Microbiology (2004), 150, 1687–1697
DOI 10.1099/mic.0.26893-0
Formation of ‘non-culturable’ cells of
Mycobacterium smegmatis in stationary phase in
response to growth under suboptimal conditions
and their Rpf-mediated resuscitation
Margarita Shleeva,1 Galina V. Mukamolova,1,2 Michael Young,2
Huw D. Williams3 and Arseny S. Kaprelyants1
1
Bakh Institute of Biochemistry, Moscow, Russia
Correspondence
Arseny S. Kaprelyants
2
[email protected]
3
Institute of Biological Sciences, University of Wales, Aberystwyth, UK
Department of Biological Sciences, Imperial College, London, UK
Received 6 November 2003
Revised
19 February 2004
Accepted 24 February 2004
Conditions were investigated that promote the formation of ‘non-culturable’ (NC) cells of
Mycobacterium (Myc.) smegmatis in stationary phase. After cultivation in a rich medium, or under
conditions that may be considered optimal for bacterial growth, or starvation for carbon, nitrogen or
phosphorus, bacteria failed to enter a NC state. However, when grown under suboptimal
conditions, resulting in a reduced growth rate or maximal cell concentration (e.g. in modified
Hartman’s–de Bont medium), bacteria adopted a stable NC state after 3–4 days incubation
in stationary phase. Such conditions are not specific as purF and devR mutants of Myc.
smegmatis also showed (transient) loss of culturability following growth to stationary phase in an
optimized medium, but under oxygen-limited conditions. The behaviour of the same mutants in
oxygen-sufficient but nutrient-inappropriate medium (modified Hartman’s–de Bont medium) was
similar to that of the wild-type (adoption of a stable NC state). It is hypothesized that adoption of a
NC state may represent an adaptive response of the bacteria, grown under conditions when their
metabolism is significantly compromised due to the simultaneous action of several factors, such
as usage of inappropriate nutrients or low oxygen availability or impairment of a particular
metabolic pathway. NC cells of wild-type Myc. smegmatis resume growth when transferred to a
suitable resuscitation medium. Significantly, resuscitation was observed when either recombinant
Rpf protein or supernatant derived from a growing bacterial culture was incorporated into the
resuscitation medium. Moreover, co-culture with Micrococcus (Mcc.) luteus cells (producing and
secreting Rpf) also permitted resuscitation. Isogenic strains of Myc. smegmatis harbouring
plasmids containing the Mcc. luteus rpf gene also adopt a similar NC state after growth to stationary
phase in modified Hartman’s–de Bont medium. However, in contrast to the behaviour noted above,
these strains resuscitated spontaneously when transferred to the resuscitation medium,
presumably because they are able to resume endogenous synthesis of Mcc. luteus Rpf.
Resuscitation was not observed in the control strain harbouring a plasmid lacking Mcc. luteus rpf. In
contrast to wild-type, the NC cells of purF and devR mutants obtained under oxygen-limited
conditions resuscitate spontaneously, presumably because the heterogeneous population contains
some residual viable cells that continue to make Rpf-like proteins.
INTRODUCTION
It has been estimated that one-third of the Earth’s
population is ‘latently’ infected with Mycobacterium
(Myc.) tuberculosis, the causative pathogen of tuberculosis
(Dye et al., 1999). Although there is variation from one
geographical area to another, the reactivation of latent
Abbreviations: MPN, most probable number; NC, ‘non-culturable’.
0002-6893 G 2004 SGM
infections accounts for a significant proportion of the global
burden of active tuberculosis cases. This suggests that Myc.
tuberculosis cells can assume a persistent (possibly dormant?) state in the human host, during which growth is
either very slow or non-existent (Gangadharam, 1995;
Grange, 1992). Opinions differ on the precise nature of this
persistent state and the mechanisms by which it is controlled
(Parrish et al., 1998). Many authorities consider that it is
primarily controlled by the host immune system, whereas
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M. Shleeva and others
others consider that it is the clinical manifestation of an
intrinsic property (i.e. dormancy or ‘non-culturability’) of
the bacteria themselves.
Information on the ability of mycobacteria to produce
dormant or ‘non-culturable’ (NC) forms either in culture or
inside macrophages is very limited. In the in vitro model
developed by Wayne, cells of Myc. tuberculosis are subjected
to gradual oxygen depletion. This results in the formation of
an anaerobic, drug-resistant, non-replicating state (Wayne,
1994; Wayne & Hayes, 1996; Wayne & Sohaskey, 2001).
Under these conditions, the bacteria shut down protein
synthesis, but it restarts immediately after the reintroduction of oxygen (Hu et al., 1998). Similar quiescent
non-replicating states have recently been described for
Mycobacterium smegmatis (Lee et al., 1998) and
Mycobacterium bovis (Lim et al., 1999). Since none of
these bacteria require a period of resuscitation, they should
not be considered as dormant (Kaprelyants et al., 1993).
In the in vivo Cornell model, the eventual reappearance of
culturable bacilli long after the cessation of chemotherapy of
infected mice (they are absent for several weeks posttherapy) is often considered to be indicative of the presence
of dormant forms of Myc. tuberculosis in tissues in vivo
(Gangadharam, 1995; Grange, 1992). The important
difference between the Wayne model and the Cornell
model is that in Wayne’s in vitro model the bacteria remain
culturable, whereas in the Cornell model in vivo they do not.
Although bacterial ‘non-culturability’ has been known for
many years (McCune et al., 1966a, b), there is considerable
debate about the mechanisms that enable cells to become
NC and the very existence of the phenomenon is not
universally accepted (Barer, 1997; Barer & Harwood, 1999;
Barer et al., 1998; Mukamolova et al., 2003; Nystrom, 1995,
2001, 2003).
We have shown that several members of the Actinomycetales
[Micrococcus (Mcc.) luteus, Rhodococcus rhodochrous and
Mycobacterium tuberculosis) can enter a NC state when
grown to and maintained in stationary phase in batch
culture (Kaprelyants & Kell, 1993; Kaprelyants et al., 1994;
Shleeva et al., 2002). These NC organisms may be described
as dormant, since they have extremely low metabolic activity
and they require specialized treatment to promote their
resuscitation. Although the precise conditions under which
each of the above-mentioned bacteria lost culturability were
different, a common feature seems to be that they were
grown and maintained under non-optimal conditions (e.g.
in a medium that supports only poor growth). In the present
study we test the hypothesis that Myc. smegmatis can enter a
NC state under non-optimal growth conditions and that the
Rpf family of proteins plays a role in promoting the
resuscitation of NC bacteria.
Starvation conditions. Oxygen limitation experiments were performed exactly as described by O’Toole et al. (2003). To this end,
cultures were grown under agitation in sealed 250 ml flasks containing 150 ml HdeB medium. N-, C- or P-limitation was achieved by
reducing the concentrations of the relevant nutrients tenfold in
unmodified HdeB medium.
Spent medium preparation. Supernatant was obtained from Myc.
smegmatis cultures grown in Sauton’s medium harvested at various
times, as indicated in Results. After centrifugation (12 000 g,
20 min), supernatant was sterilized by passage through a 0?22 mm
filter (Whatman) and used immediately.
Bacterial counts. The total number of Myc. smegmatis cells was
determined microscopically using a Helber’s chamber. Cells in a
minimum of 10 large fields were counted. Where there was significant clumping, each aggregate was counted as one ‘cell’.
Viability estimation. Bacterial suspensions were serially diluted in
fresh Sauton’s medium and then 100 ml from each dilution was
METHODS
Organism and media. Myc. smegmatis strain mc2155 and deriva-
tives harbouring plasmid pAGM0, pAGR or pAGH (Mukamolova
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et al., 2002b) were pre-cultured for 30 h at 37 uC on an orbital
shaker (250 r.p.m.) in 150 ml of the rich medium Broth E LabM
[NBE, containing (per litre): 5 g peptone, 5 g NaCl, 1?5 g beef
extract, 1?5 g yeast extract] supplemented with 0?05 % (v/v) Tween
80 in 750 ml flasks. Bacteria were then subcultured into modified
Hartman’s–de Bont (mHdeB) medium containing (per litre): 11?8 g
Na2HPO4.12H2O, 1?7 g citric acid, 20 g (NH4)2SO4, 30 ml glycerol,
0?05 % Tween 80 and 10 ml of trace elements solution. The trace
elements solution contained (per litre): 1 g EDTA, 10 g MgCl2.6H2O,
0?1 g CaCl2.2H2O, 0?04 g CoCl2.6H2O, 0?1 g MnCl2.2H2O, 0?02 g
Na2MoO4.2H2O, 0?2 g ZnSO4.7H2O, 0?02 g CuSO4.5H2O and 0?5 g
FeSO4.7H2O. The components of the trace elements solution were
added in this order to 1 litre distilled H2O. Sodium phosphate and
citric acid were mixed and adjusted, if necessary, to pH 7?0 with
citric acid before adding the other components. This is essentially
the medium as described and used by Smeulders et al. (1999),
except that potassium phosphate was omitted, the sodium phosphate concentration was increased 14-fold and citrate was added. In
some experiments, HdeB medium as used by Smeulders et al. (1999)
was differently modified by omitting the trace elements and increasing the concentration of sodium/potassium phosphate sevenfold
[DHdeB, containing (per litre): 7?75 g K2HPO4, 4?25 g NaH2PO4,
20 g (NH4)2SO4, 30 ml glycerol, 0?05 % Tween 80]. Cultures
(150 ml) were inoculated with 1 ml taken from pre-cultures of different ‘age’ (normally taken from the end of exponential phase or
the beginning of stationary phase) and incubated at 37 uC on an
orbital shaker (250 r.p.m.) in flasks with glass protrusions for better
mixing. For plasmid-containing strains, growth media were supplemented with hygromycin (50 mg ml21). Sauton’s medium (0?5 g
KH2PO4 l21, 0?5 g MgSO4 l21, 4 g L-asparagine l21, 60 ml glycerol,
0?05 g ferric ammonium citrate l21, 2 g citric acid l21, 0?1 ml of
1 % ZnSO4 l21; Connell, 1994) was also used in some experiments.
devR and purF mutants of Myc. smegmatis mc2155 were initially
grown statically in 5 ml unmodified HdeB medium [containing per
litre: 1?55 g K2HPO4, 0?85 g NaH2PO4, 20 g (NH4)2SO4, 30 ml glycerol, 0?05 % Tween 80, 10 ml trace elements solution (the trace elements solution contained (per litre): 1 g EDTA, 10 g MgCl2.6H2O,
0?1 g CaCl2.2H2O, 0?04 g CoCl2.6H2O, 0?1 g MnCl2.2H2O, 0?02 g
Na2MoO4.2H2O, 0?2 g ZnSO4.7H2O, 0?02 g CuSO4.5H2O and 0?5 g
FeSO4.7H2O)] containing kanamycin (10 mg ml21) or hygromycin
(50 mg ml21) in 30 ml culture tubes for 48 h as described by Keer
et al. (2001). Kanamycin (10 mg ml21) was also added during
growth of the mutants in mHdeB medium.
spread on agar-solidified NBE and incubated at 37 uC. The number
of colony-forming units (c.f.u.) was determined after 5 days (limit
of detection 5 c.f.u. ml21).
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Microbiology 150
Rpf-mediated resuscitation of Myc. smegmatis
Resuscitation of NC Myc. smegmatis cells. Resuscitation and
most probable number (MPN) assays were performed in 48-well
plastic plates (Corning) containing 0?5 ml Sauton’s medium, or
0?5 ml filter-sterilized supernatant taken from Myc. smegmatis cultures. Some wells contained recombinant Rpf [final concentration
125 pM, prepared as described previously (Mukamolova et al.,
1998b)]. All wells were supplemented with 0?05 % yeast extract
(LabM). Appropriate serial dilutions of Myc. smegmatis cells (50 ml)
were added to each well. Plates were incubated at 37 uC with agitation at 150 r.p.m. for 5 days. Wells with visible bacterial growth
were counted as positive and MPN values were calculated using
standard statistical methods (de Man, 1975).
Membrane energization and permeability barrier. Cells were
stained with propidium iodide (4 mM in phosphate buffer) to assess
the state of the membrane permeability barrier. Rhodamine-123 was
used to monitor membrane energization as described previously
(Kaprelyants & Kell, 1992). Rhodamine-123 accumulation was sensitive to the uncoupler CCCP. Cells were examined under a Nikon
fluorescence microscope (excitation at 510–560 nm and emission at
590 nm for propidium iodide; excitation at 450–490 nm and emission at 520 nm for Rhodamine-123). In some experiments a
BacLight live/dead kit (Molecular Probes) was employed; cells with
an intact membrane show green fluorescence (SYTO9), whereas
those with a damaged membrane show red fluorescence (propidium
iodide).
Preparation of recombinant Mcc. luteus Rpf. The Rpf protein
of Mcc. luteus (histidine-tagged recombinant form) was obtained as
described by Mukamolova et al. (1998b). MonoQ ion-exchange purification was omitted in some experiments. The purified protein was
stored in 10 mM Tris/HCl pH 7?4 containing 50 % glycerol at
220 uC for up to 2 weeks and the protein concentration was determined spectrophotometrically. Before use, all preparations were
screened for growth-promoting activity using a small inoculum of
Mcc. luteus, as described previously (Mukamolova et al., 1998a).
Preparations with poor activity were discarded; only those with substantial activity were employed for these experiments.
Transformation of Myc. smegmatis. Myc. smegmatis mc2155 was
transformed with plasmids pAGM0, pAGH and pAGR using previously established procedures (Snapper et al., 1990). Plasmids
pAGM0, containing a functional copy of rpf transcribed from the
amidase promoter, and pAGH (vector control) have been described
previously (Mukamolova et al., 2002b). To construct pAGR, pAGH
was digested with XbaI, treated with T4 polymerase and ligated with
a 1375 bp SmaI fragment of Mcc. luteus genomic DNA. The rpf gene
in this plasmid can potentially be transcribed either from its native
promoter or from the vector-encoded amidase promoter that lies
upstream.
Co-cultivation procedure. NC cells of Myc. smegmatis were resuscitated as described above, except that 104–105 Mcc. luteus cells (i.e.
a sample from an exponentially growing culture in NBE diluted
1000-fold in Sauton’s medium) were added to the wells at the time
of inoculation. (Mcc. luteus grows poorly, if at all, in Sauton’s
medium.)
ELISA method. Culture supernatant (0?2 ml) was added to plastic
96-well plates (Costar), which were incubated at 37 uC for 1 h. The
wells were then washed three times with PBS-T (PBS containing
0?05 % Tween 80). The primary antibody, rabbit anti-Rpf
(1 : 10 000), was added and incubation was at 37 uC for 45 min.
After washing three times with PBS-T, the secondary antibody, antirabbit–alkaline phosphatase conjugate (1 : 5000; Sigma), was added.
After washing three times again with PBS-T, phosphatase substrate
(p-nitrophenyl phosphate tablet set; Sigma) was added and incubation was at room temperature for 30 min. Staining intensity was
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determined by scanning (405 nm) plates in a Labsystem optical
reader. The assay was calibrated using different concentrations of
recombinant Rpf protein in the relevant culture medium.
RESULTS
Myc. smegmatis cells can enter an NC state
A wide variety of culture conditions were explored to
determine whether it is possible for viable cells of Myc.
smegmatis to be converted into NC cells during stationary
phase. They included variation of the medium composition,
starvation for different nutrients, oxygen availability and
cultivation temperature. Cultivation of Myc. smegmatis
under conditions of N-, C- or P-limitation (achieved by
tenfold reduction of the concentrations of the relevant
nutrients in unmodified HdeB medium) did not result in
the formation of a stable population of NC cells. In all three
cases, growth stopped rather rapidly once the limiting
nutrient was exhausted, giving a population of between 107
and 108 c.f.u. ml21. Upon further incubation, viability
decreased and was unstable with time, fluctuating between
105 and 106 c.f.u. ml21. Staining with propidium iodide
revealed that many of these cells had a compromised
permeability barrier (data not shown).
In contrast, cultivation of the bacteria under suboptimal
conditions led to the formation of NC cells in stationary
phase. This could be achieved either by suitable alteration of
the medium composition, growth temperature, or oxygen
availability or by the use of mutant strains.
In the standard HdeB medium, bacteria grew in much the
same way as in Sauton’s medium, forming a more or less
stable population of between 108 and 109 cells ml21 during
stationary phase (Fig. 1a). In DHdeB medium, the trace
elements were omitted and the concentration of sodium/
potassium phosphate was increased to provide additional
buffering capacity during stationary phase (see Methods).
As a result, the bacterial growth rate (estimated by
monitoring the OD600) was reduced to about half of that
observed in the control. Moreover, the maximum cell
density (~107 ml21) attained as the culture entered
stationary phase ~48 h post-inoculation was reduced
about tenfold compared with that of the control. Further
incubation of the bacteria in stationary phase resulted in a
rapid decrease in viability to a value of 103 c.f.u. ml21 after
4 days and viability continued to decline more slowly over
the next 4 days (Fig. 1a). In contrast, the total cell count
remained essentially constant once the bacteria had entered
stationary phase (not shown). This state of ‘non-culturability’ was maintained until 11 days after onset of
cultivation; thereafter the cells become stainable with
propidium iodide and eventually died.
In another similar experiment, potassium phosphate was
replaced with sodium phosphate (mHdeB; see Methods).
This also resulted in a decrease in the maximum
concentration of cells measured by c.f.u. or total count
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M. Shleeva and others
should stress that there was significant clumping associated
with the transition to the NC state (see below). For
simplicity, we recorded one cell aggregate as one ‘cell’ for
estimating the total count. Assuming no loss of culturability,
the total count should therefore correspond to the
estimation of viable cells by c.f.u. Evidently, this will lead
to an underestimation of the total cell number and the c.f.u.
Clumping is probably responsible for the observed fall in
these parameters (one order of magnitude) after 3–4 days
incubation (Fig. 1). Microscopic examination indicated
that clumping could only account for a c.f.u. decrease of one
to two orders of magnitude.
As reported previously for Mcc. luteus (Mukamolova et al.,
1998a) and R. rhodochrous (Shleeva et al., 2002), rather
strictly defined culture conditions must be maintained to
establish a rapid transition of significant numbers of cells to
an NC state. Apart from medium composition (see Fig. 1),
the ‘age’ of the inoculum obtained after growth in rich
medium (NBE) was a critically important variable. There
was an optimum inoculum ‘age’. For inocula grown for
30–32 h in NBE, essentially the entire bacterial population
became NC – this occurred after 6 days in the experiment
shown in Fig. 2. An inoculum grown for 48 h in the rich
medium took much longer to lose culturability, whereas
inocula grown for only 24 h or 72 h showed no loss of
culturability over the entire 14 days of the experiment.
(Note that the all these cultures were inoculated to a
standard density of ~105 cells ml21.)
Fig. 1. Formation of NC cells of Myc. smegmatis following
growth in mHdeB medium. Myc. smegmatis was initially grown
in NBE (with 0?05 % Tween 80) for 30 h and then employed
as inoculum (106 cells ml”1) for growth in: (a) Sauton’s medium
(n) or HdeB medium (.) or DHdeB medium ( ); (b) Sauton’s
medium (#), mHdeB medium ( ), mHdeB+potassium phosphate (2 g l”1; m) and the same medium with citrate replaced
by succinate (n). The number of c.f.u. was monitored with
time by plating on agar-solidified NBE starting ~50 h postinoculation (%). The data shown are the results of a typical
experiment (four replicates for Fig. 1a; two replicates for
Fig. 1b), except for the experiment denoted by closed circles,
which was repeated 46 times). The SD values for c.f.u. determination were between 10 and 30 %.
N
N
(Fig. 1b). Very similar results were obtained, with the viable
count falling to a level below the limit of detection after
6 days in this particular experiment. (The medium pH
remained stable – at ~pH 7– throughout the incubation
period.) Again, the total cell count remained essentially
constant once the bacteria had entered stationary phase
(Fig. 1b). However, there was no loss of culturability during
an extended stationary phase when mHdeB was further
modified by replacing citrate with succinate (Fig. 1b). We
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Fig. 2. The transition of Myc. smegmatis to the NC state
depends on the age of the inoculum. Bacteria were grown in
NBE for various times (24 h, ; 30 h, .; 48 h, %; 72 h, &;
27 h, n) and then used to inoculate mHdeB medium at an
initial density of 105 cells ml”1. Samples were removed at intervals for measurement of c.f.u. by plating on agar-solidified NBE.
The results of one experiment out of two are shown. The SD
values for c.f.u. determination were between 10 and 30 %.
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Microbiology 150
Rpf-mediated resuscitation of Myc. smegmatis
Characteristics of NC cells
Microscopic examination of the bacteria revealed that
during the transition to the NC state, cells of Myc. smegmatis
have a tendency to clump together, forming aggregates with
a rounded shape at the time of ‘non-culturability’ (Fig. 3).
Cells in such aggregates had a reduced size (two to three
times less than the size of exponentially growing cells) and
they maintained an intact permeability barrier (according to
fluorescence microscopy of bacteria stained with propidium
iodide; Fig. 3). The majority of these cells contained DNA
and had an intact membrane, since they showed green
fluorescence when stained with a live/dead kit (see
Methods). However, their metabolic activity was low; they
showed weak fluorescence after staining with Rhodamine123, indicating poor membrane energization (Kaprelyants &
Kell, 1992) and their endogenous respiratory rate was also
negligible when measured polarographically (not shown).
Further incubation of Myc. smegmatis in stationary phase
resulted in the accumulation of cells with a damaged
permeability barrier and, after 11 days incubation, almost
all cells were stainable with propidium iodide.
Transient ‘non-culturability’ of mutant strains
Adjustments to bacterial metabolism accompany growth in
suboptimal environments. We therefore examined whether
(a) Exponential cells
(d) 120 h
(b) 60 h
mutant strains, in which metabolic adjustments are
necessitated by genetic change, are more susceptible to
loss of culturability than the wild-type. Williams and his
colleagues (Keer et al., 2001; O’Toole et al., 2003) have
reported what appeared to be a transient decrease in
culturability of Myc. smegmatis mutants defective either in
purine metabolism (purF) or in a two-component regulation system (devR) when they were grown under oxygenlimited conditions. We also found that these mutants
showed a significant decrease in c.f.u. when they were grown
in unmodified HdeB medium under oxygen limitation, but
not under aerobic conditions (Fig. 4a, b). In contrast, the
wild-type strain grown under anaerobic (or aerobic)
conditions did not show the same loss of culturability
(Fig. 4c). Much longer incubation times (10–15 days) were
needed for the c.f.u. of the mutant strains to decline to the
minimum value under oxygen limitation than for wild-type
(5–6 days) c.f.u. to reach its minimum value in mHdeB
medium in aerobic conditions. In addition, the state of
‘non-culturability’ for the mutant cells was transient and an
increase of c.f.u. occurred after the minimum value was
attained (Fig. 4).
In contrast to the significant aggregation of wild-type cells
during the NC state (Fig. 3), mutant cultures with reduced
viability were well dispersed and mainly contained short
(c) 96 h
(e) 120 h, +propidium iodide
Fig. 3. Microscopic analysis of Myc. smegmatis cells during
the transition to the NC state. Bacteria were grown in mHdeB
medium under conditions similar to those described in
Fig. 1(b). (a–d) Phase-contrast images; (e) fluorescence microscopy of bacteria (shown in d) stained with propidium iodide.
Bars, 6 mm.
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M. Shleeva and others
rods. These cells maintained an intact permeability barrier,
as they were not stained by propidium iodide (not shown).
Aerobic cultivation of the mutants on mHdeB medium
resulted in behaviour very similar to that observed for the
wild-type (a decrease in c.f.u. after 3–4 days incubation,
with no subsequent increase over the next 8 days; not
shown). During growth of the mutant strains, the pH of the
culture medium remained essentially constant (between
pH 6?5 and 7).
Resuscitation of NC cells
Resuscitation of Myc. smegmatis cells grown in mHdeB
medium and taken from the period of ‘non-culturability’
was performed as described previously for Mcc. luteus and R.
rhodochrous in Sauton’s medium supplemented with 0?05 %
yeast extract using a MPN protocol in 48-well plates
(Mukamolova et al., 1998a; Shleeva et al., 2002). The best
resuscitation was obtained when the NC cells were
incubated in the presence of supernatant (50–100 %)
taken from a culture of viable cells grown in Sauton’s
medium for 26 h (Fig. 5). The addition of purified
recombinant Rpf resulted in less-pronounced resuscitation.
However, this effect was highly variable for different batches
of recombinant protein, suggesting that the observed effect
was probably limited by the concentration of biologically
active molecules in the protein preparation (see below). In
contrast to the NC cells of the wild-type strain, those of the
purF and devR mutants were able to resuscitate spontaneously in liquid medium without the addition of either
supernatant or Rpf (Table 1). Administration of Rpf had
little effect on the final MPN count.
The recombinant Rpf used for this work was unstable during
storage and degraded rapidly at room temperature or above
(unpublished observations). This instability is probably
responsible for the observed variation of Rpf activity seen
in different resuscitation experiments with NC cells of
the wild-type strain. We therefore employed two separate
procedures to maintain a suitable concentration of biologically active Rpf molecules in the resuscitation medium,
in the expectation that this would result in more
reproducible resuscitation. To this end, strains of Myc.
smegmatis were obtained harbouring plasmids encoding a
secreted form of Rpf. Plasmid pAGM0 contains rpf under
the control of an amidase promoter (Parish et al., 1997),
whereas pAGR contains rpf under the control of its native
promoter (with the amidase promoter also lying upstream).
A third strain that harbours the vector, pAGH, lacking rpf
was employed as a control.
Fig. 4. Transient loss of culturability of Myc. smegmatis
mutants in stationary phase. Myc. smegmatis devR (a), purF (b)
and wild-type (c) strains were grown in HdeB medium with
aeration (#) or under oxygen limitation ( ). Cultures were
inoculated with 105 cells ml”1. Samples were removed at intervals, for determination of c.f.u. values by plating onto agarsolidified NBE. The results of one experiment out of two (a) or
three (b) are shown. The SD values for c.f.u. counts were
between 10 and 30 %.
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Strains of Myc. smegmatis harbouring these plasmids
showed similar behaviour to that of the wild-type
(harbouring the plasmid vector, pAGH) with respect to
the formation of NC cells (not shown). However, NC cells
harbouring either plasmid pAGM0 or pAGR resuscitated
spontaneously in Sauton’s medium in the absence of either
exogenous Rpf or supernatant after an apparent lag of 3 days
(Fig. 6; Table 2). The control cells (harbouring the vector
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Microbiology 150
Rpf-mediated resuscitation of Myc. smegmatis
Fig. 6. Spontaneous resuscitation of NC Myc. smegmatis cells
harbouring plasmids and of the wild-type co-cultivated with
Mcc. luteus. Myc. smegmatis mc2155 derivatives containing
plasmids pAGH (#), pAGM0 (n) and pAGR ( ) were grown
under conditions as shown in Fig. 1. Four-day-old cells with
zero c.f.u. counts were inoculated in Sauton’s medium supplemented with 0?05 % yeast extract and cultivated with aeration.
Samples were withdrawn periodically for measurement of the
OD600. For co-cultivation experiments, between 104 and 105
exponential cells of Mcc. luteus cells grown in NBE were
added to the medium inoculated with Myc. smegmatis containing pAGH (.).
Fig. 5. Resuscitation of NC cells of Myc. smegmatis. Myc.
smegmatis cells were harvested during the period of minimum
culturability (see Fig. 1b, mHdeB medium) and the total count
and c.f.u. determined by plating onto agar-solidified NBE (see
Methods). MPN assays (columns with bold outline) were performed in Sauton’s medium containing 0?05 % yeast extract
with and without recombinant Rpf (125 pM), or with supernatant (SN) taken from logarithmic cultures of Myc. smegmatis
grown for 26 h on Sauton’s medium or with 105 viable Mcc.
luteus cells ml”1. The results of 12 different experiments are
summarized.
N
resuscitation medium and serial dilutions of Myc. smegmatis
suspensions. Since Mcc. luteus grows very poorly, if at all, in
Sauton’s medium, the presence of this organism did not
interfere with the assay. When grown in the presence of Mcc.
luteus, the final MPN count for Myc. smegmatis was several
orders of magnitude greater than the c.f.u. number,
revealing significant resuscitation of NC cells in the presence
of Mcc. luteus producing Rpf (Figs 5 and 6).
plasmid, pAGH, lacking rpf) failed to resuscitate spontaneously, as was previously observed with the wild-type
(Fig. 6).
Another approach was employed to maintain adequate
levels of biologically active Rpf molecules in the resuscitation medium. NC cells of Myc. smegmatis were co-cultivated
with Mcc. luteus – the natural producer of Rpf. Cells from an
actively growing culture of Mcc. luteus cells were added
(final number 104–105 ml21) to each MPN well containing
ELISA estimation of the accumulation of Rpf-like proteins
in supernatant taken from cultures of different strains shows
Table 1. Resuscitation of NC cells of Myc. smegmatis mutants
The SD values for c.f.u. counts were between 10 and 30 %, for MPN assays they were between 25 and
50 % and for total counts they did not exceed 80 %.
Mutation
purF
devR
Culture age (days)
10
15
19
22
12
15
http://mic.sgmjournals.org
Total count
8
2?0610
1?56108
1?76108
1?56108
1?56108
1?56108
C.f.u.
MPN
3
1?0610
2?06103
1?06104
2?06106
6?06103
6?46103
MPN+Rpf
7
1?3610
1?86105
1?86105
2?06106
2?76106
2?66105
1?36107
1?16106
9?06106
2?06106
3?96107
3?36105
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1693
M. Shleeva and others
Table 2. Resuscitation of NC cells of Myc. smegmatis
strains harbouring plasmids
Plasmid harboured
pAGH (control)
pAGR
pAGM0
pAGM0+acetamide
Total
count ml”1
7
1?0610
1?06107
1?06107
1?06107
C.f.u. ml”1
0*
0*
0*
0*
MPN ml”1
0*
2?66106
4?66106
2?46105
*Less than 5 cells ml21.
that there is maximum accumulation of the proteins in the
exponential phase. The concentration of Rpf-like proteins
was similar for the strains harbouring pAGM0 and no
plasmid (wild-type), but it was higher in supernatant of the
strain harbouring pAGR (Fig. 7).
DISCUSSION
Whilst the formation of NC bacteria was described many
years ago (McCune et al., 1966a, b), the mechanism whereby
cells lose their ability to grow (either on solid or in liquid
media) under conditions that are demonstrably adequate
for the growth of normal cells remains unknown. Two very
different models have been proposed to explain the
phenomenon. One suggests that the loss of culturability is
due to bacterial senescence and cell deterioration and
therefore that transition to the NC state is stochastic in
nature. The other suggests that the formation of NC cells
reflects an adaptive strategy of cell survival and therefore
that it should be considered as genetically ‘programmed’
and deterministic in nature. Arguments in favour and
Fig. 7. Accumulation of Rpf-like proteins in Myc. smegmatis
culture supernatants. Myc. smegmatis mc2155 wild-type (%; no
plasmid present) and derivatives containing plasmids pAGM0
(,) and pAGR (#) were grown in Sauton’s medium with aeration. Samples were withdrawn periodically, centrifuged and Rpflike proteins in the supernatant were quantified by ELISA (see
Methods). &, OD600 for Myc. smegmatis, wild-type.
1694
against both models have been adduced (Barer & Harwood,
1999; Mukamolova et al., 2003; Nystrom, 2001, 2003) but
the question still remains unresolved.
We have shown previously that a variety of different
cultivation regimes may be employed to induce ‘nonculturability’ of various actinobacteria during the stationary
phase of growth in batch culture (Mukamolova et al., 1995;
Shleeva et al., 2002). In the present study, using a new model
organism, Myc. smegmatis, we attempted to determine
whether these diverse conditions share a factor in common,
since this might help to reveal which of the two models,
stochastic or deterministic, is more plausible.
As we found previously, for two other fast-growing
organisms (Mcc. luteus and R. rhodochrous), conditions
that support good growth of Myc. smegmatis did not result
in the formation of NC cells in stationary phase. After
cultivation in a rich medium, or in two different defined
media (Sauton’s and HdeB), a high level of viability was
maintained during a protracted stationary phase (Fig. 1).
Similarly, when the defined HdeB medium was modified
such that the cells suddenly experienced starvation for either
carbon or nitrogen or phosphorus (i.e. after growth for a
reduced period at their maximum rate), NC cells were not
produced. Under these conditions, cell lysis accompanied by
cryptic growth occurs during stationary phase, as has been
reported for other bacteria (Mukamolova et al., 2003).
However, the situation changed dramatically if the medium
was modified such that it could only support growth at a
reduced rate during exponential phase. Populations of
bacteria grown under such conditions to a final cell density
of ~108 ml21 lost viability very substantially (in some
experiments completely) during stationary phase. The
observed aggregation of cells found at this particular
phase of growth (see Fig. 3) could only account for a
drop in the viable count of up to two logs, to
~107 c.f.u. ml21 (assuming each aggregate represents a
c.f.u.). The NC cells obtained under these particular
conditions were characterized by low metabolic activity,
suggesting that they had become dormant (Mukamolova
et al., 1995; Shleeva et al., 2002).
Apparently, specific growth conditions are not required for
cells to make the transition to an NC state. Significantly, a
period of ‘non-culturability’ ensued when the chemical
composition of the medium was modified (e.g. decreased
availability of microelements or substitution of a less readily
metabolized/assimilated carbon source) such that the
bacteria grew at a suboptimal rate. It is most likely that a
reduction of the bacterial growth rate by other means may
also enable the bacteria to become NC in stationary phase
but, in each case, it may be important to manipulate other
conditions, like the inoculum age or size, as was found here
(Fig. 2) and in previous work with R. rhodochrous (Shleeva
et al., 2002). We do not presently understand why several
hours difference in the age of the inoculum (with similar
numbers of viable cells) resulted in such dramatically
different behaviour of the culture.
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Microbiology 150
Rpf-mediated resuscitation of Myc. smegmatis
If the transition to a NC state in stationary phase is indeed
connected with a previous period of growth under
suboptimal environmental conditions, then genetically
determined alterations to bacterial metabolism might also
contribute to the formation of NC cells. In this connection,
we studied the behaviour of two Myc. smegmatis mutants (a
purF mutant with impaired purine metabolism and a devR
mutant that lacks a stationary-phase regulator required for
adaptation to oxygen starvation). Both strains revealed
suboptimal growth as the maximum cell concentration
reached by these cultures is much less than that reached by
the wild-type (Fig. 4). It has been shown previously that
these mutants lose culturability in stationary phase after
growth under oxygen-limited conditions (Keer et al., 2001;
O’Toole et al., 2003). This behaviour was reproduced in the
present study (Fig. 4). The evident difference between the
wild-type and the mutants is the very transient character of
the NC state for the mutants, whereas that of the wild-type is
much more prolonged. The transient nature of this state in
the mutants may be connected with the presence of some
residual viable cells at the point of minimum culturability
(Fig. 4). These viable cells could promote resuscitation of
the rest of the bacterial population (Votyakova et al., 1994).
Similarly transient behaviour of viability in stationary phase
was obtained for R. rhodochrous and Myc. tuberculosis when
some cells in the population remained culturable (Shleeva
et al., 2002). In the absence of any residual viable cells the
culture assumes a stable NC state – see Fig. 1 and Shleeva
et al. (2002).
Significantly, the Myc. smegmatis mutants behaved similarly
to the wild-type (adoption of a stable NC state) after
cultivation under oxygen-replete conditions. The transition to the NC state appears to be triggered differently
by applying different combinations of conditions. Nevertheless, we propose that the necessary result of the conditions
imposed is a period of cultivation of the bacteria under
suboptimal conditions. In this study, suboptimal conditions
are characterized by decreased growth rate (Myc. smegmatis
wild-type in modified media) and (or) lowered maximal
concentration of cells achieved during growth (Myc.
smegmatis mutants). Formation of dormant and NC cells
of Mcc. luteus after cultivation in a chemostat at a very low
dilution rate could illustrate this proposal (see Kaprelyants
& Kell, 1992).
As we have suggested elsewhere (Mukamolova et al., 2003),
bacteria may lose culturability under conditions in which
they cannot initiate a starvation survival programme. Loss of
culturability may occur if the stress encountered is too
severe, or if bacteria are simultaneously subjected to a
combination of several different stresses or even as a result of
prolonged exposure to conditions that are not ideal for
survival. The adoption of an NC state could be regarded as
an adaptive response. Its reversibility and the presence of an
intact membrane permeability barrier in the majority of NC
cells are in favour of this suggestion. The possible existence
of a specialized survival programme is compatible with the
http://mic.sgmjournals.org
transient character of the NC state, which will otherwise
eventually lead to cell deterioration and death (at least
in vitro as observed for wild-type Myc. smegmatis).
Alternatively, some cells in the population could ‘spontaneously’ resuscitate after which they may show cryptic
growth.
The existence of a survival programme that leads to an NC
state will remain controversial until the mechanistic details
and the structural elements involved have been elucidated. It
has been proposed that DevR regulates survival of Myc.
smegmatis in stationary phase (O’Toole et al., 2003). In
particular, DevR is required for bacterial adaptation to
oxygen starvation. However, the authors did not exclude a
role for DevR under other kinds of starvation conditions
(O’Toole et al., 2003). Perhaps the formation of NC cells of
the devR mutant under oxygen starvation reflects a role
played by DevR in stationary phase in modulating cell
metabolism and viability. This could result in either
maintenance or induction of an NC state depending on
the stringency of negative environmental factors (some of
which may not necessarily be connected with oxygen tension
per se).
It is clear that cells with an NC phenotype could be of some
considerable medical or microbiological significance if they
are able to resuscitate to form normal, viable organisms
(Barer, 1997; Barer & Harwood, 1999; Barer et al., 1993,
1998; Kaprelyants & Kell, 1996; Kaprelyants et al., 1999; Kell
et al., 1998, 2003; Mukamolova et al., 2003). In this study, we
resuscitated NC bacteria in an appropriate medium in the
presence of supernatant taken from a bacterial culture in
exponential phase. To make numerical estimations of the
effectiveness of resuscitation we performed MPN assays
(Kaprelyants et al., 1994; Mukamolova et al., 1998a; Shleeva
et al., 2002). As was observed previously with Mcc. luteus,
R. rhodochrous and Myc. tuberculosis, NC cells of Myc.
smegmatis (wild-type) were very efficiently resuscitated in
the presence of culture supernatant (Fig. 5). However NC
cells of the purF and devR mutant strains were able to
resuscitate spontaneously by simple incubation in liquid
medium without any additions (Table 1). NC cells of Myc.
tuberculosis obtained under similar oxygen-limited conditions as the Myc. smegmatis mutants studied here also
showed spontaneous resuscitation in liquid medium
(Shleeva et al., 2002). Such behaviour may reflect different
degrees of dormancy in NC cells obtained in different
experimental models. Two possible explanations have been
proposed to explain the recovery in viability of purF and
devR mutants of Myc. smegmatis: (a) resuscitation of NC
cells and (b) regrowth of surviving cells (Keer et al.,
2001; O’Toole et al., 2003). The present study, in which
MPN conditions were employed, confirms the first
possibility. However, we cannot exclude a possible contribution of regrowth in the process of restoration of
viability of heterogeneous populations as studied by these
authors.
We reported previously that a secreted protein called Rpf
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1695
M. Shleeva and others
(resuscitation promoting factor) was responsible for the
observed activity of Mcc. luteus culture supernatants
(Mukamolova et al., 1998b). Rpf homologues have subsequently been found in a number of GC-rich Gram-positive
bacteria, including Myc. tuberculosis (five rpf-like genes) and
Myc. smegmatis (Kell & Young, 2000; Mukamolova et al.,
2002a). Some of these proteins were tested for growth
stimulatory activity and cross-species reactivity was
reported. For example, Rpf from Mcc. luteus was active in
respect to NC cells of R. rhodochrous and Myc. tuberculosis
(Shleeva et al., 2002) and several of the Rpf-like proteins of
Myc. tuberculosis were active in assays with Mcc. luteus and
Myc. smegmatis (Mukamolova et al., 2002a; Zhu et al.,
2003). In the present study, we also found an enhancement
of resuscitation of NC cells of Myc. smegmatis by
administration of recombinant Rpf. However, Rpf was
less active than culture supernatant (Fig. 5) as was noted
previously with resuscitation of NC cells of R. rhodochrous
and Myc. tuberculosis (Shleeva et al., 2002). Recombinant
(histidine-tagged) proteins of the Rpf family are not stable;
they undergo degradation during storage (M. Telkov & A. S.
Kaprelyants, unpublished results) that could explain the
comparatively low activity of Rpf observed in our experiments. To circumvent this problem, we transformed Myc.
smegmatis with two plasmids carrying the rpf gene. Both
plasmids expressed the secreted form of Rpf, one from its
native promoter and the other from the amidase promoter
(Parish et al., 1997). In both cases, the bacteria showed
almost complete spontaneous resuscitation (Fig. 6 and
Table 2). We hypothesize that in contrast to NC cells of the
wild-type strain, those of the plasmid-containing strains are
able to express and secrete Rpf, leading to resuscitation.
Estimation of the amounts of Rpf-like proteins in culture
supernatants indeed showed that cells containing pAGR
secreted elevated concentrations of Rpf as compared with
the control. The amount of Rpf in the culture supernatant
of the strain harbouring pAGM0 was similar to that of the
control. In contrast to the native Rpf-like proteins of Myc.
smegmatis, Rpf contains a C-terminal LysM module, which
is believed to mediate the interaction of protein
molecules with the bacterial cell envelope. Molecules
bound in this way would not have been detected by our
assay procedure.
Co-cultivation of NC cells of Myc. smegmatis with viable
cells of Mcc. luteus also leads to significant resuscitation of
cells of the former organism (Figs 5 and 6). This is also
consistent with the suggestion that continuous production of biologically active Rpf molecules is important for
effective resuscitation.
The establishment of a model for the quantitative transition
of entire populations of fast-growing mycobacteria to an
NC (and possibly dormant) state may prove to be of value
for further studies of mechanisms of ‘non-culturability’
and dormancy in mycobacteria including pathogenic
Myc. tuberculosis, the causative agent of the latent form of
tuberculosis.
1696
ACKNOWLEDGEMENTS
We wish to thank the programme ‘Molecular and Cellular Biology’,
Russian Academy of Sciences, The Russian Foundation for Basic
Research (grant 03-04-89044), the International Science and
Technology Centre (Project 2201), the UWA Research Fund and the
UK BBSRC for financial support.
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