ELSEVIER
FEMS Microbiology Letters 139 ( 1996) 7 1-76
Immunocytochemical detection and location of the
membrane-bound nitrite oxidoreductase in cells of
Nitrobacter and Nitrospira
Eva Spieck a,*, Jens Aamand b, Sabine Bartosch a, Eberhard Bock a
’ Unicersirijt Hamburg, Insiitutftir Allgemeine Boranik, Abteilung Mikrobiologie, Ohnhorsrstr. 18, D-22609 Hamburg, Germany
b Geological Surrey of Denmark, Thorarej 8, DK-2400 Copenhagen NV, Denmark
Received 2 1 March 1996; accepted 26 March 1996
Abstract
The intracellular location of the membrane-bound
nitrite oxidoreductase
of Nitrobacter hamburgensis
Xl4 was
determined by electron microscopic
immunocytochemistry.
Post-embedding
labeling performed on ultrathin sections
revealed that the enzyme was located at the cytoplasmic membrane and at the intracytoplasmic membranes. As shown by
immunonegative staining of isolated membranes the labeling of the monoclonal antibodies named Hyb 153-2 and Hyb 153-3
was located at the cytoplasmic side of the membranes. Using Hyb 153-2, which recognize the a-subunit of nitrite
oxidoreductase, the labeling was found at the surface of the particulate membranes. In contrast, Hyb 153-3, recognizing the
P-subunit of nitrite oxidoreductase, bound preferably at the periphery of the membrane fragments. Besides Nitrobucter, Hyb
153-3 also recognized the nitrite-oxidizing system in two strains of Nitrospiru. No reaction was found with Nitrospinn and
Nitrococcus.
Keywords: Nitrite oxidoreductase; Nitrobacrer; Nirrospira; Monoclonal antibody; Subunit-specific labeling; Electron microscopy
1. Introduction
Chemolithotrophic
bacteria of the genus Nitroderive their energy from the oxidation of
nitrite to nitrate. This reaction is catalyzed by a
membrane-bound
nitrite oxidoreductase (NOR) [ 1,2].
Experiments
with NOR purified by heat treatment
revealed an enzyme consisting of two subunits with
batter
* Corresponding author. Tel.: +49 (40) 82282 426; Fax: +49
(40) 82282 43 1.
molecular weights of 115 000 ((Y) and 65 CKKl( p)
[3]. Furthermore, when the enzyme was purified with
detergents, cytochromes a, and c, were found to be
associated with NOR [1,2,4]. Cytochrome
c, has
been described as the y-subunit of NOR [2].
In ultrathin sections, cells of Nitrobucter
can be
identified easily by their characteristic intracytoplasmic membranes (ICMs), arranged as a polar cap of
paired membranes. In nitrite oxidizing cells an electron dense layer on the cytoplasmic
side of the
membranes has been observed. By negative staining
of isolated cytoplasmic membrane (CM) and ICMs
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PII SO378-lO97(96)00123-1
72
E. Spieck rt al. / FEMS Microbiology
the surface of the layer becomes visible. It forms a
crystalline lattice composed of 7-10 nm particles
which are supposed to represent the NOR [5- 101.
This is in agreement with electron microscopic examinations of the purified enzyme revealing uniform
particles with a size of 8- 10 nm [3, lo]. The two-dimensional
structure of the crystalline
lattice was
further characterized by digital image processing of
negatively stained membranes. The lattice was shown
to consist of particle dimers [lo]. During cell rupture
or at a high ionic strength the particles were released
into the aqueous phase [9,10]. If the NOR is repressed, as described for heterotrophically
grown
cells, the electron dense layer is not detectable 191.
Membrane-bound
particles similar to that of Nitrobacter have been observed in cells of Nitrococcus [ 1 l] and Nitrospira [ 12,131. In the latter case
they were located on the periplasmic side of the CM
[ 131. Membrane-bound
particles in Nitrospina have
not been described so far [l 11. In this report the
particulate structure and location of NOR is further
characterized
by use of monoclonal
antibodies
(mAbs) recognizing
the (Y- and the P-subunit of
NOR [ 141.
2. Materials and methods
2.1. Bacterial strains
Nitrobacter hamburgensis strain Xl4 was isolated
from soil of the old Botanical garden in Hamburg
[ 151. Nitrospira moscoviensis
strain M-l (DSM
10035) originates from an iron pipe of the heating
system in Moscow [ 131. The marine species Nitrospira marina (strain 2951, Nitrococcus mobilis (strain
231, ATCC 35380), and Nitrospina gracilis (strain
3/211) were described by [ 161. All strains are stored
in the culture collection of the Institut fur Allgemeine Botanik, Abteilung Mikrobiologie, Universitat
Hamburg.
2.2. Culture media
Nitrobacter
hamburgensis
was grown
in a
mixotrophic medium [ 151 and Nitrospira moscoviensis in an autotrophic medium [ 131. For Nitrospira
marina, Nitrococcus mobilis and Nitrospina gracilis
the marine medium according to [ 1 l] was used.
Letters I39 (IYY6I
2.3. Isolation
membranes
71-76
of cytoplasmic
and intracytoplasmic
Cells were harvested at the end of the exponential
growth phase by centrifugation. They were disrupted
by gentle sonication as described by [lo]. CM and
ICMs were isolated from the crude extract by sucrose density gradient centrifugation
according to
[171.
2.4.Isolation of NOR
NOR was purified by heat treatment
as described
by [31.
2.5. Enzyme acticities
Nitrite oxidase activity was measured by the consumption of nitrite with NaClO, as artificial electron
acceptor [31.
2.6. Preparation
of the mAbs
Two mAbs, named Hyb 153-2 and Hyb 153-3,
recognizing the (Y- and the P-subunit of NOR from
Nitrobacter
respectively,
were purified from hybridoma culture supernatants [ 141. The protein concentrations amounted to 1.02 mg ml-’ (Hyb 153-2)
and 0.66 mg ml-’ (Hyb 153-3).
2.7. Dot immunobinding
test
Drops of diluted crude extracts were put on nitrocellulose strips and washed twice with phosphate
buffered saline (PBS: 0.14 M NaCl, 2.7 mM KCl,
1.5 mM KH,PO,,
8.1 mM Na,HPO,,
0.1% Naazide, pH 7.4). After blocking for 30 min (5% milk
powder and 1% bovine serum albumin (BSA) in
PBS) the strips were incubated for 1 h with the
mAbs in a dilution of 1:500 or 1: 1000. After two
washes the strips were incubated for 1 h with alkaline-phosphatase
conjugated goat anti-(mouse IgG)
(Sigma), diluted 1: 1000. Staining was performed
with nitroblue tetrazolium (1 mg ml-’ in 0.1 M
NaHCO,, 0.05 M Na,CO,, 4 mM MgCl,, pH 9.5)
and 5-bromo-4-chloro-3-indoylphosphate
(5 mg ml- ’
in dimethyl sulfoxide) by a procedure modified from
[ 181. All dilutions were performed in a dilution
E. Spieck et al./FEMS
Microbiology Letters 139 (19%) 71-76
buffer (0.025% Tween 20, 5% milk powder, 0.05%
BSA in PBS). Nitrocellulose strips incubated with
preimmune serum instead of mAbs served as control.
2.8. Electron microscopy
Cell fixation, embedding and ultrathin sectioning
were carried out as described by 1191. Poststaining
was done for 7 min with 4% (w/v) uranyl acetate in
70% (v/v) aqueous methanol and for 10 min with
10% (w/v) lead citrate in 0.1 M aqueous NaOH of
pH 12.0 [20,21]. Micrographs were taken with a
Philips EM 420 electron microscope.
2.9. Post-embedding labeling
Cells were fixed with 2.5% formaldehyde for 1 h
on ice. After washing several times in PBS containing 10 mM glycine (pH 7.4) the cells were dehydrated by incubation in increasing amounts of ethanol
at decreasing temperature: 15% and 30% for 15-30
min at 0°C 50% for 60 min at -2O”C, and 70, 90,
and 100% for 60 min at - 35°C. Embedding in the
low temperature resin Lowicryl K4M was performed
as described by [22]. The samples were incubated for
polymerization under 366 nm light at - 30°C for 18
h and at room temperature for 24 h. Ultrathin sections were taken with a diamond knife and mounted
onto movital-covered nickel grids. Incubation with
the mAbs in a dilution of 1:10 was performed for 1 h
at room temperature. The sections were rinsed in
PBS several times, followed by incubation on drops
of goat anti-(mouse IgGl-gold complexes (GAM 10
nm) in a dilution of 1: 10 for 1 h at room temperature. Finally, the sections were washed in distilled
water and post-stained with uranyl acetate and lead
citrate. In control experiments incubation with the
mAbs recognizing NOR was omitted.
2.10. Immunonegative staining
Isolated CM and ICMs were labeled with the
mAbs by the electron microscopic on-grid technique.
The labeling was performed as described for the
ultrathin sections. As a modification, the mAbs and
the GAM-gold complexes were diluted 1: 100. Membranes were negatively stained by a method modified
from [23] using sodium phosphotungstate (2%, pH
7.0).
73
3. Results and discussion
For the application of the post-embedding labeling it was necessary to clarify if NOR of Nitrobacter
hamburgensis was still reactive against the mAbs if
formaldehyde was used for fixation. Using dot immunobinding a positive reaction was found with the
mAbs and crude extract of Nitrobacter treated with
0.25-2.5% formaldehyde. Ultrathin sections revealed that the use of 2.5% formaldehyde resulted in
a better structural preservation of the cells than the
use of 0.25% formaldehyde (not shown).
In Fig. 1 the intracellular location of NOR on the
CM and ICMs of Nitrobacter hamburgensis is
demonstrated by post-embedding labeling applied on
ultrathin sections. In experiments with both Hyb
153-2 recognizing the o-subunit of NOR (Fig. la)
and Hyb 153-3 recognizing the @subunit of NOR
(Fig. lb) electron dense gold particles were dominant
at the CM and ICMs at the cell periphery. Only few
gold particles were present in the interior of the cells
or in the background. Similarly, no or only a few
gold particles were present in ultrathin sections of
Nitrobacter hamburgensis incubated with the GAMgold complex alone (Fig. lc). The results confirm
the assumption that NOR is a membrane-bound enzyme [2,6].
Using post-embedding labeling it could not be
shown on which side of the membrane the NOR was
located, because the best resolution using secondary
antibodies and gold complex with a size of 10 nm as
markers is 30 nm [24]. Therefore, the location of
NOR was studied by immunonegative staining of
isolated membranes with a nitrite-oxidizing activity
of 4.4 mm01 NO; min- ’ mg protein-‘. On the
surface of the membrane fragments the regularly
arranged NOR particles were visible. In experiments
with both Hyb 153-2 and Hyb 153-3 an enrichment
of gold particles on the particulate membranes was
observed (Fig. 2). Therefore, the localization of the
NOR on the cytoplasmic side of the membrane was
verified as earlier supposed by [2,10,25,26]. Furthermore, different labeling patterns were obtained with
the subunit-specific mAbs. Hyb 153-2 (recognizing
the cl-subunit) bound specifically to the membrane
surface (Fig. 2a) while Hyb 153-3 (recognizing the
P-subunit) bound most frequently at the periphery of
the membrane fragments (Fig. 2b). Only a few gold
74
E. Spieck et 01. / FEMS Microhiolo,cp
Letters
I.39
CIYWi)71-76
particles were present in the background or in the
control incubated without the mAbs (Fig. 2~). These
results may be interpreted as follows. At the periphery of the membrane fragments the NOR particles
were far apart from each other. whereas they were in
close contact at the surface of the membranes. The
binding of the mAbs Hyb 153-3 to the P-subunit
may have been restricted in the crystalline lattice due
to steric hindrance. whereas the mAbs Hyb 153-2
could react freely with the a-subunit.
While the
Q
Fig. 2. Localization
of the LY- and P-subunit of NOR from
X14. Isolated cytoplasmic and intracytoplasmic membranes were incubated with the subunit-specific
mAbs and negatively stained with sodium phosphotungstate
to
show the surface of the particulate membranes. (a) Incubation
with mAbs Hyb 153-2 recognizing the cu-subunit. (b) Incubation
with mAbs Hyb 153-3 recognizing the P-subunit. (c) Control:
incubation with buffer instead of mAbs followed by incubation
with CAM-gold complex (10 nm). The bars correspond to 0.1
Nitrobacter
hanlburgensis
pm.
Fig. 1. Localization of nitrite oxidoreductase
(NOR) shown by
immunogold labeling of Nitrobrrcter
hamburgensis
X13. Cells
were embedded in Lowicryl K4M and the sections incubated with
monoclonal antibodies (mAbs) recognizing NOR and with secondary antibodies (GAM) labeled with gold complexes (10 nm).
(a) Incubation with mAbs Hyb 153-2 recognizing the a-subunit.
(b) Incubation with mAbs Hyb 153-3 recognizing the P-subunit.
(c) Control: incubation with GAM-gold complex (10 nm) alone.
cm = cytoplasmic
membrane, om = outer membrane,
g = gold
particle. The bars correspond to 0.25 Km.
antigenic reactive site of the a-subunit supposedly
faces the cytoplasmic space, the P-subunit seems to
be located in the near vicinity of the membrane.
To understand the differences in labeling patterns
of the membranes, the structure of the isolated enzyme was investigated. Recently, it was shown that a
single NOR particle represents an a), P,-heterodimer
[IO]. The enzyme particle revealed a nearly globular
to slightly elongated shape. consisting of two protein
masses of different size and shape [lo]. The small
mass was covered on one side by the big mass.
These masses may represent the a- CM, 115 000)
and the P-subunit of the NOR (M, 65 000). It is
assumed that only a small surface area of the small
E. Spieck et al./ FEMS Microbiology Letters 139 (19%) 71-76
75
References
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Fig. 3. Dot-immunobinding
of Nitrospira moscociensis with mAbs
against NOR of Nitrobacter. The mAbs Hyb 153-3 (recognizing
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mAbs Hyb 153-2 and with preimmune serum as a
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moscouiensis did not react (Fig. 3b,c). Similar results
were obtained with Nitrospira marina, whereas none
of the mAbs gave a positive reaction with the marine
species Nitrospina gracilis and Nitrococcus mobilis
(not shown).
Acknowledgements
We wish to thank Irmhild Wachholz for excellent
technical assistance.
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