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FEMS Microbiology Letters 167 (1998) 7^11
Increased alanine dehydrogenase activity during dormancy in
Mycobacterium smegmatis
Bernd Hutter, Thomas Dick *
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore
Received 5 June 1998; revised 22 July 1998; accepted 4 August 1998
Abstract
The aerobic fast-growing Mycobacterium smegmatis has, like its slow-growing pathogenic counterpart M. tuberculosis, the
capability to adapt to anaerobiosis by shifting down to a drug resistant dormant state. Here, we report the identification of the
first enzyme, L-alanine dehydrogenase, whose specific activity is increased during dormancy development in M. smegmatis.
This mycobacterial enzyme activity was previously identified as the 40-kDa antigen in M. tuberculosis and shows a preference
for the reductive amination of pyruvate to alanine at physiological pH. The determination of the temporal profile of alanine
dehydrogenase activity during dormancy development showed that the activity stayed at a low baseline level during the initial
aerobic exponential growth phase (0.7 mU mg31 min31 ). After termination of aerobic growth, alanine dehydrogenase activity
increased rapidly 5-fold. As oxygen becomes more and more limiting, the enzyme activity declined until it reached a level about
two-third that of the peak value. The strong induction immediately after deflection from aerobic growth suggests that alanine
might be required for the adaptation from aerobic growth to anaerobic dormancy. As alanine synthesis is coupled to NADH
oxidation, we propose that the induction of alanine dehydrogenase activity might also support the maintenance of the NAD
pool when oxygen as a terminal electron acceptor becomes limiting. z 1998 Federation of European Microbiological
Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Mycobacterium smegmatis ; Dormancy ; Anaerobiosis
1. Introduction
Mycobacterium tuberculosis is a pathogen capable
of causing both an acute disease and an asymptomatic latent infection. In the latent infection, dormant tubercle bacilli persist for years before reviving
and resulting in reactivation of tuberculosis. Our
current arsenal of drugs for treating active tuberculosis is relatively ine¡ective against the latent form
* Corresponding author. Tel.: +65 (874) 3745;
Fax: +65 (779) 1117; E-mail: [email protected]
([1], for review). Lawrence Wayne has conducted
pioneering studies of the dormancy of M. tuberculosis ([2] and references therein). In the Wayne model,
cultures of M. tuberculosis are subjected to gradual
oxygen depletion by incubation in sealed containers
with controlled agitation. Growth under such conditions leads to a physiologically well-de¢ned anaerobic, drug resistant, non-replicating synchronised state
of the tubercle bacillus [3]. The Wayne model has
clinical correlations with human anaerobic latent lesions containing dormant bacilli [4].
Recently, we demonstrated that the physiological
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 3 6 0 - 7
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B. Hutter, T. Dick / FEMS Microbiology Letters 167 (1998) 7^11
behaviour of the fast-growing saprophyte M. smegmatis, under the oxygen-depletion conditions of the
Wayne model, is strikingly similar to that shown by
the slow-growing pathogenic M. tuberculosis [5].
Furthermore, pulse labelling experiments indicated
that the bacilli in the dormant state show low level
RNA [5] and protein synthesis (Dick, unpublished
observations). This indicates that the dormant bacilli
are not metabolically inactive but maintain a low
level metabolism.
We are interested in the molecular mechanisms of
oxygen depletion-induced dormancy and we use M.
smegmatis as a fast-growing non-pathogenic model.
Our present knowledge of the machinery involved in
dormancy is limited [6^9]. One of the central questions is how the bacilli recycle NADH, derived from
their catabolic activities, under oxygen limiting conditions. M. tuberculosis L-alanine dehydrogenase (EC
1.4.1.1, 40-kDa antigen [10,11]) catalyses the reductive amination of pyruvate to alanine under physiological pH [12,13]. We report here that the speci¢c
activity of alanine dehydrogenase during dormancy
development was increased. We propose that the enzyme, whose suggested function is the generation of
alanine for protein and peptidoglycan synthesis, may
play an additional role in maintaining the NAD pool
under oxygen limiting conditions.
2. Materials and methods
2.1. Strain and cultivation
All experiments were conducted with M. smegmatis mc2 155 [14] in Dubos Tween-albumin broth (Difco) at 37³C. Anaerobic dormant and aerobic growing M. smegmatis cultures were prepared and
monitored as described previously [5,3]. Brie£y,
screw-cap test tubes, 20U125 mm, with a total £uid
capacity of 25.5 ml were used as containers. An early
exponential phase preculture was diluted to 106 c.f.u.
ml31 in a total volume of 17 ml. For sealed dormancy cultures, solid caps with latex liners were
tightly screwed down and the cultures were gently
stirred at 170 r.p.m. Self-generated oxygen depletion
was monitored via the decolorisation of the oxygen
indicator dye, methylene blue. For aerated exponentially growing cultures, the caps were loosely tight-
ened to allow air to enter and the tubes were incubated under vigorous shaking at 250 r.p.m. until they
reached early exponential phase (2U107 c.f.u. ml31 ).
Growth and survival of the bacterial populations
were monitored by viable count measurements. Microscopic examination of the culture samples before
plating showed that signi¢cant clumping a¡ecting
c.f.u. determination did not occur.
2.2. Preparation of extracts, assay of protein, in situ
and photometric enzyme assays
Protein extracts were prepared according to Basu
et al. using glass beads to break up the cells [15].
Protein concentrations were determined using a
Bio-Rad kit as recommended by the supplier. Alanine dehydrogenase activity was visualised in nondenaturating polyacrylamide gels using nitroblue tetrazolium and phenazine methosulfate as described
previously [11]. For the detection of other NAD-dependent amino acid dehydrogenase activities, gels
were incubated as described for the alanine dehydrogenase with the respective amino acid, in a bu¡er of
20 mM Tris-HCl, pH 8.0 [16]. The photometric alanine dehydrogenase assay is based on measurement
of the rate of reduction of NAD to NADH that
accompanies the oxidative deamination of alanine
to pyruvate. Units of speci¢c activity of alanine dehydrogenase are expressed as micromoles of NAD
reduced per min per milligram of protein [17]. For
the photometric determination of the reductive amination of pyruvate to alanine the reaction mixture
contained 20 mM Tris-HCl, pH 8.0, 20 mM pyruvate and 0.5 mM NADH. In order to determine the
pH dependence of the reactions the following bu¡ers
were used: 40 mM Na2 HPO4 /NaH2 PO4 , pH 7.0; 20
mM Tris-HCl, pH 8.0; 20 mM Tris-HCl, pH 9.0;
125 mM glycine-KOH, pH 10.2. For the photometric measurement of proline dehydrogenase activity
alanine was substituted by proline and 20 mM
Tris-HCl, pH 8.0, was used as bu¡er. To detect glycine dehydrogenase activity, an assay based on optical measurement of the rate of oxidation of NADH
to NAD that accompanies the reductive amination
of glyoxylate to glycine was used [18,19]. The activities of all amino acid dehydrogenases were measured
against a cuvette containing the reaction mixture
without the respective substrate.
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B. Hutter, T. Dick / FEMS Microbiology Letters 167 (1998) 7^11
Fig. 1. Demonstration of alanine and proline dehydrogenase activity in growing and dormant cultures by native gel electrophoresis. Activity-stained 7.5% non-denaturing polyacrylamide gels
after electrophoresis of extracts from aerobic growing and 10
days old anaerobic dormant cultures are shown. Amino acid dehydrogenase activities are visualised as dark bands. A: Control
staining without amino acid substrate. B: Alanine dehydrogenase
activity was detectable in aerobic growing cultures (lane 1) and
showed an increase in anaerobic dormant culture (lane 2). C :
Proline dehydrogenase activity was detectable in aerobic growing
cultures (lane 1) and anaerobic dormant culture (lane 2) at similar levels. Loading was 10 Wg of total protein. In addition to
amino acid dependent activities an amino acid independent positive staining in the extracts from anaerobic dormant culture was
detected (upper smear in A, B, C, lanes 2). Furthermore, several
culture independent negative staining bands could be detected
which were also amino acid independent. Omitting NAD from
the reaction mix gave the same results demonstrating that the
amino acid independent positive and negative stainings were also
NAD independent (not shown). The nature of the responsible reactions is not known.
9
ution without alanine. No reaction was observed,
showing that the bands detected in Fig. 1B are alanine dependent. These results demonstrate that M.
smegmatis contains alanine dehydrogenase and that
this activity is upregulated in the anaerobic non-replicating persistent culture.
In order to analyse the temporal pro¢le of alanine
dehydrogenase activity during dormancy development, we determined the enzyme activity under
sealed dormancy culture conditions at various time
points. Fig. 2 shows that the activity stayed at a low
baseline level during the initial aerobic exponential
growth phase (0.7 mU mg31 min31 ). After termination of aerobic growth, alanine dehydrogenase activity increased rapidly to a level 5-fold greater than the
baseline level. As oxygen became more and more
limiting, the enzyme activity declined until it reached
a level about two-third that of the peak value. These
results show that alanine dehydrogenase activity is
strongly upregulated immediately upon oxygen limitation-induced termination of growth. Then the level
declines but stays 3.5-fold above the baseline level
seen during aerobic growth. The strong induction
3. Results and discussion
3.1. Alanine dehydrogenase activity is upregulated
during dormancy
To assess whether L-alanine dehydrogenase activity can be detected in M. smegmatis and whether
there is a di¡erence in the enzyme levels between
aerobic growing and anaerobic dormant cultures,
protein extracts from both cultures were analysed
by an in situ activity assay after electrophoresis in
non-denaturing polyacrylamide gels. Fig. 1B (lanes 1
and 2) shows that alanine dehydrogenase activity
was detectable in the extract from the aerobic growing culture and that the activity was increased in the
extract from the anaerobic dormant culture. Fig. 1A
shows an identical gel incubated in the substrate sol-
Fig. 2. Temporal pro¢le of alanine dehydrogenase activity in
sealed dormancy cultures. Aerobic logarithmic preculture was diluted to 106 c.f.u. ml31 and incubated in sealed tubes under gentle stirring conditions as described in Section 2. Protein extracts
were prepared at various time points and speci¢c alanine dehydrogenase activities were determined using a photometric assay
(circles). Growth and survival of the sealed culture was monitored by determination of the viable counts (c.f.u. ml31 ; squares)
and was as described previously [5]. Mean values and standard
deviation are shown from two experiments. Methylene blue was
used as the indicator for oxygen depletion. The times at which
cultures containing 1.5 Wg of methylene blue per ml exhibited
fading (f) and complete decolorization (d) of the dye are indicated [5].
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B. Hutter, T. Dick / FEMS Microbiology Letters 167 (1998) 7^11
immediately after de£ection from aerobic growth ^
compared to the less pronounced induction during
anaerobic dormancy ^ suggests that the enzyme
might play a role in the adaptation from aerobic
growth to anaerobic dormancy. What might this
physiological role be?
Alanine dehydrogenase from M. tuberculosis catalyses the reductive amination of pyruvate at physiological pH [12,13]. To demonstrate that the M.
smegmatis enzyme shows similar pH dependence, activity measurements at various pHs (pH 7.0^10.2)
were carried out. These analyses showed that the
velocity of oxidative deamination of alanine at physiological pH was only about 10% compared to the
velocity at pH 10.2. In contrast, the pH optimum for
the reductive amination of pyruvate was pH 7^8.
This suggests that the metabolic function of the enzyme is the generation of alanine. Alanine is required
for protein and peptidoglycan synthesis. Therefore,
the induction of alanine dehydrogenase activity
might indicate the requirement of protein and/or
peptidoglycan synthesis for the development of the
persistent dormant form of the bacilli. The generation of alanine is accompanied by the oxidation of
NADH. This aspect of alanine synthesis might play
a role under the oxygen limiting conditions encountered after de£ection from aerobic growth and later
during anaerobic dormancy. Therefore, we propose
that the induction of alanine dehydrogenase activity
might also support the maintenance of the NAD
pool when oxygen as terminal electron acceptor becomes limiting.
3.2. Proline dehydrogenase activity is growth phase
independent and glycine dehydrogenase activity
is not detectable
In addition to alanine dehydrogenase, two other
NAD-dependent amino acid dehydrogenases have
been identi¢ed in M. tuberculosis. A putative proline
dehydrogenase gene (EC 1.5.99.8) was discovered in
the genome project [20]. Glycine dehydrogenase activity (EC 1.4.1.10) was discovered by Goldman and
Wagner [18]. To assess whether proline and glycine
dehydrogenase activities can be detected in M. smegmatis, extracts from aerobic cultures and anaerobic
dormant cultures were analysed in non-denaturing
polyacrylamide gels. In addition the extracts were
tested for NAD-dependent dehydrogenase activities
for the other 17 amino acids found in proteins. The
only amino acid dehydrogenase detectable was proline dehydrogenase showing the same staining in
growing and dormant culture (Fig. 1C). Photometric
quanti¢cation of proline dehydrogenase revealed a
growth phase independent activity of 0.3 mU
min31 mg31 . Photometric glycine dehydrogenase assays con¢rmed the absence of glycine dehydrogenase
activity in M. smegmatis.
Wayne and coworkers demonstrated recently an
increase of glycine dehydrogenase activity during
oxygen depletion induced dormancy in M. tuberculosis and the authors proposed that the reductive
amination of glyoxylate to glycine could provide
one of the mechanisms used by the bacilli to recycle
their NADH under oxygen limiting conditions [3,19].
In this work we demonstrate the upregulation of
alanine dehydrogenase activity during oxygen depletion in M. smegmatis. Whether alanine dehydrogenase activity is increased during dormancy in M. tuberculosis or whether this enzyme activity substitutes
the missing glycine dehydrogenase in M. smegmatis
during oxygen limitation remains to be elucidated.
Acknowledgments
We would like to thank Marianne Eleuterio for
comments on the manuscript. This study was supported by the Institute of Molecular and Cell Biology (IMCB).
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