
sequence from the 16S rDNA that is related to known
AOB sequences.
All sequences retrieved with the amoA primers
were positive, whereas only 20% of the clones re-
Fig. 6. Phylogenetic tree based on 16S rDNA nucleotide sequences using a distance matrix algorithm. Representative sequences from the h-Proteobacterial subclass are included.
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203198
trieved with the CTO primer set fell into the expected
AOB lineages.
3.10. Sequence variation vs. mobility in DGGE
By sequencing several amoA and some CTO-16S
rDNA clones with identical mobility patterns, it was
found that the sequences obtained could differ consid-
erably despite identical mobility. However, in most
cases, bands with equal mobility gave identical se-
quences.
By comparing the phylogenetic distance (percent
base difference) and the mobility differences by
DGGE (percent denaturant difference at band position)
of the amoA sequences, it seems that some information
regarding sequence similarity can be revealed by
DGGE analysis. Fig. 7, based on S and T clones, show
that a large difference between band mobility on the
DGGE gel implies at least a minimal amount of
sequence divergence. On the other hand, nothing can
be said about sequences with very similar mobility.
Furthermore, there was a separation of the Nitro-
somonas- and the Nitrosospira-like sequences on the
DGGE gel, where Nitrosomonas-like sequences had a
melting point in the range f 30% to f 46% and the
Nitrosospira-like sequences had a melting point in the
range f 50% to f 60%.
4. Discussion
The strength of DGGE as a screening method for
diversity is the ability to monitor changes in com-
munity structure in response to changes in environ-
mental parameters over space and time. Studies where
we seek to evaluate the stability of community struc-
ture in relation to perturbations of the environment
may benefit from using the DGGE approach because
of the high resolution and the large amount of samples
that can be analyzed in a short time.
The reproducibility of the initial PCR is critical for
the reliability of the subsequent DGGE analysis and
the PCR protocol should, therefore, be optimized for
the environmental system investigated. Detailed
knowledge of primer specificity is also essential for
proper interpretation of DGGE analysis results. The
methane monooxygenase (pmmo) gene of methano-
trophic bacteria from the g-subclass of Proteobacteria
and the amoA gene of g-subclass AOB are more
closely related than the amoA gene from the g-sub-
class is to the h-subclass amoA gene (Holmes et al.,
1995; Klotz and Norton, 1998; Rotthauwe et al.,
1997). It is, therefore, not possible to target the g-
subclass amoA, together with the h-subclass amoA,
without targeting the pmmo from the methane oxidiz-
ing bacteria at the same time. The amoA primer pair
and the 16S rDNA CTO primers, therefore, only
target the h-subclass AOB. However, presently, onlytwo AOB species belonging to the g-subclass of
Proteobacteria have been described and both of these
are from marine environments. The vast majority of
the known autotrophic AOB still belong to the h-subclass. Finally, it should be emphasized that the
heterotrophic AOB are not targeted by the amoA-
primer set, nor with the 16S rDNA primers (Kowal-
chuk et al., 1997; Rotthauwe et al., 1997). It is, thus,
likely that the amoA-based approach underestimates
the functional diversity.
In the present study, the choice of reverse primer
AmoA-2R-TC was based on both experimental and
theoretical considerations. The original reverse primer
Fig. 7. Correlation between amoA sequence similarity and band
position in a DGGE gel. For each pair of sequences, the percent
sequence divergence was plotted against the difference in band
mobility measured as percent denaturant. Bands with similar DGGE
mobility could have very different sequences. However, bands with
very dissimilar mobility in the DGGE gel (e.g. > 17% denaturant)
were also always shown to have very different nucleotide sequences
(e.g. < 80% amoA sequence similarity).
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203 199
sequence described by (Rotthauwe et al., 1997) had
two ambiguous positions (Table 1). As the DGGE
analysis is able to discriminate between sequences that
only differ at a single base position (Myers et al.,
1987), degenerate primer sequences can result in addi-
tional bands, or produce a smear of bands in a DGGE
analysis, even in environmental samples with low
diversity. This was found to be the case for the original
AmoA-2R primer as shown in Fig. 2. Similar obser-
vations have been made by Kowalchuk et al. (1997)
when using the CTO primers. Degenerate primers can
potentially amplify a wider range of sequences since it,
in theory, is able to anneal to more targets. However,
investigations by Bjerrum et al. (submitted for pub-
lication) indicate that the degeneracies in the AmoA-
2R reverse primer sequence are not critical for efficient
amplification of different target sequences. They
showed using competitive PCR that amplification
efficiency for templates with each of the four possible
priming sites did not differ significantly when using
the nondegenerate AmoA-2R-TC primer. Conse-
quently, we find that the primer pair AmoA-1F and
AmoA-2R-TC is a reasonable choice for PCR-DGGE
analysis of environmental amoA sequences.
The amoA gene is present in three copies in Nitro-
sospira sp. NpAV (Norton et al., 1996) and two copies
in N. europaea (McTavish et al., 1993). The copies of
the amoA gene is supposed to have arisen from gene
duplication and not by horizontal gene transfer, as
there is a higher similarity between different gene
copies in each investigated strain than between genes
from different strains (Klotz and Norton, 1998; Norton
et al., 1996). A hypothesis is that the high similarity
between gene copies is maintained through homolo-
gous recombination since third codon mutations are
almost absent (Norton et al., 1996). However, as single
base mutations may be readily detected by DGGE,
even highly similar gene sequences from a given or-
ganism may give rise to multiple bands in a DGGE gel.
In our study, DGGE analysis of N. multiformis amoA
amplicons produced four bands, whereas N. europaea
amoA amplicons only gave one band (Fig. 1). It is
possible that the strains we were working with had a
different number of gene copies than the ones inves-
tigated by Norton et al. (1996) and McTavish et al.
(1993). Alternatively, some of the gene copies could
be identical. Finally, it is possible that some of the se-
quences could contain several mutations, which in
combination has no net effect on the melting behavior.
Multiple gene copies and heteroduplex formation dur-
ing the last PCR cycles is a large potential problem
when using the complexity of a DGGE band pattern to
evaluate the biodiversity present in any given sample.
We are generally skeptical about attempts to use
DGGE for this purpose as the nature of each band
and the relationship between number of species and
band numbers are rarely known. However, when using
DGGE to evaluate gradual changes in community
structure over space and time following a perturbation,
in which case reproducible changes in band intensities
are more interesting than the band pattern itself, the
DGGE approach seems appropriate. Still, the number
of PCR cycles applied should always be as low as
possible.
Several cloned amoA sequences produced bands
that were not directly visible in DGGE analysis of the
environmental samples. Most of these sequences dif-
fered only by a few bases from sequenced bands that
were found in the same environmental sample. These
clones may also have been present in the environ-
mental PCR amplicon, but of such low abundance that
they were not visible as bands on the DGGE gel.
Alternatively, these sequences could have been pro-
duced by PCR errors in amplification of the more
dominant environmental bands. The general impor-
tance of using a polymerase which perform proof-
reading, e.g. have 3V–5Vexonuclease activity, has beenstressed before (Simpson et al., 1999). Yet, in the
present study, a Taq polymerase without the 3V–5Vexonuclease activity was used, which could have
produced erroneous sequences with slightly different
migration patterns in the DGGE. However, as the re-
producibility of the DGGE pattern of environmental
samples was always evaluated and found to be excel-
lent, random sequence mutations during amplification
was not a likely explanation for our findings.
The CTO-16S rDNA primer-based sequence anal-
ysis of the Kysing Fjord K samples (Fig. 6) revealed
two clusters of sequences related to the Nitrosomonas
group, one of which was more closely related to N.
marina. This result was remarkably similar to what
was found for the retrieved amoA sequences and a ge-
neral agreement between the results of the two
approaches was observed. Additional sequences be-
longing to other AOB lineages were found by the
amoA primers, indicating that a higher diversity was
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203200
revealed by this primer pair. The results from the R
sample was quite different as there was only one
sequence from the 16S rDNA approach branching
among known AOB sequences. The amplification of
R sequences outside the Nitrosomonas–Nitrosospira
clade by the CTO-16S rDNA primers illustrates the
difficulties to be expected when attempting to make a
specific primer pair for selective amplification of an
ultraconservative gene, such as the 16S rDNA gene,
from particular organisms (see also Kowalchuk et al.,
1997; Rotthauwe et al., 1997). The apparently higher
diversity revealed with these primers compared to
amoA primers for the R sample (Fig. 3) was, therefore,
not a correct picture of the actual AOB diversity found
in the sample.
To illuminate a possible discrepancy between the
two approaches, more sequences from this environ-
ment are required. With the amoA primers, even se-
quences belonging to novel phylogenetic lineages are
likely to be from strains capable of doing the process,
assuming that no paralogous sequence is detected.
It has previously been assumed very difficult to
infer phylogenetic relatedness from DGGE band mobi-
lity (Kowalchuk et al., 1997, 2000; Muyzer et al.,
1993). However, distantly related sequences always
displayed very different melting behavior and, con-
sequently, different mobility in a DGGE gel (Fig. 7).
On the other hand, identical mobility did not imply
sequence identity, which has also been confirmed in
other DGGE studies (Kowalchuk et al., 1998). Purk-
hold et al. (2000) have through a thorough analysis of
the phylogeny of AOB pure cultures suggested that
20% difference in amoA nucleic acid sequence could
be a reliable indication of a novel species in environ-
mental samples. For all amoA amplicons whose cor-
responding band position in a DGGE gel differed by
more than 17% denaturant, the nucleic acid sequence
difference was found to be larger than 20% (Fig. 7).
Indications of new species can, therefore, also be
obtained from DGGE gels directly in that amoA bands
separated by more than 17% denaturant are not likely
to be from the same species. However, it should be
stressed that for bands separated by less than 17% de-
naturant, no direct conclusions can be made. Another
important finding of this study was a marked differ-
ence in mobility between clones related to Nitroso-
spira vs. clones related to Nitrosomonas. The mobility
of the former was much larger, producing bands within
a denaturant concentration gradient from 50% to 60%,
whereas cloned Nitrosomonas sequences ceased to
move at much lower concentrations of denaturant
(30–46%). This observation has subsequently been
confirmed in our lab with cloned amoA sequences
from a broad range of habitats and is consistent with
the data obtained and described by Oved et al. (2001).
The amoA primer set has previously been used to
study AOB diversity in environmental samples (Ste-
phen et al., 1999). In that study, only sequences related
to Nitrosospira were detected and it was mentioned
that this could suggest that the amoA primer set was
not compatible with the Nitrosomonas present in the
soil. Most sequences retrieved in this study from the
four habitats were from the Nitrosomonas cluster 6a;
our findings do not support the suggestion that Nitro-
somonas sequences might be less amplifiable by the
amoA primers. Furthermore, sequences retrieved from
a particular habitat were often strongly related; thus,
they formed a cluster in the subsequent phylogenetic
analysis (Figs. 5 and 6). The fact that environmental
sample sequences appear to be genetically related
indicates that environmental factors select for partic-
ular strains of AOB. This ecological impact of the
environment on AOB community structure has pre-
viously been reported by other investigators (e.g.
Kowalchuk et al., 1998; Rotthauwe et al., 1997; Ste-
phen et al., 1998).
We find that the DGGE analysis of amoA ampli-
cons is a reliable screening tool to investigate AOB
diversity in a wide range of environments. Changes in
band pattern and relative intensities are informative
and may well contribute to a quantitative understand-
ing of the system. However, general limitations of
DGGE-based approaches also apply to this setup. The
limitations have been reviewed by Muyzer et al.
(1998).
Even though molecular tools are important in the
investigation of AOB, attempts to obtain pure cul-
tures, especially from novel lineages not yet repre-
sented in culture collections, should remain a priority.
To exploit the molecular information collected in
recent years, it is necessary to allocate more time to
obtain environmental strains in pure culture. Informa-
tion from pure culture studies of environmental strains
will illuminate the relative potential and weaknesses
of the 16S rDNA approach, as well as the amoA
approach, in investigating AOB diversity.
M.H. Nicolaisen, N.B. Ramsing / Journal of Microbiological Methods 50 (2002) 189–203 201
Acknowledgements
This study was supported by RUF, Danish Interna-
tional Development Agency. We want to thank Dorte
Ganzhorn and Pernille Vester Thykier for their ex-
cellent technical assistance, Lotte Bjerrum for her in-
put through many useful discussions, and the anony-
mous reviewers for their helpful comments. We would
also like to thank Prof. Wolfgang Reichardt and co-
workers for inspiring discussions and a hospitable
collaboration.
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