FEMS Microbiology Letters 14 (1982) 47-49
Published by Elsevier Biomedical Press
47
Nitrapyrin inhibits the obligate methylotrophs
Methylosinus trichosporium and Methylococcus capsulatus
Edward Topp and Roger Knowles
Department of Microbiology, Macdonald Campus of McGi/l Universi(~', 21 1l I Lakeshore Rd., Ste Anne de Bellevue,
Quebec, H g x 1 CO Canada
Received and accepted 18 January 1982
1. INTRODUCTION
Nitrapyrin [2-chloro-6-(trichloromethyl)pyridine] inhibits chemoautotrophic ammonia oxidizers [1-3], methanogenesis in marine sediments
[4], sulphate reduction in sediment and in pure
cultures of Desulfovibrio desulfuricans [5], and a
denitrifying pseudomonad [6]. It is non-toxic to a
large number of heterotrophic bacteria, actinomycetes and fungi [3,7,8]. Similarities between chemoautotrophic ammonia oxidizers and obligate
methylotrophs [9] suggested that nitrapyrin might
inhibit methane oxidation and carbon dioxide incorporation by these organisms.
In this paper we report that nitrapyrin markedly
inhibits the oxidation of methane and the incorporation of CO 2 by two obligate methylotrophs,
Methylosinus trichosporium OB3b and Methylococcus capsulatus (Texas).
2. MATERIALS AND METHODS
M. trichosporium OB3b (obtained from Dr. T.
Yoshinari) and M. capsulatus (Texas) (obtained
from Dr. R.S. Hanson) were grown in nitrate
mineral salts medium [10]. Both organisms were
grown in 2-liter batches agitated by magnetic
stirrers and sparged continuously with 30% C H 4 in
air. M. trichosporium OB3b was grown at 30°C, M.
capsulatus (Texas) at 37°C. Cells were harvested
by centrifugation at 16000 × g for 12 min. Cells
were washed and resuspended to 150 #g
protein/ml in nitrate mineral salts medium for
experiments.
Nitrapyrin (a gift from Dow Chemicals of
Canada Ltd.) was dissolved in dimethyl sulfoxide
(DMSO) for addition to cell suspensions. The final
concentration of DMSO in nitrapyrin-amended
flasks and DMSO controls was 70 #1/1. All suspensions were incubated with nitrapyrin for 2h
before addition of CH 4 or [14C]bicarbonate.
Experiments were carried out with 5-ml aliquots
of cell suspension in nitrate mineral salts medium
in 60-ml serum bottles sealed with grey butyl
rubber stoppers (Wheaton Scientific). Ambient
oxygen concentrations were used in all experiments. In [14C]bicarbonate uptake experiments,
ambient CO 2 was removed by repeated evacuation
and backfilling with helium. Ambient 02 concentrations were re-established by injection of pure
02. The initial gas phase concentration of C H 4
was 4%, established by syringe injection after removing an equivalent volume of the gas phase. M.
trichosporium OB3b was incubated at 30°C, M.
capsulatus at 37°C, and both organisms were
shaken at 250 rev./min on a gyrotary shaker.
Gas phase samples were removed by syringe
and methane was determined by gas chromatography as previously described [11]. Incorporation of
0378-1097/82/0000-0000/$02.75 © 1982 Federation of European Microbiological Societies
48
[J4C]bicarbonate ( A m e r s h a m , spec. act. 0.1
m C i / m m o l , final concentration 1 m M ) into acidstable products was determined by injecting a
100-#1 aliquot directly onto a 0.22 /~m type GS
millipore filter in a filtration assembly to which
100 /~1 4 N HC1 and 1 ml H 2 0 had been added.
After approx. 15 s, the filter was washed with 9 ml
H 2 0 . Filters were air-dried and counted in Aquasol scintillation cocktail (New England Nuclear).
Protein was estimated by the method of Lowry
[12] with bovine serum albumin as standard.
i
i
¢
'-~,~o
~'
I00
i
,o
,~
o
:a,
0.5
---~:--
5
Hours
Fig. 2. Carbon dioxide incorporation by cell suspensions of M.
trichosporium OB3b from the same batch as was used in the
experiment of Fig. I. Treatments and symbols are as described
in Fig. I.
time in Fig. 2 was presumably due to dilution of
the 14C as C H 4 was oxidized to C O 2.
We conclude that nitrapyrin is an effective inhibitor of obligate methylotrophs. Results to be
published elsewhere show that nitrapyrin does not
inhibit oxidation of the intermediates of the C H 4
oxidation pathway, methanol, formaldehyde and
formate. It therefore appears that nitrapyrin inhibits specifically C H 4 oxidation rather than other
metabolic processes. By inhibiting C H 4 oxidation,
the source of formaldehyde and energy is removed
Effect of nitrapyrin on rates of CH 4 oxidation and CO2 incorporation by M. capsulatus (Texas) a
\ ~ o
Treatment
CH4 oxidized
/~mol/(mg
protein)/h
CO2 incorporated nmol/
(rag protein)/h
None
DMSO
DMSO + nitrapyrin
Heat killed cells +
DMSO + nitrapyrin
10.27
10.26
3.07
160
149
10
o
E
~" 50
O0
i
Table 1
150
i
i
o
"
Nitrapyrin at a concentration of 10 # g / m l (43.3
/~M) effectively inhibited both C H 4 oxidation
(Fig. 1) and C O z incorporation (Fig. 2) by M. trichosporium. These processes in M. capsulatus were
also significantly inhibited by the same concentration of nitrapyrin (Table 1). Both organisms were
significantly inhibited by nitrapyrin concentrations d o w n to 2 / ~ g / m l (8.66 # M ) (data not shown).
T h e D M S O used to dissolve the nitrapyrin had no
significant effect, as shown by the controls receiving only D M S O (Figs. 1 and 2 and Table 1).
T h e decrease in rate of C O z incorporation with
i
2.0
~.t.5
°o
3. R E S U L T S A N D D I S C U S S I O N
200
E
i
I
5
I
I0
r
15
0
0
Hour=
Fig. 1. Methane oxidation by cell suspensions of M. trichosporium OB3b alone (~), and supplemented with 70 ~1
DMSO/1 (©), or DMSO plus 10 ~g nitrapyrin/ml (0). Heatkilled cells also received DM$O and nitrapyrin ([]).
a Final concentrations of nitrapyrin and DMSO were 10 #g/ml
and 70 #1/1, respectively. Methane oxidation and CO2 incorporation were determined in parallel flasks prepared from the
same cell suspension.
49
thus p r e v e n t i n g C O 2 i n c o r p o r a t i o n via the serine
p a t h w a y [13] or C a l v i n - B e n s o n cycle [ 14].
Activity of chemoautotrophic
ammonia
oxidizers in a q u a t i c systems is often e s t i m a t e d as
n i t r a p y r i n - s e n s i t i v e [14C]bicarbonate i n c o r p o r a tion [5,15-17]. C i r c u m s t a n t i a l evidence suggests
that m e t h y l o t r o p h s in freshwater systems m a y be
r e s p o n s i b l e for h e t e r o t r o p h i c nitrification. T h e y
oxidize a m m o n i a to nitrite via h y d r o x y l a m i n e [ 1 8 20], p r o d u c e nitric oxide [21] a n d n i t r o u s oxide (E,
T o p p a n d R. Knowles, u n p u b l i s h e d ; T. Y o s h i n a r i
u n p u b l i s h e d ) a n d are associated with nitrite m a x i m a in stratified lakes [22]. Large n u m b e r s of
Methylomonas methanica a n d M. trichosporium
h a v e been r e p o r t e d in s e d i m e n t a n d water c o l u m n
[23]. W e suggest that m e t h y l o t r o p h s p r e s e n t in
s a m p l e s t a k e n for the [ l a C ] b i c a r b o n a t e assay m a y
c o n t r i b u t e to the o b s e r v e d n i t r a p y r i n - s e n s i t i v e C O 2
i n c o r p o r a t i o n a n d lead to an o v e r e s t i m a t i o n of
c h e m o a u t o t r o p h i c nitrification activity.
ACKNOWLEDGEMENTS
W e t h a n k J. F r e a , R.S. H a n s o n a n d T. Y o s h i n a r i
for gifts of m e t h y l o t r o p h s . This w o r k was supp o r t e d b y the N a t u r a l Sciences a n d Engineering
R e s e a r c h C o u n c i l of C a n a d a a n d the I n l a n d W a t e r s
D i r e c t o r a t e of E n v i r o n m e n t C a n a d a .
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