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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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. ml1).
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 510560 nm and emission at 590 nm for propidium iodide; excitation at 450490 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 104105 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-rabbitalkaline 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.
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RESULTS |
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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 ml1 during stationary phase (Fig. 1a). In
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 ml1) 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. ml1 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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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 3032 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 ml1.)
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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 (50100 %) 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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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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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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DISCUSSION |
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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 ml1 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. ml1 (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.
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ACKNOWLEDGEMENTS |
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Received 6 November 2003;
revised 19 February 2004;
accepted 24 February 2004.
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