FEMS MicrobiologyLetters 66 (1990) 157-162 157

Pubhshed by Else,aer

FEMSLE03785

Energy conservation in Nitrobacter

A. Freitag and E. Bock

Inslttut fur AIIgememe Bolamk. Mtkrobwlogw,HamburgF R G

nccetvoc117July 1989
Rev:slonre~tved 28 July 1980
Accepted 31 July 1989

Key words: Nnrobacter sp, Nitric oxide metabohsm: NADH synthesis; ATP generation

1. SUMMARY The use of mmte as an energy source IS the

The generation of ATP and NADH In total
ceils of N~trobacter was measured under aerobic
and anaerobic condmons. NADH synthesis was
driven by an ATP independent reaction with mtnte
or nitric oxade as electron donors. The rate of
NADH formation was about 200 Umes ingher, ff
nitric oxide instead of mtnte served as electron
donor. Approximately 2 tool ntmc oxide were
nc.ded for the reduction of I mol NAD + NRnte
caused an end-product minbmon of the mine
oracle reduced NADH synthesis. ATP was synthe-
sized by NADH oxadatlon with oxygen and mtrate
as terminal electron acceptors.
2. INTRODUCTION
Bacteria of the genus Nurobacter were thought
to be obhgate aerobic organisms. However, Fret-
tag t al. [1] demonstrated that Nurobacter could
grow anaerobic~qy by disstrmlatory mtrate reduc-
tion wah acetate, pyruvate or glycerol as electron
donors. Nlmte oxidation as well as mtrate reduc-
tion support slow and inefficient growth.
Correspondence to E Book. Insatut fhr AIIgememeBotamL
MJkroblolog:e,Ohnhorststrasse 18. 2000 Hamburg 52. F R G
0378-1097/89/$03 50 1989Federationof European Microbtolo~calSooeues
subJeCt of several reviews [2,3], but a general con-
cept for the generation of ATP and reducing power
is rmssmg [4-7]. Wetzstem and Ferguson [7] ex-
plained the coupling of nitrite oxadation to ADP
phosphorylatton by proton pumping actwlty of
N~trobacter membranes, but Hollocher et al [6]
and Sone et al. [8] fmled to demonstrate proton
pumping activity neither in nimte oxidizing cells
nor in reconsmuted cytochrome oxldase vesicles.
In addition to the mtme oxidizmg system
Nnrobacter was shown to possess a NADH oxidiz-
ing system, winch was stimulated by 2,4-dt-
mtrophenol (2,4-DNP) or carbonyl cyanide m-
chlorophenylhydrazone (CCCP) (9.10).
In tins arucle we demonstrate, that NADH
synthesis is the primary energy conservmg reac-
tion m mmte consuming cells of Nurobacter. We
show for the first ume, that mtric oxide is an
efficient substrate for NADH generation. NADH
is used for ATP synthasls.
3 MATERIALS A N D METHODS
3 1. Orgamsms
The experiments were done with Narobacter
wmogradsky~ strata Engel, and N:trobacter vulgans
strata A b l [11]. The orgamsms are kept m the
culture collection of the Instltut fur Allgemelne
158
Botanik (Umverslty of Hamburg). Nitrobacter was
grown nuxotrophically m 20-1 bottles according to
[121.
3.2. Short term experiments
In order to measure ATP and NADH genera-
tion, short term experiments were earned out as
described m [13]. Anaerobic tests were done m a
glove-b0x with a gas nuxture of 88~ N:, 10~ H : ,
and 2~ CO 2. After premcubatton for 30 nun,
mtrate (15 raM) or mtnte (15 raM) m Tfis-buffer
(75 raM, pH 7) were added to the cell suspcnston.
Aerobic resplratton was reduced by the addltmn
of air saturated Tns-buffer (75 mM, pH 7.5)
without substrates (endogenous resplratton) or by
the addmon of 15 mM mtme (mtnte oxidation) or
h',trate (aerobic mtsate reduction). Oxidation of
nitrtc oxide was achieved under anaerobic condi-
tions by the addttion of mmc oxide saturated
Tns-buffer (75 raM, pH 7) Under aerobic condi-
tions mtrtc oxide-gas (5000 vpm, Messer Gnes-
hetm, F.RG.) was continuously bubbled through
the cell suspenston wtth a rate of 10 m l / m m .
Aerobtc and anaerobic resplraUon were stopped
by the addmon of ethanol to a final concentranon
of 75(g. Nttnc oxade (NO) consumptmn dunng
these experiments was calculated from the ac-
cumulatton of the end-products mtnte and mtrate,
which were estimated in 5 nun intervals within 30
nan. The chermcal mtnc oxide oxidation was de-
termined in control experiments with boded cells.
These values were subtracted from those which
were measured wtth active cells.
3 3 In wvo measurements of NADH
In order to demonstrate in vivo NADH forma-
tmn, the NADH pool of intact Nttrobacter cells
was measured by difference spectra [9]. Before
starting the experiments, poly-fl-hydroxybutyrate
(PHB) was removed from the cells by prcincuba-
tion for 48 h without any substrate. The protein
content of the cell suspension was adjusted to 0 5
m g / m l The NADH production was recorded as
an increase of extinction at 340 nm on a computer
controlled Shimadzu photometer. NADH forma-
tion was initiated by the addition of 15 mM nitrite
or NO gas (5000 vpm. 10 m l / m i n ) The reference
euvettes contained inactivated cells, which were
premeubated wtth 1 mM thiocyanate. N,N'-dl-
eyclohexy) carbodiimide (DCCD, S~gma, F.R.G.)
was used as ATPase inhibitor. In accordance with
[14] cells were premcubatcd for 30 nun with 0.3
mM DCCD. 2,4-&mtrophcnol (2,4-DNP, Sigma,
F.R,G.) was used as an uncoupler that did not
affect the mtrite oxldatton activity [15]. The opti-
mal concentration was 0.1 raM. The premcubat|on
time was 30 nun.
3.4. Analyucal procedures
ATP and NADH were extracted from intact
cells by the method of [16]. ATP was quannfied
by the luclferin-lucifcras test [17]. Quantitative
N A D H analysis was carried out by the method of
[18]. Protein measurcmems were accomphshed by
the method described m [19]. Oxygen was mca-
sured with an oxygen electrode (WTW, F.R.G.).
Nttritc and mtratc were quanufied by HPLC tech-
mque according to [20]. Poly-fl-hydroxybutyrate
was quantified by the method of [21].
4. RESULTS
4 1 ATPproductlea
Anaerobic -t, chtloas In Fig. la, b changes of
the ATP and NADH pools are presented aftes the
addinon of nitrate or mtnte to resting cells of
Nttrobacter wmogradskyJ under anaerobic condi-
tions.
If mtratc was added as the electro:-, acceptor,
an increase of the ATP pool was mea~ lred at the
expense of the endogenous I~ADH pool (Fig. la).
The NADH content dropped rapidly within the
first two seconds and then more slowly. Nitrate
reducing cells produced 5.5 nM m t r i t e / r m n . m g
protein (not shown). If nlt ~.~was used as electron
acccptor neither ATP production nor NAD_,,TM con-
sumption (Fig. lb) were ,'~easurable. The ATP
pool remained constant bu, the NADH pool in-
creased slightly. 2 6 nM n i t n t e / m i n . n ~ g protein
were reduced to NO and N20 (not shown)
Aerobic conditions When oxygen was added to
anaerobically resting cells, the ATP content of the
~otal cells increased within 5 s while the NADH
content decreased. When oxygen together with
nitrate were used as electron aceeptors, ATP
 159

ATP NADH ATP NADH

triM/rag Protmn] [nM/mg Protein] [nil/rag Protein] [nH/mg Protmnl

]b] 3

3 TP '2

1 1 NAOH " I
L . . . . . . . . A_TP
0 5 10 [sec] 5 10 [sec]
Fig la.b Anaerobic ATP formation and NADH consump- Fig 2a.b Aerobic ATP formation and NADH consumpt,on in
hon/fermalmn m resting cells of Nitrobacler wmogradskyl (a) rcst)ng cells of Nlrrobacter wmogradskyt (a) nitrate reduction,
nnrat reducllon, ATP (0), NADH (O) (b) nitrite reduction. ~TP (). NADH (o) (b) endogenous resptratm0n, ATP (el,
ATP (el, NADH (3) NADH {ra), mmt o~adauon, ATP (&). NADH (A)

synthesis w a s achieved at a h i g h e r level than w l , h

o x y g e n a l o n e ( F i g 2a). W h e n cells were i n c u b a t e d Ext 0z
w i t h air s a t u r a t e d nitrite s o l u t i o n ( F i g 2b), the 340 nrn ]% ]
level o f A T P synthesis w a s the s a m e as m the
c o n t r o l w i t h e n d o g e n o u s r e s w n n g cells

4.3. N A D H productwn
NJtrtte as a substrate. Ntzeobaczer wmogradskyt
w a s d e p l e t e d o f P H B as d e s c r i b e d in 3 3 T h e

]
e x p e r i m e n t w a s started b y the a d d i t i o n of m t m e
to aerobically r e s p i r i n g cells In Fig. 3 c h a n g e s m
the N A D H p o o l are s h o w n as c h a n g e s m extrac-
t i o n a t 340 rim. I n a m t r l t e o x i d ~ n g cell s u s p e n -
sion w R h 0.5 m g / m l p r o tein , the dissolved o x y g e n [1NP
t e n s i o n d r o p p e d w l t l u n t h e first 4 n u n to less t h a n 80
4 ~ s a t u r a t i o n . D u r i n g the first 5 n u n a de c rea s e
a n d t h e n a n increase o f N A D H w a s m e a s u r a b l e . -02
T h e m a x i m u m level w a s reached a ft er 2 - 2 5 h. 20
W h e n the s a m e e x p e r i m e n t w a s carried o u t u n d e r L. . . . . ~
anaerobic conditions, the NADH pool increased 10 30 [mln]
w i t h o u t a lag p h a s e o f 5 rain ( n o t shown). Pre- Fig 3 Effects of uncouplers on anaerobic NADH formauon m
i n c u b a t i o n w i t h 2 A - D N P i n h i b i t e d the N A D H Nurobacter wmogradskyn mtnte (0), mtnt plus DCCD (o),
synthesis totally, w h ile p r e i n e u b a t i o n w i t h D C C D mmte plus 2.4-DNP (A). dissolved oxygen t erosion I - - - --)
160

cally with N O gas. The results are presented m

[3~,0] , ~ ' a ~- Ftg. 4a, b.
An immedtate increase in the N A D H pool was
o,lo [a] oncleroblc [b] aerobic measurable, when nitric oxide was added. The
maximum level was reached witlun 30 s. N A D H
synthesis did not occur if nitnc omde or mtrite
were absent.
In the presence of nitrite, the nitric oxide re-
o,o5 duced N A D H synthesis was inhibited. Under
anaerobic conditions mtrite caused an average m-
babition of 75~; m the presence of oxygen only
30% inlubttion was observed.
In order to measure the stolchrometry between
nitric oxide oxtdatmn and N A D H formation, senes
1 2 i 2 [mini of short term experiments were done as described
F~g 4 Stimulationof NADH synthes~s by NO under anaerobic in 3.2. The results from 6 experiments with two
(a) and aerobic(b) condmons m Nitrobacter wmogradskyl NO
different species of Nttrobacter are summarized in
(O), NO plus nutnte (A). without NO and mtnte (~)
Table 1. Wttlun the first 10 s the N A D H content
of cells increased with a constant rate. The maya-
mum level was reached after 2 5 - 3 0 s (not shown).
Nitrite and mtrate concentrations mcreased con-
tmuously throughout an incubation time of 30
enhanced the N A D H formation for approramately min (not shown). The rates of N A D H productmn
30% (Fig. 3) were calculated to t,e 55 mol. 1 0 - 1 2 / s . m g pro-
In a series of additional expenments the p H tern in active cells of Nitrobacfer wmogradskyJ and
optimum of the N A D H formation was deternuned 5 tool. 1 0 - ~ 2 / s - mg protein m inactive cells (sta-
to be pH 6.8-7.0 (not shown) tionary phase). The rates of biological mtric oxide
Nitric oxide as a substrate. Nttrobacter wtno- oradat]on vaned between 137 and 6 tool. 1 0 - ~ 2 /
gradskyt was depleted of PHB and incubated s. mg protein. The ratio of oxidized nitric oxide to
anaerobically with N O saturated buffer or aerobt- reduced N A D vaned between 1 and 3,

Table 1
Rates of mlnle, nitrate and NADH formation and of mtnc oxide onsumplton by Nttrobacter wmogradskyl and NJtrobacter vulgarts
dunn8 nltnc oxide oxidation
Species Rates(tool 10-]a s-Lmsprotem -;) Ratios
Nttnte Nitrate Ntmc oxide NADH NO/NADH
formation formaUon consumpaon * formation
N vulgans a 39 4 43 lg 24
N wlgarls a 36 4 40 15 2?
N wmosradskyl a 76 8 84 42 2C
N wmogradskyt " 127 9 136 55 25
N wmogradsk_m = 127 f0 137 44 30
N wmogradskyl b 2 4 6 5 I2
Rmms of mmc olude consumption to NADH formation are expressed as NO/NADH The results represent exclusivelyblolo~cal
activities,Chenuca| nitric oxide oyadatton rates have been subtracted
Nitric oxide consumption was ealculatexlfrom mtrtte plus nitrate formaUon
= calls from losanthralc growth phase, b cells from stauonary growth phase

5. DISCUSSION
According to an accepted working model
[3,9,10], ATP syntbeSlS ra Nttrobacfer cells is driven
by the nitrite oxidation and N A D H is produced at
the expense of ATP by the so called reversed
electron transport.
ATP formation in whole cells of Ntfrobacter
was shown to be achieved by two different reac-
tions, provided that NADH was available. First,
in the absence of oxygen ATP synthesis was driven
by nitrate reduction. Second, in the presence of
oxygen ATP was produced by aerobic respiraUon.
The highest rate of ATP production was achieved,
if both, nitrate and oxygen, were added as electron
acceptors, These results indicate that nitrate serves
as an electron acceptor, even when oxygen ts
competing for electrons. WRh respect to the ATP
synthesis Nttrobacter s ~ m s to be able to demmfy
under aerobic conditions. Due to the mtnte
oxidizing actxvity of Nttrobacter, an accumulation
of the end product nitrite was not measurable.
In contrast to the findings of Aleem et at. [10]
and O'KeUy and Nason [22], we dicl not detect
any direct link between nitrite oxidation and ATP
synthesis. Our results are in accordance with the
findings by Sundermeyer and Buck [23]. NADH
but not ATP was the first detectable product of
nitrite oxidation.
Using difference spectra measurements, ldesow
[9] demonstrated m vtvo NADH synthesis m mtrite
oxidizing cells. When the experiments were repro-
duced by the same method, the NADH content of
the cells increased only after the oxygen content
had dropped to about hl~ saturation. Due to these
results we conclude that the NADH formation ts a
consequence of an anaerobic reaction. After the
addition of 2,4-DNP in a concentration, which
mhihited growth of Narobacter but did not in-
fluence the mtnte oxidation activity [15l, NADH
was not generated. This proved that NADH was
produced by a reactaon, which depended on en-
ergized membranes but not on the hydrolysis of
endogenous reserve material. In addition, NADH
was synthesized, when ATP synthesis was in-
hibited by DCCD. As proved for Th:obacdhu'
ferrooxMans [24}, cells of Narobacter are able to
synthesize NADH when ATP generation is m-
hiblted.
161
Furthermore, we were able to demonstrate that
mtric oxide had a stimulatmg effect on the NADH
synthesis. The NADH formation reduced by nitric
oxide was an oxygen independent renetlon. Com-
pared to mmte, mtric oxide was the. more efficient
electron donor. The average ratio of NOox~dtz~l to
NAD~.duc~ was 2.3, whereas the ratio of NO~-o.,i~ d
to NAD~duc~d was assumed to be 5 [25].
From the thermodynatmc point of view nitric
oxide ts a better substrate for N A D H synthesis
than mtnte Compared to E~ 7 + 420 mV for the
N O 2 / N O ~- couple, the redox potential of the
NO/NO~- couple is E ~ + 374 mV [26], If water
is the reactant or more negative tf hydroxyl ions
are the reactants [27]. At the moment these find-
ings are verified by different techniques e.g. EPR
spectroscopy of an hypothetical highly electro-
negative intermediate. On the other hand we are
testing whether nimc oxide stimulates lithotrophic
growth of Natrobacter.
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