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Infection and Immunity, July 2000, p. 4084-4091, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
High Intracellular Level of Guanosine Tetraphosphate in
Mycobacterium smegmatis Changes the Morphology of
the Bacterium
Anil K.
Ojha,1
Tapan K.
Mukherjee,1 and
Dipankar
Chatterji2,*
Centre for Cellular and Molecular Biology,
Hyderabad, 500007,1 and Molecular
Biophysics Unit, Indian Institute of Science, Bangalore
560012,2 India
Received 6 December 1999/Returned for modification 14 February
2000/Accepted 30 March 2000
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ABSTRACT |
Almost one-third of the world population today harbors the tubercle
bacillus asymptomatically. It is postulated that the morphology and
staining pattern of the long-term persistors are different from those
of actively growing culture. Interestingly, it has been found that the
morphology and staining pattern of the starved in vitro population of
mycobacteria is similar to the persistors obtained from the lung
lesions. In order to delineate the biochemical characteristics of
starved mycobacteria, Mycobacteria smegmatis was grown in
0.2% glucose as a sole carbon source along with an enriched culture in
2% glucose. Accumulation of the stringent factor guanosine
tetraphosphate (ppGpp) with a concomitant change in morphology was
observed for M. smegmatis under carbon-deprived conditions.
In addition, M. smegmatis assumed a coccoid morphology when
ppGpp was ectopically produced by overexpressing Escherichia coli
relA, even in an enriched medium. The Mycobacterium
tuberculosis relA and spoT homologue, when induced in
M. smegmatis, also resulted in the overproduction of ppGpp
with a change in the bacterium's growth characteristics.
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INTRODUCTION |
Mycobacteria have emerged as a major
threat to humankind, for as many as one-third of the world's
population (1.7 billion) harbors the tubercle bacillus asymptomatically
(18). The latent bacilli can persist in a somewhat
ill-defined physiological state in pulmonary and extrapulmonary lesions
for years after infection (30). These bacilli are
opportunistic and can reactivate themselves when the host is
immunocompromised. To add to the misery, persistors require prolonged
therapy, and Mycobacterium bovis BCG vaccination has little
effect in blocking reactivation of the bacteria. Hence, for improved
control of tuberculosis, it is imperative to develop effective drugs to
cease the propagation and persistence of these latent bacilli. This
will be greatly facilitated by a better understanding of the
physiological state of these latent bacteria. Although several in vitro
models suggest low extracellular concentrations of oxygen to be an
important cause for mycobacterial dormancy (8, 10, 42), the
effect of this state on cellular metabolism is not clear.
It has been shown that the morphology and staining pattern of an in
vitro culture of mycobacteria differ from those of persistors which are
obtained from the lung lesion and are chromophobic to the conventional
acid-fast staining (25, 26). The former is an acid-fast and
long rod-shaped bacillus, as opposed to the latter, which is non-acid
fast and granular. However, these persistors can be stained after
oxidizing the cell surface with periodic acid. In yet another important
observation, it has been shown that the morphology and staining pattern
of such persistors can be obtained in vitro by starving the
Mycobacterium tuberculosis, Mycobacterium
kansasii, or Mycobacterium pheli cultures on agar plates without any nutrients (26). This key observation
suggests that the natural persistors may be physiologically similar to bacteria in nutritionally starved cultures. Thus, studying the physiology and morphology of such starved cultures could provide some
important clues towards understanding the mechanism of latency. This
hypothesis prompted us to take up the study of starving mycobacteria.
Bacteria adapt to nutritional stress for their survival, predominantly
through a mechanism termed the stringent response. The hallmark of the
stringent response is the accumulation of guanosine tetraphosphate
(ppGpp), also called the stringent factor, and downregulation of stable
RNA (rRNA and tRNA) synthesis (3). It appears that RNA
polymerase is the ultimate target of ppGpp (6), although the
exact mode of selective downregulation of the gene expression is not clear.
Many bacteria can assume a well-defined physiological state under
starvation conditions, which facilitates their survival (23, 27,
38). The role of ppGpp in the developmental process of these
physiological states has been a subject of interest for many
researchers over the years. It has been extensively studied in
Myxococcus xanthus, in which accumulation of ppGpp has been observed to be an important requirement for the formation of the fruiting body (16). In Streptomyces coelicolor,
ppGpp has been implicated in the synthesis of antibiotics in the
stationary phase of the bacteria (5). Though ppGpp has been
detected in various other prokaryotes during starvation, e.g.,
Bacillus subtilis (28), Bacillus
stearothermophilus (12), Staphylococcus spp.
(4), Streptococcus equisimilis (24),
and Salmonella enterica serovar Typhimurium (20,
35), its function in these organisms is yet to be assigned.
Although Mycobacterium smegmatis is nonpathogenic, it shares
many biosynthetic pathways with M. tuberculosis and may
serve as a good model system. In addition, its higher growth rate makes it a suitable candidate for starvation studies. In this study we have
shown that ppGpp accumulation is accompanied by morphological change in
M. smegmatis under carbon starvation conditions.
Furthermore, we have shown that M. smegmatis assumes the
coccoid morphology (similar to the persistors) when ppGpp is
ectopically produced by overexpression of Escherichia coli
relA in an enriched nutritional medium. We have also characterized
the in vivo function of the M. tuberculosis relA/spoT
homologue in M. smegmatis.
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MATERIALS AND METHODS |
Strains, media, and growth.
M. smegmatis, strain
mc2155, was grown in MB7H9 (Difco) with 2% glucose and
0.05% Tween 80 for enriched culture. In the carbon-starved medium the
glucose concentration was reduced to 0.2% without any Tween 80 in the
medium. For comparative studies of pMatt1 and pMatt2, samples of the
culture stock from 
70°C were subcultured once before being
inoculated in the experimental culture. For acetamide-induced
expression, bacteria were grown in MB7H9 having 2% succinate with
0.05% Tween 80 and the culture was induced with 2% acetamide. For the
plate culture the same composition was used with 1.5% agar. The growth
kinetics of the culture was studied by measuring the culture's optical
density (OD) at 600 nm. E. coli strain CF3120 is
relA-overexpressing strain which bears relA under
the control of the Ptac-lacIq promoter-operator
system on a multicopy plasmid, pALS10. The gene is induced by the
addition of 0.5 mM isopropyl-
-D-thiogalactopyranoside (IPTG) to Luria-Bertani medium.
Plasmids.
For overexpression of E. coli relA,
pMV261 (40) bearing Phsp60 was converted into an integrative
vector, pMatt1, by removing its NotI fragment containing
oriM and ligating the backbone with the SalI
fragment of pDK20 (9), which consists of the integrating signal of mycobacteriophage L5. Then the
EcoRI-HindIII relA fragment from
pALS10 (a gift from Mike Cashel) was subcloned into the
EcoRI-HindIII site of pMatt1 to generate
pMatt2, thus generating a transcriptional fusion of relA
with Phsp60.
The M. tuberculosis relA/spoT homologue (Rv2583c) along with
its ribosome binding site (RBS) was obtained from the
KpnI-EcoRV fragment of the cosmid pY227
(7) and subcloned into the KpnI-XbaI (end-filled with Klenow fragment) ends of pAGAN90 (29). The recombinant plasmid, pMtrel2, has an acetamide-inducible 2.2-kb transcriptional apparatus fused to the RBS and open reading frame (ORF)
of the gene.
ppGpp detection.
A 25-ml M. smegmatis culture was
grown to an OD of 0.2, and then
[32P]H3PO4 (BRIT, Hyderabad,
India) was added to it to a final concentration of 100 µCi/ml. The
labeled cells were harvested at an OD of 0.8, washed once with 10 mM
Tris (pH 8.0), resuspended in 50 µl of buffer, treated with 1 mg of
lysozyme per ml on ice for 20 min, and lysed with 1% sodium dodecyl
sulfate (SDS), and ppGpp was extracted with an equal volume of 2 M
formic acid. After centrifugation at a high speed at a cold temperature
for 10 min, 5 µl of the supernatant was loaded on a polyethyleneimine
(PEI)-coated thin-layer chromatography (TLC) plate (Merck). The plate
was developed in 1.5 M KH2PO4 (pH 3.4) in one
dimension. It was air dried and exposed to X-ray film (Konika) for 18 to 24 h at 70°C for autoradiography. For alkaline hydrolysis
of ppGpp, the formic acid extract was immediately neutralized with
NH4OH, treated with 0.3 M KOH, and then treated with 0.1 M
BaCl2 at 37°C for 1 h.
In order to detect ppGpp in a
relA or
relA/spoT-overexpressing system, the
32P-labeled
culture was induced at an OD of 0.4 and harvested at
an OD of 0.8.
Microscopy.
The heat-fixed smear of the culture was stained
with carbol-fucshin (Loba Chemicals, Mumbai, India), washed with 20%
H2SO4, counterstained with methylene blue (Loba
Chemicals), and observed under a light microscope (Olympus)
(magnification, ×1,000) in either phase-contrast or bright-field mode.
Complementation analysis.
For functional complementation of
M. tuberculosis relA/spoT in E. coli, the
relA strain of E. coli (MC4100) was transformed with pMtrel2 and selected on Luria-Bertani agar containing kanamycin (50 µg/ml). The transformants were streaked on glucose M9 minimal agar plus 100 µg of serine, methionine, and glycine (SMG) per ml or
glucose M9 minimal agar. The reversion of the relA strain to
relA+ strain was observed by the loss of
sensitivity to SMG.
Miscellaneous.
All the cloning experiments and
immunodetection were carried out as described earlier (32).
The immunoblot was analyzed by a densitometer (Bio-Rad). DNA was
electroporated into M. smegmatis using a Bio-Rad
electroporator at 1.5 kV/mm (17), and transformants were
selected on MB7H9 agar containing 20 µg of kanamycin per ml.
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RESULTS |
Growth kinetics, ppGpp accumulation, and morphology.
M.
smegmatis, strain mc2155, was grown in an enriched
(2% glucose) and carbon-deficient (0.2% glucose) medium. The MB7H9
medium contains L-glutamic acid, which can be used as a
carbon source for M. smegmatis, albeit inefficiently.
However, the total carbon coming from 0.2% glucose and glutamic acid
was much less in comparison to that from an enriched medium. The
bacteria followed altered growth kinetics when grown in
carbon-deficient medium. The carbon-deficient culture showed earlier
entry into stationary phase at a lower cell density, with an average
generation time of 2.8 h, whereas the normal culture, which
doubled every 2.0 h, had a very high cell density in the
stationary phase (Fig. 1). This
observation is consistent with an earlier report on growth kinetics and
stationary phase entry of M. smegmatis in carbon-limited
medium (37). Based in this observation we termed the 0.2%
glucose medium as carbon-starved medium for the bacteria.
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FIG. 1.
Comparative growth kinetics of normal ( ) and
carbon-starved ( ) cultures of M. smegmatis. The
generation time of a normal culture grown in enriched medium (2%
glucose) is 2.0 h, whereas that of a carbon-starved culture (0.2%
glucose) is 2.8 h. An early entry into the stationary phase at low
cell density of the latter is evident from the profile.
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Following the observation of the growth kinetics the question of
whether mycobacteria accumulate ppGpp upon carbon starvation
was
raised. This question stems from the fact that ppGpp is almost
a
universal growth regulator in starved prokaryotes. In order
to detect
the ppGpp accumulation in
M. smegmatis,
32P-labeled mid-log-phase cells of equal OD (0.8) in
enriched and
carbon-starved medium were subjected to the extraction
procedure
(see Materials and Methods). It can be seen from Fig.
2 and comparing
lanes 1 and 2 that the
formic acid extracts from the cells grown
in carbon-deficient medium
clearly showed accumulation of ppGpp,
in contrast to the
cellular extract from enriched medium. The
authenticity of ppGpp
was demonstrated by its comigration with
ppGpp from the
32P-labeled formic acid extract of
E. coli
overexpressing
relA (CF3120)
(Fig.
2, lane 3). The alkaline
hydrolysis of ppGpp, as reported
earlier (
2,
31), was
carried out to further confirm the existence
of the nucleotide.
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FIG. 2.
Accumulation of ppGpp in carbon-starved M. smegmatis. Five microliters of 32P-labeled formic acid
extract of normal culture grown in MB7H9-2% glucose-0.05% Tween
80 (lane 1) and carbon-starved culture grown in MB7H9-0.2% glucose
(lane 2) were loaded on a PEI-coated TLC plate and resolved as
mentioned in the text. To confirm the authenticity of the spot, a
32P-labeled formic acid extract of E. coli
strain (CF3120, overexpressing the ppGpp synthase gene,
relA, was loaded as a control (lane 3).
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In order to compare the morphologies of the normal and carbon-starved
bacteria, the acid-fast stains of the two cultures at
an OD of 0.8 were
prepared and observed under phase-contrast microscope
at a
magnification of ×1,000. It was observed that the cells under
carbon
starvation showed reduction in length (almost like a coccoid)
in
comparison to the normal bacilli (Fig.
3). In order to rule
out any effect of
Tween 80 on the growth kinetics and morphological
changes, the
carbon-deficient medium was supplemented with 0.05%
Tween 80, and the
observations were repeated (not shown). Moreover,
the morphology of the
bacteria was also observed from the plate
culture that was devoid of
Tween 80.
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FIG. 3.
Morphological difference between normal (a) and
carbon-starved (b) cultures of M. smegmatis. The two
cultures were grown in the same way as mentioned in the legend for Fig.
2. The heat-fixed smear of the cells was stained with carbol-fuchsin
and observed under a light microscope in the phase-contrast mode at
×1,000 magnification.
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Such a morphological change has been reported previously in
late-stationary-phase cultures of
M. smegmatis
(
37), in which
the length of the stationary-phase bacterium
is reduced to half.
There is indirect evidence to suggest that ppGpp
regulates the
cell division through FtsZ (a protein required for septum
formation)
in
E. coli (
41). Overexpression of
relA in
E. coli leads to
enhanced septum
formation and reduced cell size (
33). The reduced
cell size
with concomitant accumulation of ppGpp in
M. smegmatis suggests a possible role of ppGpp in the morphological changes
in
mycobacteria.
Overexpression of E. coli relA.
In order to understand
the correlation between morphological changes and accumulation of
ppGpp, we tried to overexpress relA (ppGpp synthase) from
E. coli in M. smegmatis. An attempt to
overexpress relA from a multicopy plasmid (pMV261)
containing the BCG Phsp60 promoter was not successful. This could be
explained by the fact that the gene driven by Phsp60 from a multicopy
vector would produce a high level of ppGpp, which would perhaps be
toxic for the organism. Since cellular response to ppGpp is dose
dependent, a single-copy vector was chosen. An integrative vector
(pMatt1) was constructed by replacing oriM from pMV261 with
att-int (the attachment site and integrase protein, from
mycobacteriophage L5). A transcriptional fusion of E. coli
relA and Phsp60 was constructed by subcloning the gene along
with its translational signal downstream of Phsp60 in pMatt1 to
generate pMatt2. Both pMatt1 and pMatt2 were electroporated into
M. smegmatis. The expression of relA at the
translational level was confirmed by immunoblotting against
anti-E. coli relA antibody (Fig.
4A), and the bands were
quantitated using a densitometer. Although the basal level of protein
at 30° was high, the expression was temperature dependent. There was
an almost 2.5-fold increase in expression upon shifting the culture
from 30 to 44°C. However, there was only a 30% increase in protein
level when the culture was shifted from 30 to 37°C. Hence, for all
the subsequent experiments on pMatt1 and pMatt2 strains the cultures
were maintained at 30°C and induced by shifting to 44°C. The
intracellular levels of ppGpp in the strains containing pMatt1 and
pMatt2 were compared in mid-log phase. As can be seen from Fig. 4B, the
strain containing pMatt2 showed accumulation of ppGpp in contrast to
the strain containing pMatt1, in which there was no such accumulation.
It confirmed the expression and function of E. coli relA in
M. smegmatis.
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FIG. 4.
(A) Expression of E. coli relA in
M. smegmatis. Equal amounts of protein from whole-cell
lysate of pMatt1 (lanes 1 to 3) and pMatt2 (lanes 4 to 6) grown at
30°C (lanes 1 and 4), 37°C (lanes 2 and 5), and 44°C (lanes 3 and
6) were subjected to SDS-8% PAGE, transferred to nitrocellulose, and
probed with anti-E. coli relA antibody. (B) Accumulation of
ppGpp upon overexpression of E. coli relA in
M. smegmatis. The strain overexpressing
relA, pMatt2, was induced along with the control strain,
pMatt1, by shifting the culture (at an OD of 0.4) from 30 to 44°C.
Five-microliter aliquots of 32P-labeled formic acid extract
of pMatt1 (lane 1) and pMatt2 (lane 2) were loaded on PEI-coated TLC
plates, and spots were resolved as mentioned in the legend for Fig. 2.
Comigration of purified cold ppGpp (not in picture) confirmed the
labeled ppGpp spot. (C) Effect of high intracellular levels of ppGpp on
growth kinetics of M. smegmatis. The generation time of
the strain overexpressing E. coli relA, pMatt2 (), was
2.6 h, compared to 2.1 h for the control, pMatt1 (). Both
the cultures were grown in MB7H9-2% glucose-0.05% Tween 80 at
30°C till mid-log phase and were induced by shifting to 44°C. The
arrow indicates the time of induction.
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Since ppGpp is known to be a growth regulator in various prokaryotes,
the effect of ppGpp accumulation on the growth kinetics
of
M. smegmatis was studied. As expected, we observed a slow growth
rate
for the strain harboring pMatt2 in comparison to the one
with pMatt1
(Fig.
4C). Although the time of entry into stationary
phase for the two
cultures was the same, the OD of the pMatt2
culture was lower
than that of the pMatt1 culture. This observation
has been
reported previously in other organisms (
34,
36).
The
observed growth kinetics, as expected, suggest that ppGpp
is likely to
have a mechanism of operation in mycobacteria similar
to that in other
prokaryotes. However, the overall change in growth
kinetics in this
case is quantitatively different (the generation
time of pMatt1 and
pMatt2 are 2.1 and 2.6 h, respectively) from
the one
noticed under carbon starvation (Fig.
1). This can be
explained by the
fact that global metabolism of the cell would
be affected more
by nutritional depletion than by accumulation
of one regulatory factor.
Furthermore, poor regulation of expression
and subsequent outgrowth of
suppressor variants would further
reduce the effect of the regulatory
molecule on the growth rate
of the bacterial
population.
In order to observe the morphological change as a consequence of ppGpp
accumulation, the two strains (pMatt1 and pMatt2) were
grown at 30°C
in enriched medium to an OD of 0.4 and then were
shifted to 44°C.
Then heat-fixed smears were stained with carbol-fuchsin.
The slides
were observed under a bright-field microscope (×1,000
magnification).
The strain carrying pMatt2 appeared as short cocci,
in contrast to the
pMatt1 strain, which appeared as long, thin,
rod-shaped bacilli (Fig.
5). These microscopic observations again
indicate that ppGpp plays a crucial role in the morphological
changes
in
M. smegmatis.
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FIG. 5.
Morphological difference as a consequence of ppGpp
accumulation in M. smegmatis. The strain harboring
pMatt2 (overexpressing E. coli relA) has a spherical
morphology (B) compared to the elongated rod shape morphology of the
strain carrying pMatt1 (empty vector) (A). The two cultures were grown
at 30°C till an OD of 0.4 was reached and then was induced by
incubating at 44°C. After 4 h of induction the
carbol-fuchsin-stained bacteria were observed with a light microscope
in the bright-field mode at ×1,000 magnification.
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Functional Characterization of M. tuberculosis
relA/spoT in vivo (i) Overexpression of M. tuberculosis relA/spoT homologue in M. smegmatis.
Upon analyzing the genome sequences, a putative ppGpp synthase has been
identified in M. tuberculosis (7) and
Mycobacterium leprae (11). Interestingly, the
single gene identified has almost 50% homology to both relA
and spoT (encoding ppGpp hydrolase) of E. coli.
Hence, it is possible that mycobacteria have only one gene for both
synthetic as well as hydrolytic activity of ppGpp. Furthermore, the
relA/spoT homologues from the two species of mycobacteria
are 97% identical in their amino acid sequence, which suggests that
the gene is functionally conserved across the species in mycobacteria.
Since the relA/spoT homologue of M. smegmatis is not yet identified, we decided to characterize the
function of M. tuberculosis relA/spoT using
M. smegmatis as a surrogate host. In order to achieve
this, another widely used mycobacterial promoter, Pamidase,
was chosen. This promoter cassette is a 2.2-kb sequence with three ORFs
and several consensus promoter sequences and is induced by addition of
actamide to a medium having a poor carbon source (succinate)
(29). However, the exact mechanism of induction is
unknown. The EcoRV-KpnI fragment (ORF
Rv2583c, cosmid MTCY227) consisting of the relA/spoT ORF
with its RBS was subcloned downstream of the 2.2-kb acetamide-inducible
region of pAGAN90 (29), resulting in a transcriptional
fusion of relA/spoT with an acetamide-inducible promoter.
The recombinant plasmid (pMtrel2) thus obtained was electroporated into
M. smegmatis, and transformants were selected on MB7H9
agar containing kanamycin. The induction of the gene was seen
when the
strain bearing pMtrel2 was grown in MB7H9 broth containing
2%
succinate till mid-log phase, with a subsequent addition of
2%
acetamide. The Coomassie blue-stained gel used for
SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) showed induced
expression of one
89-kDa protein after 2 h of addition of the
inducer (acetamide)
(Fig.
6). The
cellular content of the protein increased even after
20 h of
induction, which indicates a long half-life of the protein.
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FIG. 6.
Coomassie-blue stained polyacrylamide gel showing
acetamide-induced expression of M. tuberculosis
relA/spoT in M. smegmatis. The mid-log phase of
the culture harboring pMtrel2 was induced with 2% acetamide. A 1-ml
aliquot was taken out at the indicated time interval after induction.
Equal amounts of the total cellular proteins were resolved by SDS-8%
PAGE. Lane 1, marker; lanes 2 to 8, 0, 2, 4, 6, 8, 10, and 20 h,
respectively, after induction; lane 9, total cellular protein of a
saturated culture of a strain harboring empty vector, pAGAN90.
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(ii) Complementation of E. coli relA by M. tuberculosis relA/spoT.
Since the 2.2-kb inducible region of
pMtrel2 has appropriately placed E. coli consensus 35 and
10 sequences (21), it was assumed to be transcriptionally
active in E. coli. Hence, the upstream regulatory region of
pMtrel2 was thought to be sufficient for complementation in E. coli. Thus, the plasmid pMtrel2 containing M. tuberculosis relA/spoT was transformed into a relA
strain of E. coli (MC4100). The transformant was checked for
the relA+ phenotype on a minimal medium plate
with SMG. Since relA strains of E. coli are
defective in derepression of the amino acid biosynthetic genes
in amino acid-limiting medium, the cells are rendered sensitive to the
presence of amino acids (through end product inhibition) in
minimal medium (3). As can be seen in Fig.
7A, MC4100, which is sensitive to SMG,
could form colonies on SMG plates when transformed with pMtrel2. This
indicates that the gene coding for the 89-kDa protein can complement
the RelA phenotype in E. coli and thus is a relA
homologue.
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FIG. 7.
(A) complementation of relA E. coli (MC4100)
by the M. tuberculosis relA/spoT homologue. The
reversion of relA to relA+ was
assayed by loss of SMG sensitivity. The transformant harboring empty
vector (pAGAN90) was streaked on the left sector, whereas the one
harboring vector with the gene (pMtrel2) was streaked on the right
sector. Panel (A) Minimal medium plus SMG; panel (B) minimal medium.
(B) ppGpp synthetic activity of the M. tuberculosis
relA/spoT homologue in M. smegmatis. The
32P-labeled cells were grown in MB7H9-2% succinate till
mid-log phase and then induced for 4 h with 2% acetamide.
Five-microliter aliquots of the formic acid extract of
strains pAGAN90 (lane 1) and pMtrel2 (lane 2) were loaded on
PEI-coated TLC plates and the spots were developed as described in the
text.
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(iii) ppGpp accumulation and its effect on cell growth upon
induction of relA/spoT homologue.
In the next
experiment relA/spoT in pMtrel2 was induced in M. smegmatis as described above. The elevated intracellular level of
ppGpp as a consequence of induction was observed (Fig. 7B). There was
no detectable level of ppGpp either in the uninduced state
of pMtrel2 or the empty vector, pAGAN90 (data not shown). The
result indicates the ppGpp synthetic activity of the
relA/spoT homologue. Although the synthesis of pppGpp by the
same gene has been reported in an in vitro experiment
(1), such a product could not be detected unambiguously
in the formic acid extract because of comigration of some unknown spot.
The growth kinetics of the strains having pMtrel2 and pAGAN90
were compared in culture as well as on plates (Fig.
8). The generation time
of pMtrel2 was 4.1 h, compared to 2.5 h for pAGAN90,
after induction. An enhanced reduction in growth rate compared to that
in the pMatt1-pMatt2 system can be attributed to the controlled
regulation of Pamidase. The slow growth of the strain
having pMtrel2 upon induction is consistent with our previous
observation that ppGpp downregulates the growth rate in M. smegmatis. The morphology of the two strains could not be compared
because of severe clumping of the cells in the medium containing
succinate and acetamide.
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FIG. 8.
Effect of ectopic expression of the M. tuberculosis relA/spoT on growth of M. smegmatis. (A)
Reduction in colony size of the strain overexpressing
relA/spoT (pMtrel2) as compared to the control (pAGAN90)
when grown on MB7H9-2% succinate-2% acetamide. (B)
Comparison of growth kinetics in liquid culture. The arrow indicates
the time at which the inducer (2% acetamide) was added.
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DISCUSSION |
The experimental observation reported here indicates that
morphological changes, from an elongated rod to a spherical coccus, in
carbon-starved M. smegmatis may be due to elevated
intracellular levels of ppGpp. It has been observed that other
bacteria, like S. enterica serovar Typhimurium
(13), Vibrio vulnificus (22), Arthrobacteria crystallopoietes (39), and
Pseudomonas putida (14), undergo similar changes
in low-nutrient medium, although the concomitant change in the ppGpp
pool has not been established. It appears that such morphological
change substitutes for a programmed differentiation (as seen in
sporulating bacteria) in nondifferentiating bacteria. Although the
molecular mechanism of bacterial size reduction is far from being
clear, it is held that rapid cell division without an increase in cell
mass results in the short spherical shape (19). Probably,
the increase in cell number improves the strain survival during
starvation. The fact that M. smegmatis undergoes a
similar morphological change during the period of starvation shows a
fundamentally common mechanism of bacterial survival under extreme
growth conditions.
The involvement of ppGpp in cellular differentiation in M. smegmatis may provide an important clue towards understanding the survival of the organism. It can be perceived that M. smegmatis adopts a stringent physiology during starvation which
results in a concomitant increase in the ppGpp pool. However, detailed studies on kinetics of macromolecular synthesis and degradation in a
starved culture are required before a clear picture can be conceived.
The complementation study of the relA/spoT of M. tuberculosis in E. coli suggests a functionally
conserved pathway in prokaryotes. However, the reason for the presence
of a bifunctional (ppGpp synthase and hydrolase) gene in mycobacteria
and related organisms, streptomyces, remains unanswered.
Nevertheless, a possible role of stringent pathways in the
developmental processes of even evolutionarily divergent species of
bacteria cannot be ruled out.
The studies on starvation in mycobacteria bear relevance to the
physiological state of latent tubercle bacilli. Because of similarities
in morphology between starved cultures and natural persistors, the two
can be argued to have the same metabolic activity. The change in
bacterial shape as a consequence of ppGpp accumulation suggests an
important role of the stringent factor in transformation of active
bacilli into latent bacilli.
The role of ppGpp in pathogenesis appears to be interesting, based on a
recent report showing that the nucleotide is a key switch in
transformation of an avirulent to virulent form of Legionella pneumophila (15). Upon correlating the mechanisms of
infection and natures of persistence between L. pneumophila
and M. tuberculosis, we suggest an important role for
ppGpp in the latency of the mycobacterium, and thus, studies on the
stringent pathways would answer some important questions pertaining to
the physiological transformation in this pathogen.
| |
ACKNOWLEDGMENTS |
We express our deep sense of gratitude to Bill Bishai (The Johns
Hopkins University) and Mike Cashel (NIH) for their generous help at
various stages of this work. We are also thankful to Anil K. Tyagi for
the kind gift of pDK20, Tanya Parish for the gift of pAGAN90, and
S. T. Cole for the cosmid MTCY227. We thank Faaizah Khan and Saket
Verma for performing control experiments. We also acknowledge anonymous
reviewers for their help in improving the manuscript.
A.K.O. is the recipient of a CSIR fellowship. This work was funded by
the CSIR and the Department of Biotechnology of the government of India.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Phone: 91-80-309-2836. Fax: 91-80-360-0535. E-mail:
dipankar{at}mbu.iisc.ernet.in.
Editor:
S. H. E. Kaufmann
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Abstract
Introduction
