Microbiobgy (1995), 141,2793-2800
Printed in Great Britain
Amplification of 16s ribosomal RNA genes of
autotrophic ammonia-oxidizing bacteria
demonstrates the ubiquity of nitrosospiras in
the environment
William D. Hiorns,’ Richard C. Hastings,l Ian M. Head,’t
Alan J. McCarthy,’ Jon R. Saunders,’ Roger W. Pickup2
and Grahame H. Hall2
Author for correspondence: Alan J. McCarthy. Tel: +44 151 794 4413. Fax: +44 151 794 4401.
e-mail: aj55m @ liv.ac.uk
1
Department of Genetics
and Microbiology,
University of Liverpool,
PO Box 147, Liverpool
L69 3BX, UK
2
Institute of Freshwater
Ecology, Windermere
Laboratories, Far Sawrey,
Ambleside, Cumbria
LA22 OLP, UK
Oligonucleotide sequences selected from the 163 rRNA genes of various
species of ammonia-oxidizing bacteria were evaluated as specific PCR
amplification primers and probes. The spedficities of primer pairs for
eubacterial, Nitmsospira and Nibrosomones rRNA genes were established with
sequence databases, and the primer pairs were used to amplify DNA from
laboratory cultures and environmental samples. Eubacterial rRNA genes
amplified from samples of soil and activated sludge hybridized with an
oligonucleotide probe specific for Nifmsospifa Spp.8 but not with a
Nitmsomonas-specific probe. Lakewater and sediment samples were analysed
using a nested PCR technique in which eubacterial rRNA genes were subjected
to a secondary amplification with Nifmsomnas or Nitrosospira specific
primers. Again, the presence of Nifmsospira DNA, but not Nitmsomonas DNA,
was detected and this was confirmed by hybridization of the amplified DNA
with an internal oligonucleotide probe. Enrichments of lakewater and
sediment samples, incubated for two weeks in the presence of ammonium,
produced nitrite and were found to contain DNA from both Nitmsospira and
Nitmsomonas as determined by nested PCR amplification and probing of 163
rRNA genes. This demonstrates that Nifmsospira spp. are widespread in the
environment. The implications of the detection of Nitmsomnas DNA only
after enrichment culture are discussed.
Keywords: nitrification, ammonia-oxidation, Nitrosomunas, Nitrosospira, 16s ribosomal
RNA
INTRODUCTION
Isolation of autotrophic ammonia-oxidizing bacteria from
the environment is a time-consuming task. Repeated
sequential enrichment in ammonium salts medium is the
method most frequently employed; it results in a collection of isolates that may not be representative of the
diversity that exists in the environment (Belser, 1979). For
example, laboratory enrichment would be expected to
favour those species that exhibit the fastest growth,
irrespective of their activity under natural environmental
tPresent address: Newcastle Research Group in Fossil Fuels and Environmental Geochemistry, Drummond Building, University of Newcastle,
NE1 7RU, UK.
0001-9767 Q 1995 SGM
conditions. This is a current theme in microbial ecology
and there are now a number of reports in which the
application of molecular biological techniques has revealed populations whose compositions are not represented by collections of isolates (e.g. Fuhrman et al.,
1992; Giovannoni et a!., 1990a, b; Liesack & Stackebrandt, 1992 ; Ekendahl et al., 1994). Ammonia-oxidizing
bacteria have been recovered from a wide variety of
environments (Macdonald, 1986 ; Prosser, 1989) and are
largely responsible for driving the process of nitrification
(Hall, 1986). They are classified in the 8- and y-subdivisions of the Proteobacteria (Woese et al., 1984, 1985)
and the 16s rRNA gene sequences of strains representing
this diversity have now been published (Head et al., 1993).
Phylogenetic analysis of those data led to the conclusion
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W. D. HIORNS a n d OTHERS
that the P-subdivision ammonia-oxidizerscould be accommodated in two genera, Nitrosomonas and Nitrosospira
emend. (Head e t a/., 1993).
Studies on the physiology of ammonia-oxidizing bacteria
have been almost exclusively centred on Nitrosomonas
ewopaea (Prosser, 1989), because of the comparative ease
with which this species can be grown in culture. This has
led some workers to equate ammonia oxidation in the
environment with the activity of nitrosomonads (Paerl,
1993; Sprent, 1987). There are a number of reports of the
isolation of nitrosospiras from soil and it has been
suggested that their contribution to ammonia oxidation in
the environment has been underestimated (Belser, 1979;
Prosser, 1989). In this paper, we describe the development
of techniques based on the detection of 16s rRNA genes
(16srDNA) to determine the presence and distribution of
these two groups of nitrifiers in the environment.
METHODS
Bacteria and media. The sources and designations of strains
were as described by Head e t al. (1993). Growth media were also
as reported, with the exception of the medium used for
enrichment of ammonia-oxidizers. This was as described by
Watson & Mandel (1971), except that it contained a reduced
amount of (NH,),SO, (0.76 mM). Samples prepared from
lakewater by tangential-flow filtration (see below) were inoculated into medium (1 %, v/v) and incubated for 15 d at 30 OC.
Nitrite and nitrate levels were monitored daily (Quantofix
dipstick, Macherey-Nagel).
Sampling. Esthwaite Water is a highly productive eutrophic
lake in the English Lake District, UK (Heaney et al., 1992).
Intact sediment cores were taken during June 1993 with a
Jenkin surface sediment sampler, and the surface 0.5 cm of
sediment was removed for analysis using techniques described
by Ohnstad & Jones (1982). Cores were taken from deposits
covered by 3 m ('littoral'; 17-5"C) and 15 m of water ('profundal', maximum depth of lake; anoxic, 10.2 "C). Eighty litres
of water were pumped to the surface from the oxycline (8 m)
and reduced to 1 litre by tangential-flow filtration (0.2 pm pore
size, Pellicon system, Millipore). Moist clay soil samples were
taken from adjoining land that was grazed by sheep. Activated
sludge suspension samples were taken from an aerobic tank at
the Crewe sewage treatment works (North West Water, UK).
Oligonucleotide primers and probes. The accession numbers
of 16s rRNA sequences for autotrophic ammonia-oxidizing
strains are given by Head e t al. (1993). Oligonucleotide primers
and hybridization probes were designed with an alignment of
16s rRNA gene sequences from 13 ammonia-oxidizer strains
and other Proteobacteria. Potentially useful oligomers were
compared to the GenBank database in order to screen for
unwanted cross-hybridization using the FASTA program
(Devereux e t al., 1984) (word size 6). Probe pDr (Edwards e t al.,
1989; Lane e t al., 1985) was used as a positive control for
eubacterial rrn genes. Oligonucleotides were either prepared inhouse, using /3-cyanoethyl-phosphoramidite chemistry on an
Applied Biosystems 381 DNA synthesizer, or purchased from
the King's College School of Medicine and Dentistry, London,
UK.
Nucleic acid extraction and purification. DNA was extracted
from pure strains by a 'freeze-thaw' method (Head etal., 1993).
Lakewater samples prepared by tangential-flow filtration were
processed using the method of Schmidt etal. (1991). Solids were
collected from enrichment cultures and activated sewage sludge
2794
by centrifugation (1600g, 20 min). Soil and sediment samples,
and centrifugally pelleted solids, were processed by the protocol
of Bruce e t al. (1992), a modification of the method of Selenska
& Klingmuller (1991).
Slot-blotting of DNA. The DNA samples were applied to
positively-charged nylon membranes (Hybond-N+,Amersham)
using a Minifold I1 manifold (Schleicher & Schuell). Nucleic
acids were denatured, neutralized and fixed according to the
manufacturer's instructions (' Membrane Transfer and Detection Methods ',Amersham). Whole cells were also applied using
this manifold, and cell lysis subsequently performed within the
assembled manifold. Incubation at room temperature for 30 min
in lysozyme solution [Tris/HCl pH 7-8, 100 mM; NaC1,
150 mM; MgCl,, 5 mM; bovine serum albumin, 1.5 %, w/v;
lysozyme, 40 pg ml-' (Sigma)] was followed by lysis at room
temperature for 15 min in detergent solution (SDS, 2 %, w/v;
EDTA pH 8.0, 1 mM). Manifold slots were washed with
10 x SSPE (NaC1, 1.8 M ; NaH,PO,, 0.1 M ; EDTA, 0.01 M ;
pH 7.7). The manifold was then dismantled and filters air-dried
and fixed at 80 OC for 1 h.
Enzymic amplification and electrophoresis of DNA samples.
Polymerase chain reaction (PCR) amplification (Saiki et al.,
1988) was carried out using ' eubacterial' primers pA and pHr
(Edwards etal., 1989)(94 OC, 1 min; 50 OC, 1 min; 72 OC, 2 min;
26 cycles) in 100 pl volumes [Tris/HCl pH 8.3, 67 mM; KC1,
50 mM; MgCl,, 1.5 mM; Tween 20,0-5 %, v/v; dATP, dCTP,
dGTP and dTTP, 200mM each; primers, 200 nM each;
AmpliTaq DNA polymerase, 1-2 units (Perkin Elmer Cetus)].
For nested PCR, aliquots of PCR amplifications primed with
oligonucleotides pA and pHr were diluted in fresh reaction
mixtures (final dilution, 2.5 x
containing the secondary
pairs of primers. Primers NS-85 and NS-1009r were annealed at
62 OC; NM-75 and RNM-1007r were annealed at 59 OC. The
results presented in Fig. 3 were produced using a Hybaid
Intelligent Heating Block (IHB101). All other amplifications
used ' Hot start' PCR (Chou e t al. 1992) in a Perkin Elmer Cetus
Thermal Cycler 480. DNA samples were electrophoresed
through 1.8 % (w/v) agarose gels (3 parts NuSeive GTG, 1 part
SeaKem ME, FMC Bioproducts) in Tris/acetate/EDTA buffer,
and bands visualized by UV excitation of ethidium bromide
stain. Boehringer Mannheim molecular size markers VI were
used (2176,1766,1230,1033, 653, 517,453,394, 298,234,220
and 154 bp).
Oligonucleotide probing. DNA was transferred and fixed to
Hybond-N+membrane according to the manufacturer's instructions (Amersham). Oligonucleotides were end-labelled with [y32P]ATPusing polynucleotide kinase (Boehringer Mannheim),
and unincorporated nucleotides were removed with Sephadex
G-50 NICK columns (Pharmacia). Using solutions recommended for digoxigenin non-radioactive nucleic acid detection
(Boehringer Mannheim), hybridization was performed overnight at the irreversible melting temperatures ( Ti) of the probes,
and filters washed at room temperature for 3 x 5 min. Fuji RX100 X-ray film was exposed to the filters at -80 OC using HiSpeed-X intensification screens (X-Ograph). Ti was determined
as described by Sambrook e t al. (1989). In this method, a probe
was hybridized to membrane-bound nucleic acids containing a
perfectly complementary site (in these cases, replicate slotblotted PCR products amplified from appropriate ammoniaoxidizing bacteria, using primers pA and pHr). Membranes
were trimmed so that the slot ' footprints ' would be submerged
in 1 ml liquid in glass test-tubes. Prehybridization, overnight
hybridization and washing (5 x 5 min) were performed in a glass
Petri dish at room temperature, and the filters were then
transferred to test-tubes. Labelled oligonucleotides that were
bound to the filters were removed by a series of 5 min washes
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16s rRNA probes for ammonia-oxidizing bacteria
N i trosospira sp
.
N i trosospira sp .
N i trosospira tenuis
N i trosospira sp
.
N i trosospira mu1 t i f o n n i s
N i trosQmaMs europaea
N i trosQmaMs eu tropha
N i trosococcus mobilis
NitrOSmMs
sp.
Nitrosococcus oceanus
N i trosococcus oceanus
RNM-1007
-AATAc
c-128
NV12
NVl
C-141
C-71
C-31
c-91
Nc2
C-56
C-27
C-107
..................
..................
..................
..................
..................
..................
..................
..................
..................
..................
U
N i trosospira sp.
Nitrosospira sp.
N i trosospira tenuis
Nitrososgira sp.
N i trosospira d t i f o r r mis
Nitrosamcrnas
e~vapdea
N i t r o s a a r a M s eutrqaha
N i t r o s o c O C c W mabilis
Nitrosamnas sp.
N i t r a s o c o c c u s OceMus
N i t r a 9 o c o c c u s oceanus
C-128
NV12
NVl
C-141
C-71
C-31
c-91
NCZ
C-56
C-27
C-107
c
U
P
P
GUCtGCU....GG.GGW.. G....
G.CCGCC....GG.GG.U..G....
G.CCGCU....GG.GG. C..G....
G.CCGCU....GG.GG. U..G....
G.CCGCC... .GG.GG.U..G....
.........................
.........................
.U.UC.A....U.G.W...G....
.U.U.C..........A..
.G....
C. .UCCU....GGCG.AG.. G.. ..
CU.UGCU....CGCG.AG.. G....
AAO-258
.
sp .
N i trosospira sp
N i trosospira
N i trosospira tenuis
N i trosospira sp
.
Nitrosospira mu1 tifonnis
Nitrosamanas europaea
N i trosamanas eu tropha
Nitrosococcus mabilis
Nitroscamonas sp.
N i trosococcus oceanus
N i trosococcus oceanus
G i 3 T A ? m K x m ~
c-128
NVl2
NVl
C-141
C-71
C-31
c-91
Nc2
C-56
C-27
C-107
...................
...................
...................
...................
...................
...................
...................
.........c.........
.........c.........
.....G...C.........
.....G...C.........
Ns-85
GGQZ-GcMi-c--
Nitrosospira sp.
N i trosospira sp.
N i trosospira tenuis
N i trosospira sp
Nitrosospira nu1 tifonnis
N i trosamanas europaea
Nitrosamanas eutropha
N i trosococcus mobilis
Nitrosomoms sg.
Nitrosococcus oceanus
N i trosococcus oceanus
.
c-128
NV12
NVl
C-141
C-71
C-31
c-91
Nc2
C-56
C-27
C-107
-....................
-....................
- -....................
- -....................
- -....................
....-..TRXG...
T.C.GtX
.m..
.C..
... .-.
. ...
....T..TPG.A.......-...
[
[
[
not hown ] G.CX3i.T.G
not knawn I C.AGF.GCG
not ]mown I c.AC;T.m
Fig. 1, Alignment of oligonucleotide probes against 165
rRNA nucleotide sequences from ammonia-oxidizing bacteria.
Numbers in the probe names indicate the position of the 5’
nucleotide with respect to the Escherichia co/i sequence.
Oligonucleotide AAO-258 hybridized to rDNA from all
terrestrial ammonia-oxidizers but would not hybridize to the
marine strains C-56 or Nc2 because of single nucleotide
mismatches (see Fig. 2). Probe pD (Edwards et a/., 1989) was a
positive control for eubacterial rrn genes. The sources and
designations of strains and accession numbers of the sequences
were reported by Head et a/. (1993).
(1 ml volume) at sequentially higher temperatures. After each
wash, the filters were rinsed with 1 ml volumes of solution at
room temperature, which were pooled with the corresponding
washes. The amount of radioactivity in each fraction was
measured as Cerenkov radiation, using the 3H channel of a
scintillation counter (Beckman LS1801). Ti was defined as the
temperature at which 50% of the radiation was stripped from
the membranes. To check for the influence of non-specific
binding, observed as time-dependent probe elution, the operation was repeated using a range of wash temperatures with a
different median. For some probes, Ti was also determined for
‘cell blots’, where rRNA was the target molecule.
NM-75
N i trosospira sp
.
N i trosospira sp.
N i trosospira tenuis
N i trosospira sp .
N i trosospira nu1t i f o n n i s
Nitrosamanas europaea
N i trosamanas eutrwha
N i trosococcus mabilis
N i trosamanas sp.
N i trosococcus oceanus
N i trosococcus oceanus
m-m---c-128
NV12
NVl
C-141
C-71
C-31
c-91
Nc2
C-56
C-27
C-107
c-..
c-..
c-..
..-.....Ac....-..AA- c-..
..-.....AC....-..AA- c-..
..-.....AC....-..AA..-.....Ac....-..AA..-.....AC....-..A&-
-- ....................
-....................
..-.....AC.... T....GCA..
notknown
not known
not known
[
[
[
Ns-1009
.
N i trosospira sp
N i trosospira sp
N i trosospira tenuis
.
N i trosospira sp
.
Nitrosospira m 1 t i f o h s
N i trosamaTlBs europaea
Nitrosawmas eutropha
N i trosococcus mobilis
N i trosamanas sp.
N i trosococcus oceanus
N i trosococcus oceanus
P
c-128
NVl2
NVl
C-141
C-71
C-31
c-91
Nc2
C-56
C-27
C-107
..............A.... .
....c.. .............
...............c....
....................
....c... ............
WIAXX;. ...CA.AA.A. .A.
UAAUG....CA.AA. A..A.
UUCUA..
..uAGAwA.. ..
UUA.G.. . .CA.AAAA.. ..
uu......... c.AAc;....
uu.........c.AAG....
RESULTS
Identification and evaluation of oligonucleotide
sequences for DNA amplification and hybridization
The 16s rDNA sequences for the 11 strains of ammoniaoxidizing bacteria (Head et al., 1993) were aligned with
representatives of the major subdivisions of the Proteobacteria and examined for potential diagnostic oligomers.
Thus, probe/primer sequences were chosen initially by
phylogenetic association. Candidate oligomers were then
compared to the GenBank sequence database to screen for
other organisms that may have coincidentally acquired
such motifs. This enabled the selection of six oligonucleotide sequences of varying specificities (Fig. 1).
Oligonucleotide pD is common to eubacteria (Edwards e t
al., 1989; Lane et al., 1985) and was used as a positive
control throughout. Probe AAO-258 was found to be
present in seven of the eleven ammonia-oxidizing strains
examined, being absent only in those strains of marine
origin (Fig. 1). A small number of other GenBank entries
contained the AAO-258 oligomer without mismatches.
Sequences of rDNA from the marine strains contained
single or double base mismatches with AAO-258. This is
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W. D. H I O R N S a n d O T H E R S
also true of six other 16s rRNA gene sequences from
marine strains of ammonia-oxidizing /3-Proteobacteria
isolated by McCaig e t al. (1994). This level of dissimilarity
was also found in a large number of l6S rDNA sequences
of other Proteobacteria deposited in the GenBank database. The four marine ammonia-oxidizing strains, i.e.
Nitrosococczls oceanas C-27 and C-107 (y-subdivision), and
Nitrosumonas sp. C-56 and Nitrosococczu mobilis Nc2 (j?subdivision) need not be considered further.
The other four oligonucleotide sequences evaluated were
much more specific for defined taxa of ammonia-oxidizing
bacteria, and this was confirmed by searches of the
GenBank database, where the most similar sequences
rarely contained less than three nucleotide mismatches.
These four oligomers were used as two pairs of primers
for PCR amplifications (for nitrosospiras, NS-85 and NS1009r; for non-marine nitrosomonads, NM-75 and RNM1007r);in these configurations, with confirmation by the
application of AAO-258 as an internal hybridization
probe, the likelihood of false positive results in heterogeneous environmental DNA samples is insignificant.
We wanted to define conditions under which sequences
containing single nucleotide mismatches would be discriminated, because probes were to be used ultimately for
analysis of complex natural communities. The methodology was established using AAO-258r as the probe
sequence, since a single-mismatch control was available.
Hybridization was performed at the predetermined irreversible melting temperature, Ti (Sambrook e t al., 1989)
and filters washed at room temperature. Hybridization to
cellular RNA was strongly evident for each of the eleven
strains probed with pDr (Fig. 2). Only those rRNA
sequences that had exact complementarity to AAO-258r
gave hybridization signals with that probe. Under these
conditions, strains with single or double nucleotide
mismatches to AAO-258r (the four marine strains of
ammonia-oxidizing bacteria) scored negative, and the
probe was therefore discriminatory within the range of
targets used. The results presented in Fig. 2 demonstrate
the high stringency afforded by hybridization at the
empirically-derived Ti. Having thus established a protocol for stringent hybridization of oligonucleotides, the
other probes (Fig. 1) were used at their irreversible
melting temperatures and exhibited their predicted specificities.
Screening environmental samples and cultures for
amplifiable rDNA sequences
The oligomers pA and pHr (Edwards e t al., 1989), used as
a pair of primers for PCR amplification, yield approximately 1.5 kbp of 16s rDNA when applied to any
eubacterial species. When cultures of all 11 strains of
ammonia-oxidizing bacteria were lysed and DNA amplified using these primers, a DNA band of the expected size
was easily detected on agarose gels (data not shown).
Confirmation that the amplified DNA fragment originated from 16s rDNA was achieved by Southern transfer
and hybridization with an internal probe (pD). The results
of such an experiment are presented for six strains in Fig.
2796
Fig. 2. Differentiation of ammonia-oxidizing bacterial strains by
oligonucleotide hybridization. Cells of axenic cultures were
lysed on nylon membranes and probed with oligonucleotides
pDr (a) and AAO-258r (b). Probe pDr was a positive control for
the presence of bacterial 165 rRNA. Note that there are only
single nucleotide mismatches between AAO-258r and the rrn
sequences of C-56 and Nc2, but hybridization was not detected
with these strains. Strains used were (1A) Nitrosomonas
europaea C-3 1, (1 B) Nitrosomonas eutropha C-91, (1C)
Nitrosomonas sp. C-56, (2A) Nitrosospira tenuis Nvl , (2B)
Nitrosospira sp. Nvl2, (2C) Nitrosospira sp. C-141, (3A)
Nitrosococcus oceanus C-27, (3B) Nitrosococcus oceanus C-107,
(3C) Nitrosococcus mobilis Nc2, (4A) Nitrosospira sp. C-128 and
(48) Nitrosospira multiformis C-7 1.
3(a), lanes 1-6. The application of more specific internal
probes resulted in the differentiation of ammonia-oxidizing species. Oligonucleotide probe AAO-258 hybridized
with 16s rRNA genes amplified from Nitrosumonas and
Nitrosospira strains (Fig. 3b, lanes 2-6), probe NS-85 from
Nitrosospira strains only (Fig. 3c, lanes 3-6), and probe
NM-75 from a Nitrosumonas ezlropaea strain (Fig. 3d, lane
2). Amplified rRNA genes from the marine Nitrosumonas
sp. strain C-56 hybridized only with the universal probe
pD (Fig. 3a-d, lane 1). The probes therefore behaved with
amplified DNA as predicted by the data presented above
in Figs 1 and 2.
One sample of activated sludge and two soil samples were
used to determine whether DNA could be recovered
directly and amplified by PCR with ' eubacterial ' primers
pA and pHr. The DNA extraction and purification
protocol developed for use with environmental samples
by Bruce e t al. (1992) yielded DNA of sufficient quantity
and purity for PCR amplification and detection by agarose
gel electrophoresis. This is illustrated in Fig. 3(a), lanes
7-9, where amplification products of the expected size
(1.5 kbp) hybridized to the universal internal probe, pD.
Southern hybridization to these same preparations with
more specific internal probes resulted in an interesting
distribution of positive signals. The AAO-258 probe
hybridized to amplification products from all three
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16s rRNA probes for ammonia-oxidizing bacteria
1
2
3
4
5
6
7
8
9
(a)
1.5 kbp
-
1 2 3 4 5 6 7
8
1 2 3 4 5 6 7
(a)
0.9 kbp -L
0
1-5kbp
Fig. 4. Differentiation of pure cultures of ammonia-oxidizing
bacteria using nested PCR. Products of PCR with 'eubacterial'
primers PA and pHr were amplified further using
oligonucleotide sequences found in the rrn genes of
Nitrosospira spp. (NS-85 and NS-l009r: a) and Nitrosomonas
spp. (NM-75 and RNM-1007r: b). The reaction products were
separated by agarose gel electrophoresis (left), and a Southern
transfer of the products was probed with oligonucleotide AAO258 (right). Lane 1, Nitrosomonas eutropha C-91; lane 2,
Nitrosomonas europaea C-31; lane 3, Nitrosospira multiformis
C-71; lane 4, Nitrosospira tenuis Nvl ; lane 5, Nitrosospira sp. C128; lane 6, Nitrosospira sp. Nvl2; lane 7, Nitrosospira sp. C141; lane 8, molecular size markers.
-
of the Nitrosospira probe NS-85 (Fig. 3c), whose specificity
is much less ambiguous. No hybridization was recorded
when amplified DNA from these samples was challenged
with probe NM-75, specific for nitrosomonads (Fig. 3d).
1.5 kbp
Nested PCR amplification analysis of laboratory
cultures and freshwater sediment samples
Fig. 3. Amplification and oligonucleotide probing of DNA from
pure cultures and environmental samples. Primers pA and pHr
were used to generate 1.5 kbp fragments of 'eubacterial' rrn
genes. Reaction mixtures were electrophoresed, transferred to
nylon membrane and hybridized with the oligonucleotide
probes pD (positive control) (a), AAO-258 (b), NS-85 (c) and NM75 (d) described in Fig. 1. Hybridization could be detected
between DNA amplified from environmental samples (lanes
7-9) and probes AAO-258 and NS-85. None was detected with
probe NM-75. The single mismatch between probe AAO-258
and the rrn sequence from Nitrosomonas sp. C-56 prevented
hybridization. Lane 1, Nitrosomonas sp. C-56; lane 2,
Nitrosomonas europaea C-3 1; lane 3, Nitrosospira multiformis
C-71; lane 4, Nitrosospira tenuis Nvl ; lane 5, Nitrosospira sp. C128; lane 6, Nitrosospira sp. Nvl2; lane 7, activated sludge;
lanes 8 and 9, soil.
samples (Fig. 3b), although the specificity of this probe
sequence does not provide unequivocal evidence for the
presence of ammonia-oxidizing bacteria (see above).
However, these signals were all confirmed by application
In order to be more rigorous in our identification of
ammonia-oxidizer DNA in environmental samples, we
wanted to directly amplify DNA with primers specific for
this group of bacteria. We were unable to obtain
detectable amplification products from freshwater and
sediment samples by direct amplification with specific
primers. However, a nested PCR protocol, in which this
was preceded by amplification with the 'eubacterial '
primers pA and pHr, was successful. This strategy was
first proven using a range of pure cultures of ammoniaoxidizing bacteria. In Fig. 4, the internal probe, AAO258, hybridized to DNA fragments of the expected size
(approx. 0.9 kbp) after the second rounds of amplification
with the primer pairs specific for nitrosospiras (NS-85 and
NS-l009r, Fig. 4a) and nitrosomonads (NM-75 and RNM1007r, Fig. 4b). After prolonged exposure and development, PCR products smaller than 0.9 kbp could be
detected that were not visible in ethidium-bromidestained gels. These shorter products were always associated with the intended major product, and did not falsify
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W. D. H I O R N S a n d O T H E R S
1
2
3
4
5
6
1
2
3
4
5
(a)
0.9 kbp
2
3
4
5
6
1
2
3
4
5
-
....................................................................................................................... ...................................
Fig. 5. Nested PCR and Southern analysis of DNA from
freshwater lake sediment. PCR products of 'eubacterial' primers
pA and pHr were amplified further using oligonucleotide
sequences found in the rrn genes of Nitrosospira spp. (NS-85
and NS-1009r: a) and Nitrosomonas spp. (NM-75 and RNM1007r: b). The reaction products were separated by
electrophoresis (left), transferred to nylon membrane and
probed with oligonucleotide AAO-258 (right). No amplification
of DNA from sediment samples was detected using
Nitrosomonas ampIimers, whereas a II samples clearIy contained
template for Nitrosospira primers. Lane 1, positive controls
(Nitrosomonas europaea C-3 1 or Nitrosospira multiformis C-71);
lanes 2 and 3, littoral sediment; lanes 4 and 5, profundal
sediment; lane 6, molecular size markers.
the specificity of the PCR primers. In all cases, the fidelity
of the PCR amplifications was as anticipated from the
primer specificities.
A range of sediment samples taken from littoral and
profundal sites of Esthwaite Water was subjected to the
DNA extraction protocol described by Bruce e t al. (1992)
followed by the nested PCR amplification regime described above. Pure-culture controls were also processed
throughout. Visible bands corresponding to 0.9 kbp
amplification products were detected in all environmental
samples to which the Nitrosospira primer pair was applied.
This was confirmed by Southern transfer and hybridization with probe AAO-258 (Fig. 5a). Corresponding
bands of amplified DNA could not be observed in agarose
gels of reaction products generated with the nitrosomonad primers NM-75 and RNM-l007r, nor were any
revealed by probing with AAO-258 (Fig. 5b).
2798
1
(a)
Fig. 6. Nested PCR and Southern analysis of DNA from
ammonia-oxidizer enrichment cultures. Enrichment cultures for
ammonia-oxidizing bacteria were inoculated with samples of
lakewater. After 14 d, samples were taken from which DNA was
prepared for amplification. Nested PCR products were
separated by electrophoresis (left) and hybridized to probe
AAO-258 (right). Lanes 1-5 contain DNA from five examples of
enrichment cuItures ampIified with Nitrosospira primers N5-85
and NS-1009r (a), and with Nitrosornonas primers NM-75 and
RNM-1007r (b). Lane 6, molecular size markers.
Nested PCR analysis of lakewater and enrichment
cultures of ammonia-oxidizing bacteria
Samples from lakewater, concentrated by tangential-flow
filtration, were processed by extraction of DNA using the
method of Schmidt et al. (1991) followed by nested PCR
amplification. In common with the sediment samples,
amplification products were readily obtained with Nitrosospira primers but not nitrosomonad primers, and these
results were confirmed by Southern hybridization with
AAO-258 (data not shown). The suggestion that target
DNA for amplification with NM-75 and RNM-1007r was
absent from lakewater was further investigated by preparing enrichment cultures inoculated with lakewater
samples. Ammonia oxidation to nitrite and nitrate was
monitored for 14 d, after which DNA was extracted from
active enrichment cultures and subjected to nested PCR
analysis and confirmation by Southern blot hybridization.
In many cases, amplification products from both nitrosomonad and nitrosospira primers were detected (Fig. ba,
b, lanes 1-5); this was also true of enrichment cultures
inoculated with sediment samples (data not shown). Some
cultures produced evidence of the presence of Nitrosospira
but not Nitrosumonas, while some behaved conversely
(data not shown), Thus, for the first time in this study,
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16s rRNA probes for ammonia-oxidizing bacteria
putative nitrosomonads were detected by direct amplification and probing of their 16s rRNA gene sequences.
DISCUSSION
Direct recovery of DNA from diverse environments has
now been described in a number of reports (Schmidt etal.,
1991; Fuhrman e t a/., 1988; Selenska & Klingmuller,
1991; Tsai & Olson, 1992). The ubiquitous rRNA genes
have become established as the targets of choice, especially
16s rDNA, as it contains conserved and variable sequences for which a large database now exists (Larsen e t al.,
1993). This is certainly the case where analysis of
community structure and species composition is the
objective, as there are few other genes that could provide
this kind of information. The comparison of sequence
data from both laboratory cultures and natural biomass
has supported the contention that considerable bacterial
diversity exists within functional communities (Fuhrman
et al., 1992; Giovannoni e t al., 1990a, b; Weller et al.,
1992). The comparative role of different community
members, their relative contributions to nutrient cycling,
and the partitioning of active and dormant populations
are now important questions that can be addressed using
the techniques of molecular biology. In targeting the
ammonia-oxidizing bacteria, we are attempting to apply
these techniques to a group of organisms that are active in
a particular and important environmental process. In
addition, in this case, the difficulties associated with the
isolation and identification of autotrophic bacteria provide an additional impetus for study. The first step was to
demonstrate that ammonia-oxidizer rRNA genes could be
recovered from environmental samples ; this paper describes the procedures by which this can be achieved.
Sequence information on the relatively few, but nevertheless diverse, species of ammonia-oxidizing bacteria
available in pure culture provided the starting-point
(Head e t a/., 1993; McCaig e t al., 1994).
Selection of appropriate oligonucleotide sequences to
serve as probes and primers for PCR proved relatively
straightforward. Their application was optimized with
DNA isolated from a range of pure cultures. While
individual oligonucleotides were not absolutely specific,
their application to environmental samples was justified
by the design of protocols in which combinations of
primers and probes could be applied with confidence.
Thus, while the probe sequence AAO-258 was not unique
to the ammonia-oxidizers (though perfect matches were
few), the use of this sequence as an internal probe to DNA
amplified with other primers enabled conclusions to be
drawn. These were principally that the nested PCR
technique could be used to identify the presence of
Nitrosospira rDNA in samples of lakewater and surface
sediment. The genus name Nitrosospira is, in this case,
being applied in accordance with its extended definition to
include ammonia-oxidizers previously identified with
' Nitrosolobt/s' and ' Nitromibrio ' (Head e t al., 1993).
Although previous studies on the microbiology of
ammonia-oxidation have been concentrated on nitrosomonads, and especially Nitrosomonas europaea (Prosser,
1989; Abeliovich, 1992), there is sufficient evidence now
available to suggest that other ammonia-oxidizing species
may be prevalent in the environment (Belser & Schmidt,
1978;Cooper, 1983;Ward, 1986;Ward & Carlucci, 1985;
Bock, 1986). Until we have recovered and sequenced
nitrifier rDNA from these samples, no comment can be
made on the species identification of the nitrosospiras
present. Furthermore, it will ultimately be desirable to
substantiate studies based on recovery of rRNA gene
sequences from the environment by the amplification of
genes that define functional groups. The ammonia monooxygenase gene sequence has been determined for Nitrosumonas europaea (McTavish e t al., 1993), and similar
analysis of other ammonia-oxidizers may lead to design of
appropriate amplification primers and probes.
The repeated failure of the Nitrosumonas-specific primer
pair to amplify environmental DNA was surprising. The
possibility that nitrosomonads were absent from the
lakewater samples can be excluded, as Nitrosomanas rDNA
was amplified from enrichment cultures, along with that
of Nitrosospira. It is unlikely that the protocols used for
cell lysis and DNA purification perform differently with
Nitrosospira and Nitrosomonas, since the methods were
developed for analysis of environmental samples that
contained a wide range of bacteria (Schmidt e t al., 1991;
Bruce et al., 1992). Additionally, we have not encountered
any such bias with pure strains of ammonia-oxidizing
bacteria. It is possible that organisms with differing
morphologies may occupy different sites in soil and
sediment matrices, but samples are thoroughly disrupted
by the extraction method used here. Thus, we suggest that
Nitrosomonas rDNA was present at very low levels in the
lakewater and sediment samples examined. However, in
our experience, the DNA yields from PCR amplifications
of pure cultures with the Nitrosomonas primers are
consistently lower than those obtained with the Nitrosospira primers. This differential performance of the
primer pairs in PCR reactions is an example of the way in
which bias can be introduced into the application of
molecular biological techniques to environmental microbiology. Culture-based methods for studying natural
populations are by definition biased, but new approaches
such as that described here provide only partial solutions
to this problem.
ACKNOWLEDGEMENTS
This work was funded by postgraduate studentships (W. D. H.
and R. C. H.) and research grants (I.M. H. and W. D. H.) from
the Natural Environment Research Council, and benefited from
use of the Science and Engineering Research Council SEQNET
facility .
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