FEMS Microbiology Letters 223 (2003) 227^230
www.fems-microbiology.org
Members of the genus Arthrobacter grow anaerobically using nitrate
ammoni¢cation and fermentative processes:
anaerobic adaptation of aerobic bacteria abundant in soil
Martin Eschbach a , Henrik Mo«bitz b , Alexandra Rompf c , Dieter Jahn
a
a;
Institut fu«r Mikrobiologie, Technische Universita«t Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany
b
Institut fu«r Mikrobiologie, Albert-Ludwigs-Universita«t Freiburg, Scha«nzlestr. 1, 79104 Freiburg, Germany
c
Max-Planck-Institut fu«r Terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany
Received 18 March 2003 ; received in revised form 22 April 2003; accepted 24 April 2003
First published online 27 May 2003
Abstract
Members of the genus Arthrobacter are usually regarded as obligate aerobic bacteria. The anaerobic growth and energy metabolism of
two Arthrobacter species were investigated. Arthrobacter globiformis utilized both nitrate ammonification and lactate, acetate and ethanol
producing fermentation processes for anaerobic growth. Only nitrate supported anaerobic growth of Arthrobacter nicotianae.
Anaerobically induced respiratory nitrate reductase activity was detected in both strains. Neither of the tested strains used the
alternative electron acceptors fumarate, dimethylsulfoxide or trimethylamine-N-oxide.
2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Anaerobic metabolism; Nitrate reductase ; Nitrate respiration; Fermentation ; Arthrobacter
1. Introduction
The type species Arthrobacter globiformis of the genus
Arthrobacter was initially described by Conn in 1928 as
bacteria numerous in various soils with the unusual morphological change from Gram-negative rods in young cultures to Gram-positive cocci in older cultures [1]. On the
basis of 16S rRNA cataloging the genus Arthrobacter was
identi¢ed as a member of the GC rich ‘actinomycete’
branch of Gram-positive bacteria, phylogenetically closely
related to other coryneform genera such as Aureobacterium, Cellulomonas, Curtobacterium and Microbacterium
[2,3]. The two major ‘species groups’ A. globiformis/Arthrobacter citreus and Arthrobacter nicotianae di¡er in
their peptidoglycan structure, teichoic acid content and
* Corresponding author. Tel. : +49 (531) 391-5801;
Fax : +49 (531) 391-5854.
E-mail address : [email protected] (D. Jahn).
lipid composition [4]. As originally reported by Conn [1],
many groups have shown that the genus Arthrobacter represents a numerically important fraction of the indigenous
bacterial £ora of soils from all parts of the world. Often
they are the most numerous single bacterial group in aerobic plate counts [5^8]. Arthrobacters can use a wide and
diverse range of organic substances as carbon and energy
sources including nicotine, nucleic acids and various herbicides and pesticides [4,9]. Like some Bacilli and Rhodococci some Arthrobacter species belong to the group of
Gram-positive organic solvent-tolerant bacterial strains
[10]. Recently, some Arthrobacter strains with clinical relevance have been isolated [11,12]. Members of the genus
Arthrobacter are usually regarded as obligate aerobes with
a pure respiratory, never a fermentative, mode of metabolism [4].
Changes in oxygen tension occur frequently in the upper
layers of soil, the habitat of the genus Arthrobacter. In
order to survive periods of oxygen limitation some Arthrobacter species have developed alternative, oxygen-independent growth strategies. Here, we describe a ¢rst initial
investigation of the anaerobic growth behavior and its
biochemical basis for two members of the genus Arthrobacter.
0378-1097 / 03 / $22.00 2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00383-5
FEMSLE 11023 16-6-03
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M. Eschbach et al. / FEMS Microbiology Letters 223 (2003) 227^230
2. Materials and methods
3. Results and discussion
2.1. Bacterial strains and growth conditions
3.1. The anaerobic energy metabolism of A. globiformis
and A. nicotianae
The type strains A. globiformis (DSM 20124) and
A. nicotianae (DSM 20123) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH, Braunschweig, Germany. All strains were grown
at 30‡C in Corynebacteria medium containing 1% (w/v)
casein hydrolysate (Gibco, Karlsruhe, Germany), 0.5%
(w/v) yeast extract (Difco, Detroit, MI, USA) and 0.5%
(w/v) NaCl. 25 mM glucose and 10 mM pyruvate were
added where indicated. Anaerobic growth was performed
in completely ¢lled £asks ( 6 1% gas phase) with rubber
stoppers and shaking at 100 rpm to minimize aggregation
of bacteria. The media were supplemented with nitrate,
fumarate, dimethylsulfoxide (DMSO) and trimethylamine-N-oxide (TMAO) in a ¢nal concentration of 10
mM where indicated. Nitrite was added at concentrations
of 1 mM or 5 mM where indicated. The media were not
completely anaerobic in order to allow an initial and vital
adaptation phase. After approximately 20 min of incubation all oxygen was consumed as determined by oxygen
electrode measurements and followed by redox indicators.
At that point strictly anaerobic growth conditions were
achieved [13].
2.2. Preparation of cell-free extracts and enzyme assays
Bacteria from the mid-exponential growth phase (OD578
of 0.2^0.8) were sedimented by centrifugation at 5000Ug
and washed with bu¡er, resuspended in 50 mM Tris, pH
7.4, 10 mM MgCl2 , 100 mg l31 DNase, 50 mg l31 RNase
and passed several times through a French press. The
crude extract was centrifuged at 15 000Ug and the supernatant was used for enzyme assays. All operations were
carried out under strict oxygen exclusion.
Nitrate reductase activity in cell-free extracts was measured photometrically by the nitrate-dependent oxidation
of reduced benzyl viologen in anoxic cuvettes [13]. One
unit of activity corresponds to the reduction of 1 Wmol
nitrate per minute. Reported values are the average of
three independent experiments performed in triplicate. Nitrate, nitrite, ammonia and acetoin concentrations were
determined as described before [13].
Anaerobic growth experiments in the presence and absence of nitrate were performed using two Arthrobacter
strains which represent the two major ‘species groups’
A. globiformis/A. citreus and A. nicotianae [4]. The bacteria
were ¢rst grown aerobically and subsequently shifted to
anaerobic growth conditions. Similar to observations
made for Bacillus subtilis a transition period with continuously decreasing oxygen tension is required to allow subsequent anaerobic growth [15]. The shift from aerobic to
strictly anaerobic conditions resulted in a growth lag phase
of approximately 12^24 h for both tested Arthrobacter
strains (data not shown) [15]. Oxygen electrode measurements demonstrated that all detectable oxygen had been
utilized approximately 20 min after the shift. Nevertheless,
A. globiformis and A. nicotianae showed signi¢cant anaerobic growth which was stimulated by the presence of nitrate in the medium (Fig. 1a,b). In agreement with these
¢ndings complete reduction of nitrate via nitrite to ammonia by both organisms was demonstrated via time-dependent analysis of the growth medium (Fig. 1c,d). A. globiformis converted most of the formed nitrite immediately
into ammonia (Fig. 1c) while A. nicotianae accumulated
nitrite up to levels of 5 mM prior to further reduction
(Fig. 1d). No obvious signs for denitri¢cation processes
such as the development of gas or increased pressure in
the anaerobic £asks during anaerobic growth with nitrate
2.3. High performance liquid chromatography (HPLC)
analysis of fermentation products
Fermentation products were determined from the supernatant of the cultures after removal of the bacteria by
centrifugation [14].
Fig. 1. Aerobic growth (R) of A. globiformis DSM 20124 (a) and A.
nicotianae DSM 20123 (b). A. globiformis and A. nicotianae were grown
at 30‡C anaerobically in Corynebacteria medium as outlined in Section
2 using 25 mM glucose and 10 mM pyruvate as carbon source with no
further additions (O) and with the addition of 10 mM nitrate (E).
Growth was followed by OD578 nm measurements in combination with
viable cell counts. The nitrate (b), nitrite (F) and ammonia (8) concentrations in the growth media during anaerobic growth of A. globiformis
(c) and A. nicotianae (d) were determined.
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229
Table 1
Fermentation product formation by A. globiformis
Electron acceptor
(mM)
Consumed carbon sources
(mM)
Nitrateb
Glucoseb
Pyruvateb
10
^
^
7
2
1.5
10
6
^
Concentrations of
grown cells (g (wet weight) l31 )
4.1
0.9
0.7
Formed products (mol mol31 pyruvatea ) [total (mM)]
Lactate
Acetate
Ethanol
0.50 [12]
0.50 [5]
0.30 [0.9]
0.29 [7]
0.40 [4]
0.53 [1.6]
0.17 [4]
0.05 [0.5]
0 [^]
A. globiformis was incubated anaerobically for 48 h at 30‡C in Corynebacteria medium using 10 mM nitrate as electron acceptor, 25 mM glucose and
10 mM pyruvate as carbon source where indicated. The amounts of glucose and pyruvate consumed and fermentation products formed were quanti¢ed
using HPLC analysis of the growth media as described in Section 2. Formed CO2 and redox equivalent turnover as outlined in Fig. 2 have to be added
to balance the fermentation equation.
a
The amount of pyruvate consumed includes the quantity of externally added pyruvate consumed with pyruvate formed from glucose. Even in experiments where only glucose was used, glucose metabolism proceeds via pyruvate. Therefore, to make the results easy to compare we related the formed
products to the single C3-carbon source pyruvate.
b
Compounds utilized.
were observed. Finally, benzyl viologen-dependent nitrate
reductase activity was found in cell-free extracts prepared
from anaerobically grown A. globiformis and A. nicotianae. Nitrate reductase activities were detected in extracts
prepared from A. globiformis grown anaerobically in the
presence (107 S 10 U g31 protein) and absence (92 S 10 U
g31 protein) of nitrate. Extracts prepared from aerobically
grown A. globiformis in the presence or absence of nitrate
did not contain signi¢cant nitrate reductase activities (less
than 10 U g31 protein). These results indicate an anaerobic induction of A. globiformis respiratory nitrate reductase activity independent of the presence of the utilized
electron acceptor, nitrate. In contrast, signi¢cant nitrate
reductase activity (45 S 7 U g31 protein) was only observed in extracts prepared from A. nicotianae grown anaerobically in the presence of nitrate.
The addition of 5 mM nitrite stimulated anaerobic
growth of A. globiformis, however, after a lag phase of
up to 24 h which is probably due to the toxicity of the
electron acceptor (data not shown). Our current data do
not allow to discriminate whether the analogs of the in
Gram-negative bacteria periplasmic Nap-Nrf or the cytoplasmic NarG-NirB systems [16], or even both, function in
A. globiformis. However, as discussed by Potter et al. [17]
no Nap homologs have yet been found in Gram-positive
bacteria.
A. globiformis also grew anaerobically in the absence of
nitrate and nitrite (Fig. 1a). Anaerobic growth experiments
using 25 mM glucose and 10 mM pyruvate as carbon
sources demonstrated the formation of acetate, lactate
and ethanol indicative of a mixed acid fermentation (Table
1). No obvious formate formation was observed. Again,
similar to observations made for B. subtilis fermentative
growth, only the combination of glucose with pyruvate as
carbon sources yielded su⁄cient anaerobic growth (Fig.
1a) [13,14,18]. Glucose as sole carbon source only allowed
very weak anaerobic fermentative growth of A. globiformis
(data not shown). In agreement only small quantities of
fermentation products were formed (1.6 mM acetate, 0.9
mM lactate and no detectable ethanol) after 48 h of an-
aerobic cultivation with glucose as sole carbon source.
HPLC analysis demonstrated the almost stoichiometrical
conversion of the utilized carbon sources into the detected
fermentation products (Table 1). Similar to B. subtilis,
signi¢cant fermentation product formation was also observed under anaerobic growth conditions in the presence
of the alternative electron acceptor, nitrate. Nitrate was
completely converted by A. globiformis into ammonia after
20 h of anaerobic growth (Fig. 1c). After 48 h all of the
pyruvate and about 30% of the added glucose were consumed and converted into 12 mM lactate, 7 mM acetate
and 4 mM ethanol. Based on our observations we propose
a model for the mixed acid fermentation of A. globiformis
which is shown in Fig. 2.
No fermentative growth was observed for A. nicotianae.
Fig. 2. Proposed initial model for the A. globiformis fermentation processes. Our investigation does not exclude the presence of additional fermentative pathways.
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The alternative electron acceptors TMAO (10 mM),
DMSO (10 mM), fumarate (10 mM) and thiosulfate
(2 mM, 5 mM) did not increase the anaerobic growth of
A. globiformis and A. nicotianae.
[5]
3.2. Conclusion
[6]
Not all members of the genus Arthrobacter are strict
aerobes. Anaerobic cultures of A. nicotianae reduced nitrate to ammonia. A. globiformis was even more £exible
since it can use nitrate as terminal electron acceptor and
mixed acid fermentation under anaerobic conditions.
[7]
[8]
[9]
[10]
Acknowledgements
[11]
We thank R. Brandsch, Freiburg, Germany, for the gift
of Arthrobacter strains and helpful discussions. We thank
R.K. Thauer, Marburg, Germany, for continuous support.
Financial support was provided by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
[12]
[13]
[14]
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