Formation of ‘non-culturable’ cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation

Margarita Shleeva1, Galina V. Mukamolova1,2, Michael Young2, Huw D. Williams3 and Arseny S. Kaprelyants1

1 Bakh Institute of Biochemistry, Moscow, Russia
2 Institute of Biological Sciences, University of Wales, Aberystwyth, UK
3 Department of Biological Sciences, Imperial College, London, UK

Correspondence
Arseny S. Kaprelyants
arseny{at}inbi.ras.ru


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


Abbreviations: MPN, most probable number; NC, ‘non-culturable’


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 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 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 post-therapy) 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Organism and media.
Myc. smegmatis strain mc2155 and derivatives harbouring plasmid pAGM0, pAGR or pAGH (Mukamolova et al., 2002b) were pre-cultured for 30 h at 37 °C 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 [{Delta}HdeB, 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 °C 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 µg ml–1). Sauton's medium (0·5 g KH2PO4 l–1, 0·5 g MgSO4 l–1, 4 g L-asparagine l–1, 60 ml glycerol, 0·05 g ferric ammonium citrate l–1, 2 g citric acid l–1, 0·1 ml of 1 % ZnSO4 l–1; 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 µg ml–1) or hygromycin (50 µg ml–1) in 30 ml culture tubes for 48 h as described by Keer et al. (2001). Kanamycin (10 µg ml–1) was also added during growth of the mutants in mHdeB medium.

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 µm 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 µl from each dilution was spread on agar-solidified NBE and incubated at 37 °C. The number of colony-forming units (c.f.u.) was determined after 5 days (limit of detection 5 c.f.u. ml–1).

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 µl) were added to each well. Plates were incubated at 37 °C 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 µM 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 –20 °C 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 °C 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 °C for 45 min. After washing three times with PBS-T, the secondary antibody, anti-rabbit–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 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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. ml–1. Upon further incubation, viability decreased and was unstable with time, fluctuating between 105 and 106 c.f.u. ml–1. 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 ml–1 during stationary phase (Fig. 1a). In {Delta}HdeB 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 ml–1) 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. ml–1 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.



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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 ({triangleup}) or HdeB medium ({blacktriangledown}) or {Delta}HdeB medium ({bullet}); (b) Sauton's medium ({circ}), mHdeB medium ({bullet}), mHdeB+potassium phosphate (2 g l–1; {blacktriangleup}) and the same medium with citrate replaced by succinate ({triangleup}). The number of c.f.u. was monitored with time by plating on agar-solidified NBE starting ~50 h post-inoculation ({square}). 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 %.

 
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 (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 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 ml–1.)



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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, {bullet}; 30 h, {blacktriangledown}; 48 h, {square}; 72 h, {blacksquare}; 27 h, {triangleup}) 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 %.

 
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 Rhodamine-123, 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.



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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 µm.

 
Transient ‘non-culturability’ of mutant strains
Adjustments to bacterial metabolism accompany growth in suboptimal environments. We therefore examined whether 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 oxygen-limited 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).



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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 ({circ}) or under oxygen limitation ({bullet}). Cultures were inoculated with 105 cells ml–1. Samples were removed at intervals, for determination of c.f.u. values by plating onto agar-solidified 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 %.

 
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 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.



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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.

 

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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 %.

 
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.

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 plasmid, pAGH, lacking rpf) failed to resuscitate spontaneously, as was previously observed with the wild-type (Fig. 6).



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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 ({circ}), pAGM0 ({triangleup}) and pAGR ({bullet}) 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 ({blacktriangledown}).

 

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Table 2. Resuscitation of NC cells of Myc. smegmatis strains harbouring plasmids

 
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 ml–1) to each MPN well containing 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).

ELISA estimation of the accumulation of Rpf-like proteins in supernatant taken from cultures of different strains shows 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).



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Fig. 7. Accumulation of Rpf-like proteins in Myc. smegmatis culture supernatants. Myc. smegmatis mc2155 wild-type ({square}; no plasmid present) and derivatives containing plasmids pAGM0 ({triangledown}) and pAGR ({circ}) were grown in Sauton's medium with aeration. Samples were withdrawn periodically, centrifuged and Rpf-like proteins in the supernatant were quantified by ELISA (see Methods). {blacksquare}, OD600 for Myc. smegmatis, wild-type.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 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 ‘non-culturability’ 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 ml–1 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. ml–1 (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.

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 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 (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.


   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.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 6 November 2003; revised 19 February 2004; accepted 24 February 2004.



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