FEMS Microbiology Letters 6 (1979) 297-300
© Copyright Federation of European MicrobiologicalSocieties
Pubhshed by Elsevier/North-Holland Biomedical Press
297
AMMONIA, 02 U P T A K E AND PROTON EXTRUSION BY SPHEROPLASTS OF
NITR OSOMONA S E UR OPA EA
BASANT BHANDARI and D.J.D. NICHOLAS
Department of Agricultural Biochemistry, WaiteAgricultural Research lnstztute, University of Adelaide, Glen Osmond, 5064,
South Australia
Received 13 July 1979
1. Introduction
2.2. Preparation o f spheroplasts
Rees and Nason [1] prepared spheroplasts of
Nitrosomonas europaea by suspending 1 g washed
cells in 0.25 M sucrose-0.1 M potassium phosphate
(pH 7.5) 1 mm Na2EDTA containing 50 mg of lysozyme in a final volume of 40 ml for 2 h at 28°C.
However, this preparation contained intact cells.
Suzuki and Kwok [2] modified this method by
doubling the concentrations of both Na2EDTA and
lysozyme. This procedure, in our hands, did not consistently produce active spheroplasts which oxidised
ammonia to nitrite. In this paper we report on an
improved method for preparing active spheroplasts
which utilize ammonia and 02, produce NO2 and
extrude protons over a period of 48 h, as do intact
cells [3].
Spheroplasts were prepared by a modification of
the method of Suzuki and Kwok [2] : I g washed
cells were suspended in 50 mM Tris-HC1 containing
0.2 M sucrose (pH 7.5), 2 mm Na2EDTA and 100 mg
lysozyme in a final volume of 40 ml. The cell suspension was first incubated for 2 h at 30°C with contant
stirring, then 3 mM Mg-acetate containing 1 pg DNase
added and the incubation continued for 1 h. The suspension was then centrifuged at 2000 × g for 20 min
at 0°C, yielding a pellet of spheroplasts which were
resuspended in 2 ml of the Tris-HC1 sucrose buffer
(pH 7.5).
2. Materials and Methods
2.1. Growth conditions
Nitrosomonas europaea was grown and harvested
as described elsewhere [3].
Abbreviations: mCCCP,carbonyl cyanide m-chlorophenylhydrazone; DIECA, sodium diethyldithiocarbamate; DNP,
2,4-dinitrophenol; HOQNO, 2-heptyl-4-hydroxyquinoline-Noxide; N-Serve, 2-chloro-6-trichloromethyl-pyridine; 2-TMP,
2-triebloromethyl-pyridine; TPB, tetraphenylboron.
2.3. Electrode methods
NIT4 and 02 uptake was determined by electrode
methods [3]. A suspension of spheroplasts (20 mg
protein) was added to a perspex vessel (8.8 ml) containing 8.4 ml of 0.05 M Tris-HC1 0.2 M sucrose
buffer, pH 7.5 and 5/al catalase (2 mg/ml) [3]. The
reaction mixture was continuously stirred with a magnetic flea and maintained at a constant temperature
at 30°C. The reaction was started by injecting 10 tal
of NH4C1 (1/lmol) into the vessel via a gas-tight
syringe. Oxygen was regenerated by adding 7/al, 2%
v/v H202 to the reaction mixture [3]. NO~ production was determined colorimetrically and proton
extrusion by a fluorescence technique as described
previously [3]. Protein was determined by a microbiuret method [4].
298
3. Results and Discussion
The optimum concentrations o f sucrose and buffer
for the preparation of active spheroplasts that oxidise
NH,~ to NO~ was found to be 0.2 M in 0.05 M T r i s HCI (pH 7.5). The data in Fig. 1 show that either
Mg2+ or Cu 2+ at 3 mM resulted in optimum uptake of
NH~ by the spheroplasts, but above this concentration Cu 2+ was inhibitory. The Mg2+ requirement
could be replaced by either Ca 2÷, Mn 2+ or Co 2+, each
at 3 mM. These spheroplasts take up NH: more
readily than those prepared by the method of Suzuki
and Kwok [2] and are also more stable over a longer
period, as shown in Fig. 2. The electron micrograph
(Fig. 3) indicates that the spheroplasts prepared with
Mg 2÷ are larger and more uniform in size than those
without.
The stoichiometry for NH~, O2 uptake and NO~
production was 1 : 1.5 : 1 and for hydroxylamine
oxidation a ratio o f 1 for O2 : NO~. These ratios are
idenhcal with those for washed cells [3]. The addition of hydroxylamine has been shown to eliminate
the lag phase of ammonia oxidation, based on the
production of nitrite in cells [5], spheroplasts [2]
and extracts [6] of Nitrosomonas. In our experiments, however, the addition o f hydroxylamine
(0.5/amol) did not increase the rate of NH~ uptake by
the spheroplasts, but the utilisatlon o f 02 was stimulated. Similar results were also obtained wtth washed
cells. The effects of various inhlbitors, including those
for metals, electron transport, uncouplers and lonoo
phores, on the uptake of NH,~ and 02 by spheroplasts
presented in Table I are similar to those reported for
intact cells [3]. Thus the inhibitory effect of DIECA
was completely offset by Cu 2÷ and that of CO was
reversed by exposing the treated spheroplasts to
tungsten hght for 20 min.
The results of the fluorescence technique in
Table 2 show that the inhibitors used in the electrode
experiments produced similar effects on proton
extrusion on adding ammonia to the spheroplasts.
These data again are similar to the results reported for
washed cells [3].
Thus, the spheroplasts described in this paper have
similar properties to intact cells [3] in that they have
an active NH~ translocase which is Cu and energy
dependent.
12(] "
10(
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~ 8(
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.
~
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-
/,,,
/
,
•
.
,
%
\
t!
10(]
"
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\
+ '-I"
~
Z
6(
=\
j
/
c
8(
E
,
1
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÷ff
Z
\
¢
O
'
1
I2
'
3
mM a g 2 + o r
'
4
Cu 2+
|_
I
6
Fig. 1. Effects of Mg2+ and Cu2+ on the uptake of NH,~by
spheroplasts. A suspension of spheroplasts (20 mg protein) in
the Tris-HC1 sucrose buffer (pH 7.5) was incubated for 1 h
with Mg2+ (o
o) and Cu2+ ( - - - ) respectively. Nil+4
uptake was determined by an electrode technique as
described in Methods.
I
10
I
20
l
30
k
I
40
I
50
hr
Fig. 2. Stability of the spheroplasts. NH,~uptake was determined as described m Methods. (1) spheroplasts prepared as
described in Methods (
); (2) spheroplasts prepared by
the method of Suzuki and Kwok [2] (- - -).
30
10
30
0
30
20
35
5
Nigericm
70tag
TPB
70taM
Rotenone
2~M
HOQNO
40tag
lonophores
Electron transport
90
70
mCCCP
6~M
Uncouplers
75
60
DNP
47/~M
60
40
DIECA
12#M
100
100
NaN3
2mM
Metal and others
100
100
Thlourea
10~tM
90
60
N-Serve
60uM
60
45
2-TMP
60~M
100
60
CO
(140
KN/m 2)
100
100
C2H 2
(140
KN/m 2)
45
25
47
40
65
68
47
45
Nlgencin
70t~g
TPB
01mM
Rotenone
6/~M
HOQNO
25tag
lonophores
Electron transport
a Control = 100 arbitrary umts.
b Pamally reversed by Cu 2+ (32 urn).
c Partially reversed by exposure to tungsten light for 20 min.
2 mm
5 mm
Inhlbitors
(final
concentratxon)
60
50
mCCCP
8t~M
Uncouplers
45
45
DNP
40~M
57
50
DIECA b
16t~M
60
50
NaNa
4mM
Metal and others
33
30
Thlourea
2mM
55
50
N-Serve
0.2mM
25
15
2-TMP
0.2mM
55
45
CO c
(140
KN/m 2)
55
45
C2H2
(140
KN/m 2)
0 1 ml spheroplasts (10 mg protein) was preincubated with or without the mhlbrtor for 3 mm in 125/~mol Tris-HC1 0.2 M sucrose (pH 7.5) and 0.1 #mol quinacrme in a final volume of 2.6 ml m a 1 cm cuvette before starting the reaction by adding 75/smol NH4CI , as described in Methods [3]. The lnhibltors other than
TPB, DIECA, NAN3, thlourea, were dissolved m 95% w/v ethanol. Appropriate controls were included with ethanol. The effects of the mhlbltors over periods of 2
and 5 mm after addmon of NH4CI are compared wath those without these compounds. When CO or C2H 2 were used, the spheroplasts were preincubated with the
gases for 20 mm prior to adding them to the reaction mixture.
Effects of inhlbltors on fluorescence quenching aetwaty in spheroplasts (% decrease m fluorescence a)
TABLE 2
NH~
02
lnhibitors
(final
concentratmn)
A suspension of spheroplasts (20 mg protein) was added to a perspex vessel containing 8.4 ml of 0.05M sucrose buffer pH 7.5 and 5 t~l catalase (2 mg/ml) [3]. The
reaction rmxture was continuously sttrred with a magnetic flea and maintained at a constant temperature 30°C. The appropriate inhibitor was added to the reaction
mixture vm a gas-tight syringe. After a preincubatlon period of 5 mm with the mhlbltor, 10 ~1 NH4C1 (1 tamole) was added to start the reaction. When CO or C2H 2
were used, the spheroplasts were preincubated with the gases for 20 mm prior to adding them to the reactmn mixture. 02 was regenerated in the system by adding
7 IA, 2% v/v H202 to the reactmn vessel. In the absence of the mhthltors the uptake values of NI-F4 and O2 were 100 and 150 nmol/mm/20 mg protean respectwel).
Except for DIECA NaNa, thmurea and TBS, the other inhibitors, mCCCP, DNP, HOQNO, Nlgericm, Rotenone, N-serve, 2-TMP, were dissolved m 95% w/v ethanol
and appropriate controls were included for ethanol.
Effects ofinhlbltors on the uptake of ammoma and O2 by spheroplasts (% mhibitmn)
TABLE l
300
Fig. 3. Electron mlcrograph of spheroplasts prepared m the presence of Mg2+ (3 mM) were negatively stained with 3% w/v phosphotungstlc acid (pH 6.6) and examined in an electron microscope JEOL (Model JEM-100cx) at an accelerating voltage of 60 kV,
×132 000.
Acknowledgements
References
One of us (B.B.) is grateful to the University of
Adelaide for a postgraduate scholarship. The work
was supported by a generous grant from the Austrahan Research Grants Committee.
We also thank Professor R.D. Dua, who is a visitmg fellow in the department sponsored by the
Australian Vice-Chancellors' Committee, for helpful
advice. The skilled technical assistance of Mr. M.
Byrne with the electron micrographs is gratefully
acknowledged.
[1] Rees, M. and Nason, A. (1965) Blochem. Biophys. Res.
Commun 21,248-256
[2] Suzuki, I. and Kwok, S.C. (1969) J. Bacterlol. 99, 897898.
[3] Bhandan, B. and Nicholas, D J D. (1979) Arch. Mlcroblol (in press).
[4] ltzhakl, R.F. and Grill, D.M. (1964) Anal. Blochem. 9,
401-410
[5] Hooper, A B. (1969) J. Bactenol. 97,968-969.
[6] Suzuki, I. and Kwok, S.C. (1970) Blochem. Blophys
Res. Commun. 39,950-955.