The cold-shock stress response in Mycobacterium smegmatis induces the expression of a histone-like protein



The response of Mycobacterium smegmatis to a cold shock was investigated by monitoring changes in both growth and cellular protein composition of the organism. The nature of the cellular response was influenced by the magnitude of the temperature reduction, with the shock from 37°C to 10°C having the most widespread effect on growth, metabolism and protein composition. This 27°C temperature reduction was associated with a lag period of 21–24 h before increases were seen in all the measured cellular activities. The response to cold shock was adaptive, with growth resuming after this period, albeit at a 50-fold slower rate. The synthesis of at least 15 proteins was induced during the lag period. Two distinct patterns of cold-induced synthesis were apparent, namely transient and continuous, indicating the production of both cold-induced and cold-acclimation proteins. One of these cold-shock proteins, CipMa, was identified as the histone-like protein, Hlp, of M. smegmatis, which is also induced during anaerobic-induced dormancy. The corresponding gene demonstrated transient, cold-inducible expression with a five- to sevenfold increase in mRNA occurring 9–12 h after temperature shift. Although bacterial survival was unaffected, CipMa/Hlp knock-out mutants were unable to adapt metabolically to the cold shock and resume growth, thus indicating a key role for CipMa in the cold-shock response.


Two important aspects of Mycobacterium tuberculosis pathogenesis appear to be associated with the bacterial response to stress. First, the infecting bacteria become encapsulated within a granuloma, which is formed as a result of the host's response to an M. tuberculosis infection. Once this granuloma is formed, microaerophilic/anaerobic and acidic conditions develop, which, along with the nutrient depletion, are unfavourable for bacterial survival. Despite this, some mycobacteria do survive, possibly in a dormant state (Rook and Bloom, 1994). It has been proposed that this dormant state may represent a stress-induced protected state or possibly an extreme form of the stationary phase (Young and Cole, 1993). These dormant bacteria are capable of reactivation if the immune system is compromised. Secondly, the process of transmission of M. tuberculosis may also involve a ‘stress-induced state’. Transmission of M. tuberculosis occurs through the inhalation of ‘droplet nuclei’ that are produced after evaporation of the droplets of respiratory secretions expelled from the infected patient during a cough. These nuclei are about 10 µm in diameter and can contain between three and 10 tubercle bacilli. The nuclei are extremely stable and can remain suspended in the air for extended periods (reviewed by Smith and Moss, 1994). Even though these bacteria are exposed to severe stress conditions, such as a sudden drop in temperature, a change in oxygen pressure and a limitation of nutrients, they remain viable within these droplets (Loudon et al., 1969) and are capable of causing infection upon inhalation.

Characterization of the effects of various stresses on the metabolism of mycobacteria could help to elucidate the survival mechanisms involved in both these processes. Vital protective stress proteins may also be identified that could be used in antimycobacterial treatment. It has been assumed that fast-growing, non-pathogenic strains of mycobacteria could not be used to investigate issues relating to the slow-growing, pathogenic mycobacteria, such as M. tuberculosis. However, with the recent finding that oxygen-deprived Mycobacterium smegmatis demonstrate characteristics of dormant M. tuberculosis (Dick et al., 1998; Hutter and Dick, 1998), as defined by the anaerobic model of Wayne (1977), the use of fast-growing strains to study mycobacterial responses to stress is becoming more applicable.

One of the stress responses that could be of relevance, especially in the process of transmission of pathogenic mycobacteria, is the cold-shock stress response. In Escherichia coli, the cold-shock response is an adaptive stress response, which occurs when the temperature is reduced close to the lower limit of growth. It is characterized by the induction of a specific subset of proteins, designated cold-shock proteins, which enable the cells to adapt to the lower temperature (reviewed by Jones and Inouye, 1994; Panoff et al., 1998). After a temperature reduction, there is an immediate ‘lag’ in cellular activities such as cell division and the synthesis of proteins and nucleic acids (Ng et al., 1962; Shaw and Ingraham, 1967). It is during this period that the synthesis of the cold-shock proteins is induced (Jones et al., 1992a). Many of these proteins are involved in relieving a block at the initiation of translation (Jones and Inouye, 1996; reviewed by Jones and Inouye, 1994; Panoff et al., 1998), which occurs as a result of the temperature reduction (Broeze et al., 1978). After the increased expression of these cold-shock proteins, cellular activity is restored, and growth is resumed, albeit at a reduced rate.

The aim of this investigation was to define the effects of a cold shock on M. smegmatis cultures. The effects of a temperature shift on the metabolism was investigated as well as the protein composition of the bacterium.


Metabolic investigation

M. smegmatis demonstrates rapid growth on solid media over the temperature range of 24°C to 45°C (Roberts et al., 1991), but growth at temperatures lower than 24°C has not yet been reported. As the lower limit for growth has not yet been defined, the effects of a range of temperature reductions on bacterial growth was investigated. These included shifting the bacteria from 37°C to 20°C, 15°C or 10°C. The growth of M. smegmatis after these temperature shifts was monitored by determining the culture turbidity (OD600), the number of colony-forming units (cfu) and the ATP concentration of the culture, whereas the synthesis of both protein and RNA macromolecules was measured by the incorporation of radiolabelled precursors to indicate changes in cellular metabolism.

The magnitude of the cellular response of M. smegmatis to a temperature reduction, as in E. coli (Jones et al., 1992a), was influenced by the magnitude of the temperature reduction. (Fig. 1). An immediate drop in the protein synthesis was observed after all the temperature shifts, which increased in severity as the range of the temperature shift was extended (Fig. 1A). Within 3 h of a 17°C temperature reduction (37°C−20°C), there was a threefold reduction in [14C]-leucine incorporation, whereas the magnitude of this initial reduction increased to 10-fold and 15-fold as the incubation temperatures were reduced to 15°C and 10°C respectively. All the temperature shifts resulted in a lag in cellular growth, as measured by the change in ATP concentration (Fig. 1B). A lag of 4–6 h was observed after a shift from 37°C to 20°C, whereas periods of 8–10 h and 15–18 h were required before growth resumed at 15°C and 10°C respectively.

Figure 1.

The effects of temperature downshifts on the growth of M. smegmatis. Exponentially growing cultures (20–24 h) of M. smegmatis (Middlebrook 7H9, ADC, no Tween 80) were divided into two. One portion was retained at 37°C, while the other was placed in a shaking water bath set at 20°C, 15°C or 10°C. The following were determined at 3 h intervals after temperature shift: (A) incorporation of [14C]-leucine into acid-precipitable material; (B) ATP concentration; (C) [3H]-uracil incorporation into acid-precipitable material; (D) number of colony-forming units (cfus); (E) optical density (OD) of the culture. Each observation was made in triplicate, and error bars represent intraexperimental error (standard error of the mean).

(F) Diagrammatic representation of the unbalanced growth of M. smegmatis resulting from a cold shock from 37°C to 10°C.

The temperature shift from 37°C to 10°C alone resulted in a cold-shock profile similar to that of E. coli, with a lag period in the synthesis of both RNA and protein macromolecules as well as in the growth of the organism (as measured by all the growth determinants). This is summarized schematically in Fig. 1F. An 18–21 h lag was noted for the synthesis of protein, whereas an increase in the synthesis of RNA was only observed after 21–24 h at 10°C. Over these time periods, the amount of protein being synthesized dropped 24-fold, whereas RNA synthesis was reduced sixfold. In conjunction with the 15–18 h plateau in ATP levels mentioned above, at least 15 h at 10°C was required before a notable increase was observed in the turbidity of the culture. During this period of constant culture turbidity and ATP concentration, no obvious change in the number of cfus was demonstrated, indicating that no cold-induced cell death had occurred. This pattern of resumed growth indicated that the cold-shock induced unbalanced growth in M. smegmatis (Fig. 1F).

After the lag in cellular activity (18–24 h), the cells appeared to show an increase in metabolic activity. However, the new rate of growth at 10°C, as measured by the ATP concentration, [14C]-leucine incorporation and [3H]-uracil incorporation, was greatly reduced. The generation time (g), as determined by these growth indices, changed from 3 h to 4 h (division rate v = 0.25 h−1→ 0.3 h−1) at 37°C to 20–25 h (v = 0.04 h−1→0.05 h−1) at 10°C. Synchronized cell division was not observed during the period of resumed metabolic activity (21–30 h after temperature shift). The growth recovery of M. smegmatis at 10°C was confirmed over an 18 day period of cold shock analysis (Fig. 2). The rates of growth determined during this longer period of regrowth were considerably slower than those calculated for the first 10 h of renewed growth at 10°C. As measured by the ATP concentration of the culture and cell turbidity readings, the generation time for M. smegmatis at 10°C increased to 7–8 days (v = 0.0047→0.0057). An extended generation time of 12–13 days (v = 0.0031→0.0035) was calculated by the observed increase in the synthesis of both RNA and protein. Although cell division did not appear to be synchronized, an increase in the number of cfus was observed after 9 days incubation at 10°C.

Figure 2.

Growth recovery of M. smegmatis after a 37°C→10°C cold shock. A culture of M. smegmatis was grown with aeration at 37°C in Middlebrook 7H9 (ADC, no Tween 80) until the mid-exponential phase of growth (20–24 h), when it was transferred to a shaking water bath set at 10°C. The following were then determined at 3 day intervals: ATP concentration; number of colony-forming units (cfus); incorporation of [14C]-leucine and [3H]-uracil into acid-precipitable material. Values were plotted in relation to the value at time 0, with error bars indicating intraexperimental error (standard error of the mean).

All further studies were performed on cultures cold shocked from 37°C to 10°C.

One-dimensional SDS–PAGE analysis of M. smegmatis cold-shock proteins

Protein analysis after a 37°C to 10°C temperature shift. In E. coli, the most dramatic changes in protein synthesis occur during the cold-induced lag period. For this reason, we concentrated on the initial 30 h period after a temperature shift from 37°C to 10°C to investigate the synthesis of cold-shock proteins in M. smegmatis. After the cold-shock, newly synthesized proteins were labelled by the incorporation of l-[35S]-methionine. Protein profiles were obtained hourly for the first 10 h of the cold shock and subsequently at 3 h intervals to span the first 30 h of the response. To monitor the long-term effects of the cold shock, protein synthesis was also investigated at 2, 3, 4, 5, 6 and 7 days after temperature shift. As controls, the protein profile of the culture was obtained immediately before transfer from 37°C to 10°C (−1→0 h) and from the control culture at 37°C. All initial protein analysis was performed using one-dimensional SDS–PAGE analysis. The growth of the cultures was monitored by determining the cellular ATP concentration at each time point.

Figure 3 shows the synthesis of proteins during the first 30 h of the response and indicates that, although overall protein synthesis appeared to be lower after a cold shock, the number of proteins synthesized at 37°C and 10°C was similar. Unlike E. coli, the response was somewhat delayed, with no major changes occurring during the first hour. After 3–4 h at 10°C, however, the increased synthesis of at least four proteins was evident. These included proteins of 45 kDa, 27 kDa, 22 kDa and 5–7 kDa. None of these protein composition changes was found in the control cultures (results not shown). The longer, 7 day analysis did not reveal the induction of any other significant protein bands after the first 24 h period, suggesting that the majority of the changes were limited to the 24 h lag period (results not shown).

Figure 3.

Cold-shock proteins of M. smegmatis (37°C→10°C).

Autoradiograph of a 16.5% SDS–polyacrylamide tricine gel, containing cellular proteins of M. smegmatis labelled with l-[35S]-methionine before (t = −1→0 at 37°C) and after a cold shock from 37°C to 10°C (0–28 h). At the times indicated, aliquots of cell culture (2 ml) were taken out of the bulk culture and incubated with 1 µl (15 µCi) of l-[35S]-methionine for 60 min.

A–D. Major cold-shock proteins: (A) 45 kDa; (B) 27 kDa; (C) 22 kDa; (D) 5–7 kDa. The 50 kDa control band, which showed no change in the level of synthesis, is indicated by X.

In contrast to the findings in E. coli, the most prominent cold-shock induction in M. smegmatis occurred in the synthesis of a 27 kDa protein rather than a 6–8 kDa protein (CspA) (Goldstein et al., 1990). As Fig. 4 demonstrates, this protein was induced early in the response (2–3 h after temperature shift), with the rate of synthesis increasing until 9–12 h of incubation at 10°C (10- to 12-fold induction). This resulted in the protein band representing 20–25% of the total protein synthesized at 10°C, as opposed to 1–2% of total protein at 37°C. Although the induction of the 27 kDa protein was maximal during the first 24 h period of the cold shock, longer term analysis demonstrated that the synthesis of this protein remained induced for at least 72 h. However, this protein was not readily detectable after 120 h incubation at 10°C, indicating that it was not required for continuous growth at 10°C. This pattern of expression is characteristic of a cold-shock protein rather than a cold acclimation protein (Panoff et al., 1998), as the protein appears to be required for the initial adaptation of the bacterial cell rather than for continued survival at the lower temperature. Owing to its size, the 27 kDa cold-shock protein was designated as a cold-induced protein (cold-shock protein > 10 kDa) (Graumann and Marahiel, 1996) and designated CipM (cold-induced protein of mycobacteria).

Figure 4.

Induction profile of the 27 kDa cold-shock protein. Autoradiographs resulting from the analysis of l-[35S]-methionine proteins were scanned with a densitometer, and the peak intensities (maximum intensity–background intensity) of the 27 kDa and 50 kDa (control) protein bands were calculated for each time point. The mean band intensity of three experiments was plotted at each time point. Error bars represent the standard error of the mean.

Protein analysis of the cold shock from 37°C to 25°C, 20°C or 15°C. In E. coli, the number of cold-shock proteins and their degree of induction depends on the extent of the cold-shock (Jones et al., 1992a). To investigate whether the induction of cold-shock proteins in M. smegmatis was also dependent upon the degree of cold-shock, proteins produced during the first 16 h after transfer from 37°C to 25°C, 20°C and 15°C were analysed. These were compared with the proteins produced during the same period at 37°C. No differences were observed in the proteins synthesized at 37°C, 25°C and 20°C (results not shown). After the transfer from 37°C to 15°C, only CipM was induced reproducibly. The level of induction was, however, substantially lower when compared with cultures grown at 10°C, indicating that the level of expression of cold-shock proteins in M. smegmatis is also dependant upon the degree of cold shock.

Characterization of CipMa

Identification of CipMa. N-terminal protein sequencing was used to obtain sequence data for the main protein contained within the CipM protein band. The first 15 amino acid residues of CipMa were identified as follows: Met, Asn, Lys, Ala, Glu, Leu, Ile, Asp, Val, Leu, Thr, Lys, Lys, Met, Asn. This sequence was obtained before the completion of the M. tuberculosis genomic sequence and did not show any significant homology to the available sequence data (GenBank database) at that time. To isolate the corresponding M. smegmatis gene for further characterization, the amino acid sequence was thus converted into a degenerate DNA probe, using the codon bias for mycobacteria (Dale and Patki (1990): GAGCTC/GATCGAC/TGTG/CCTG/CACG/CAAGAAGA TGAAC. This degenerate primer was end-labelled with [γ-32P]-dATP and used as a probe to screen a Sau3A restriction fragment library of the M. smegmatis (LR222) genome. A clone containing a 2.3 kb Sau3A DNA fragment (pMSA) was identified, which possessed an open reading frame (ORF) of 208 amino acids. The degree of homology (identity) between the predicted N-terminal amino acid sequence of this ORF and the N-terminal protein sequencing data was 87% and, therefore, this ORF was designated cipMa.

Subsequent database searches have revealed that CipMa is 99.5% identical (one amino acid difference in the alanine, proline, GC-rich C-terminus) to the predicted protein product of hlp (histone-like protein) of M. smegmatis (accession number AF068138) (Lee et al., 1998). A high degree of homology was also shown to the equivalent histone-like genes (hupB or hlp) in M. tuberculosis (82% identity over the first 400 bp) (accession number Z83018; gene MTCY349.01; position 274–918) (Cole et al., 1998; Prabhakar et al., 1998) and Mycobacterium leprae (83% identity over the first 376 bp) (accession number Z99263; gene MLCB637.34; position 38644–39246) (Eiglmeier et al., 1993). Figure 5 shows the degree of similarity between the three mycobacterial histone-like proteins. The N-terminal amino acid region (109 amino acids) of these mycobacterial proteins demonstrated at least 90% identity, yet although the exact sequence of the C-terminal amino acids differed, all three proteins were rich in proline, lysine, alanine, valine and threonine residues, which suggests a structural role for this terminus. All three putative proteins possess a PS00045bacterial histone-like DNA-binding protein signature and demonstrate some degree of homology to other histone-like proteins, including the histone-like DNA-binding protein II of Bacillus subtilis (HB) (48.3% identity in 89-amino-acid overlap) and the eukaryotic G777718histone H1protein (48.8% identity in 162-amino-acid overlap).

Figure 5.

Mycobacterial histone-like DNA-binding proteins. Comparison of the predicted amino acid sequences of the putative histone-like DNA-binding proteins from M. leprae and M. tuberculosis with CipMa (ORF 1) of M. smegmatis. The amino acid sequence for M. smegmatis CipMa is written in full, whereas (.) symbols indicate identical sequence, and the letters indicate sequence variations in the predicted protein sequences of the other two mycobacterial proteins. Putative O-glycosylation sites as predicted by netoglyc 2.0 (Center for Biological Sequence Analysis) are indicated by * symbols.

Expression of cipMa mRNA. Northern hybridization was used to investigate the expression of cipMa at the transcriptional level. Total RNA was extracted from cells grown at 37°C and after transfer to 10°C and then probed with [α-32P]-dCTP DNA fragments from pMSA. This analysis demonstrated that only one ORF was transcribed from the 2.3 kb mycobacterial DNA fragment, namely cipMa, producing a transcript 600–700 bp in length. Figure 6 shows the expression of cipMa mRNA during cellular growth at 37°C and after a cold shock from 37°C to 10°C. Expression of cipMa at 37°C was limited to the exponential growth phase (16–32 h), with the levels declining when the cells entered the stationary phase (36 h) (Fig. 6A). After the temperature shift from 37°C to 10°C, an increase in the quantity of mRNA could be seen after 3 h at 10°C (Fig. 6B). This is shown quantitatively in Fig. 6B(ii), which indicates the relative band densities of cipMa after the cold shock. The pattern of expression shown here represents a maximal, transient cold-shock induction of five- to sevenfold occurring at 9–12 h after temperature shift. Analysis of cipMa mRNA levels during a prolonged cold shock confirmed that increased expression was limited to the lag period (Fig. 6C), with the levels of mRNA returning to precold-shock quantities when the cells showed signs of growth recovery (48 h). These results were reproduced in two further independent experiments (results not shown).

Figure 6.

Expression of cipMa– Northern hybridization analysis.

A. RNA was extracted from cultures of M. smegmatis grown at 37°C at the indicated intervals. RNA (40 µg) was electrophoresed on a 1.5% agarose–formaldehyde gel and then transferred to Hybond N+ nitrocellulose membrane via capillary blotting. The picture shows the autoradiograph of the membrane after hybridization with a [α-32P]-dCTP cipMa 1011 bp PCR product (random primed).

B. RNA was extracted from M. smegmatis at the post-cold shock times indicated. This RNA was processed as described in (A), and the autoradiograph resulting from the hybridization with the 1011 bp cipMa probe is shown in B(i). Autoradiographs resulting from the Northern hybridization analysis of cipMa expression were scanned using a densitometer (Shimadzu CS-9000). The band densities at 10°C were compared with the band obtained pre-cold shock, and the relative increases in density were plotted as in B(ii). Error bars represent the standard error of the mean of three experiments.

C. M. smegmatis RNA was extracted at 24 h intervals after the transfer of the culture from 37°C to 10°C. The RNA was processed as described above, and the autoradiograph resulting from the hybridization with the 1011 bp cipMa probe is shown.

Growth analysis of a cipMa (hlp) knock-out mutant. Lee et al. (1998) reported the generation of an M. smegmatis hlp knock-out mutant, which was created by the insertion of a kanamycin cassette into the coding region of hlp. One-dimensional SDS–PAGE analysis demonstrated the absence of a 27 kDa protein in the protein extracts of the mutant strain, yet growth at 37°C and during anaerobic-induced dormancy was similar to the wild type. This mutant (kindly supplied by Dr T. Dick) was used in our studies to assess the relevance of CipMa (Hlp) to the M. smegmatis cold shock response. M. smegmatis strains LR222, MC2155 (hlp knock-out wild type) and hlp::km1 (Hlp mutant) were grown at 37°C until mid-exponential phase (OD600 of 0.1) and then transferred to 10°C. Growth and metabolism were assessed by monitoring the ATP concentration of the cultures, as well as the incorporation of [14C]-leucine into acid-precipitable material. MC2155 reacted in a similar fashion to LR222, with a lag period occurring before regrowth at 10°C (results not shown). Although bacterial survival at 10°C did not seem to be affected by the Hlp knock-out mutation, hlp::km1 showed no signs of adaptation/regrowth over the 5 day analysis period, as determined by the leucine incorporation (data not shown) and the ATP concentration (Fig. 7). It was also observed that frozen stock cultures of hlp::km1 had an extended lag period of 18–24 h when regrown at 37°C in liquid culture. This may indicate a reduction in cryotolerance for the Hlp mutant strain.

Figure 7.

Cold-shock adaptation: effects of CipMa/Hlp mutation. Cultures of M. smegmatis strains LR222, MC2155 (hlp knock-out wild type) and hlp::km1 (cipMa/hlp mutant) were grown with aeration at 37°C in Middlebrook 7H9 (ADC, no Tween 80) until the mid-exponential phase of growth (20–24 h). They were then transferred to a shaking water bath set to 10°C, and the ATP concentration of the cultures was determined at 24 h intervals. Values were plotted in relation to the value at time 0 = 1, with error bars indicating intraexperimental error (standard error of the mean).

Two-dimensional protein analysis of the M. smegmatis cold-shock proteins

On account of the limitations of one-dimensional protein analysis, two-dimensional analysis of the proteins synthesized after cold shock was also performed. Key time points were selected based on the one-dimensional electrophoresis data. These were: −1→0 h (37°C), 0→1 h (10°C), 3–4 h (10°C), 10–11 h (10°C), 18–19 h (10°C), 23–24 h (10°C), 28–29 h (10°C) and 48–49 h (10°C). As indicated previously by the one-dimensional analysis (Fig. 3), few changes were seen in protein synthesis during the first hour of the cold-shock response (data not shown). In contrast, the autoradiographs shown in Fig. 8 clearly show the effects of a cold shock on the synthesis of a greater number of proteins by 10–11 h after cold-shock than was originally apparent by the one-dimensional analysis. In addition to a number of proteins whose synthesis was reduced by the temperature shift, the synthesis of at least 14 proteins was induced reproducibly after the cold shock. These proteins were arbitrarily numbered 1–14, so that their synthesis could be specifically monitored during the cold shock.

Figure 8.

Two-dimensional analysis of the cold shock proteins of M. smegmatis. Autoradiographs from the two-dimensional separation of protein extracts from l-[35S]-methionine M. smegmatis cells (pH 3–10 first dimension, 10% SDS–polyacrylamide tricine gel for the second dimension). Exponentially growing cultures of M. smegmatis were transferred from 37°C to 10°C (t = 0) and the proteins synthesized at various time points were analysed. At the post-cold shock times indicated, 20 ml culture volumes were incubated with 150 µCi of [35S]-methionine for 60 min at 10°C. Proteins induced by the cold shock are circled and numbered 1–14. Spot C (60 kDa) was used as a control for quantification purposes. It must be noted that no cold-shock proteins were detected in the pH range 8–10.

It must be noted that CipMa, identified earlier, has a predicted isoelectric point of 12.13, resulting from the lysine-rich C-terminal section (DNA Man and ExPaSy pI/Mw tool; Bjellqvist et al., 1993). This protein would not have been focused under the standard isoelectric focusing conditions used in this study.

Classification of the cold-shock proteins of M. smegmatis. To analyse the expression patterns and magnitude of induction of the cold shock proteins, the protein spots were scanned and the densities calculated. To control for errors in sample loading, these densities were expressed relative to the density of an internal control spot (60 kDa), which was not obviously affected by the cold shock. Figure 9 indicates the pattern of cold-shock expression of these proteins, as obtained from the spot densities of one complete time course analysis. Similar patterns of expression were obtained when this time course analysis was repeated. Table 1 gives a summary of the nature of the cold-shock expression and magnitude of induction determined from the data presented in Fig. 9. Most of the cold-shock proteins showed induction levels in the sevenfold range. However, four proteins showed increased levels of synthesis of > sevenfold and can therefore be classified as the major cold-shock proteins of M. smegmatis: proteins 7, 9, 11 and 14. Three of these proteins, 9, 11 and 14, were not detectable at 37°C.

Figure 9.

Induction profiles of M. smegmatis cold-shock proteins. Autoradiographs resulting from the two-dimensional analysis of cold-shock proteins during the time course analysis were scanned using a densitometer, and the values were corrected according to the density of the 60 kDa control spot. The spot densities were then plotted against incubation time at 10°C, after the 37°C to 10°C temperature shift. Time 0 h = spot densities at 37°C before the shift.

Table 1. Classification of the cold-shock proteins of M. smegmatis.
Protein spotSize (kDa)Fold inductionExpression patternaClassificationb
  • a

    . Transient, peak expression during the 24 h lag period; continuous, increased expression during the regrowth period (28–29 h and 48–49 h).

  • b

    . CAP, continuous increased expression; CIP, transient expression, > 10 kDa; CSP, transient expression, < 10 kDa.

  • c

    . Exact cold-shock induction levels could not be calculated because of the lack of a detectable signal at 37°C.

920–22> 7cTransientCIP
1119–21> 7cTransientCIP
146–9> 7cTransientCSP

Two different patterns of cold-shock expression were demonstrated by these 14 proteins (Table 1). Eight proteins (1, 2, 5, 7, 9, 11, 12 and 14) exhibited transient induction, with their synthesis peaking during the metabolic lag period of 24 h. This group of proteins could therefore be classified as either cold-shock (CSP, < 10 kDa) or cold-induced proteins (CIP, > 10 kDa) depending upon their size (Graumann and Marahiel, 1996) (Table 1). The other six cold-shock proteins (3, 4, 6, 8, 10 and 13) demonstrated an expression pattern indicative of cold-acclimation proteins (CAPs). All six showed elevated levels of synthesis during the first 24 h period of the cold shock, but these proteins continued to be synthesized at elevated levels well into the growth recovery phase. This result implies that these proteins are required for continuous growth at 10°C and can be designated as CAPs (Hebraud et al., 1994; reviewed by Graumann and Marahiel, 1996; Panoff et al., 1998).

Although the number of time points was limited, the analysis of the 28–29 h and 48–49 h time points revealed that the cold-shock proteins of M. smegmatis were all induced within the first 24 h period of the cold shock. No other cold-shock proteins were detected beyond 24 h. This finding is in agreement with the results obtained with the one-dimensional analysis.


The effects of cold shock on mycobacterial metabolism and growth

To investigate the cold-shock response of M. smegmatis, the effects of temperature on the growth and metabolism of the bacterium reduction was assessed. The ultimate aim was the identification of the period during which cold shock proteins are induced. Bacteria do not respond uniformly to a cold shock and, therefore, it could not be assumed that M. smegmatis would respond as rapidly or in the same manner as E. coli. The initial metabolic studies demonstrated that the growth and metabolism of M. smegmatis were affected by temperature shifts from 37°C to 20°C and below and that the magnitude of this response was dependant upon the degree of cold shock. Notably, as in E. coli (Ng et al., 1962; Shaw and Ingraham, 1967), the synthesis of protein macromolecules was rapidly reduced as a result of the cold shock, indicating a possible block in translation. However, only a cold shock to 10°C resulted in a lag period of greater than 2 h in all the monitored cellular processes, with 21–24 h being required before an increase in the synthesis of macromolecules was seen. As with the recovery growth of E. coli at 10°C (Shaw and Ingraham, 1967), a period of rapid unbalanced growth preceded the steady-state growth at 10°C. The resulting growth rate at 10°C was, however, at least 50 times slower than that at 37°C.

Although a doubling of the number of cfus would have been expected over an 18 day period (calculated generation time of 7–8 days), this was not observed. In some bacteria, a cold shock results in the production of a protected state known as a viable-but-non-culturable (VBNC) state (Olivier, 1993). The bacteria are unable to undergo sustainable cell division in or on normal culture media, but tests for viability show that the cells are still alive and even metabolically active (Olivier, 1993). It is possible that M. smegmatis has entered a similar stress-induced protected state after the 37°C to 10°C temperature shift. This in turn may be similar to the non-replicating, persistence seen in dormant M. tuberculosis (Wayne, 1976). Further work on characterizing the expression of key cold-shock proteins will establish whether this state is similar to anaerobically induced dormancy.

Cold-shock proteins of M. smegmatis

In E. coli, the lag period is characterized by the induced synthesis of at least 20 cold-shock proteins (Jones et al., 1992a; Panoff et al., 1998). The results of the protein analysis of the cold-shock response in M. smegmatis indicated that a set of at least 15 cold-shock proteins was induced during the first 24 h of the cold-shock response. These proteins demonstrated elevated levels of synthesis against a backdrop of reduced global protein synthesis. No new cold-shock proteins were observed after this period. Most of these proteins demonstrated a sevenfold cold-shock induction in their levels of synthesis, whereas four exhibited increases of > sevenfold (proteins 7, 9, 11 and 14). Two patterns of induced expression were exhibited by these cold-shock proteins, namely transient and continuous, allowing the distinction between CSPs, CIPs and CAPs (Table 1). In E. coli, all the cold-shock proteins that have been analysed thus far demonstrate transient cold-shock induction (Graumann and Marahiel, 1996), with increased synthesis only observed during the lag period (4–6 h). Many of these proteins are ribosome-associated proteins or RNA-binding proteins that play a role in restoring translational capacity to the cell, thereby allowing growth at the lower temperature (Jones and Inouye, 1994; Panoff et al., 1998). In B. subtilis (Graumann et al., 1996), Arthrobacter globiformis (Berger et al., 1996) and Enterococcus faecalis (Panoff et al., 1997), however, some cold-shock proteins demonstrate continuous increased expression and are required for long-term survival at the lower temperature (Hebraud et al., 1994; Graumann and Marahiel, 1996). It has been suggested (Berger et al., 1996) that these cold-acclimation proteins function as proteases, removing denatured proteins whose accumulation would be deleterious to the cell, or that they are involved in maintenance membrane fluidity or the synthesis of ‘antifreeze’ substances. Once the cold-shock proteins of M. smegmatis have been identified, a clearer understanding of their role in the cold-shock response and possibly in the process of mycobacterial transmission can be established.

Characterization of CipMa

A 27 kDa protein band, CipM, was preliminarily identified as the major cold-shock protein of M. smegmatis. This protein band comprised up to 25% of the total protein that was synthesized after 10 h of incubation at 10°C. Its pattern of expression indicated a transient cold-shock induction; hence, this protein was designated as a cold-inducible protein (Graumann and Marahiel, 1996). Although this band was composed of several proteins (as seen on the two-dimensional analysis), the major protein, CipMa, within this band was identified by N-terminal sequence analysis. CipMa was shown to be identical to the histone-like DNA-binding protein (Hlp) of M. smegmatis. The predicted molecular weight of CipMa was calculated to be 21.3 kDa (208 amino acids), yet it migrated within a 27 kDa protein band. This aberrant electrophoretic mobility is typical of highly charged proteins such as histones and ribosomal proteins, as well as proteins rich in proline residues (Herbert et al., 1997), which is the case with CipMa. The five- to sevenfold, transient, cold-shock induction of cipMa mRNA, as well as the inability of the CipMa/Hlp mutant to resume active growth during cold shock, suggests an important role for this protein in the initial adaptive phase of the mycobacterial cold-shock response. It is possible that the protein has a regulatory role in controlling the expression of other cold-shock proteins that are necessary for growth at the lower temperature.

It has been suggested that hlp represents the mycobacterial hupB gene, which encodes the HUβ subunit of the HU DNA-binding protein (Cole et al., 1998). HU, which is one of the major chromosome-associated proteins in E. coli, is mainly responsible for maintaining the integrity and stability of the bacterial chromosome (Rouviere-Yaniv et al., 1979), with mutations in both the HU genes resulting in reduced cell division and cell viability (Wada et al., 1988). HU has also been shown to play a role in the initiation of DNA replication (Skarstad et al., 1990), DNA breaking and rejoining in transposition and inversion reactions (Lavoie and Chaconas, 1993), as well as in homologous recombination and recombinational repair of UV-damaged DNA (Dri et al., 1992; Li and Waters, 1998). Although HU has not yet been classified as a cold-shock protein, several lines of evidence suggest that it may play a role in the cold-shock stress response of E. coli. Malik et al. (1996) proposed that HU facilitates the action of DNA gyrase, which is one of the cold-shock proteins of E. coli (Jones et al., 1992b), and both DNA gyrase and HU have been associated with the increase in negative supercoiling that occurs after cold shock in E. coli (Mizushima et al., 1997). HU mutants in the huA and huB genes are also cold sensitive, showing reduced viability after a cold shock (Wada et al., 1988). Owing to the similarity of the CipMa/Hlp protein to HU and the highly charged C-terminus, it is probable that this protein is able to bind to DNA or RNA. This protein may therefore function in an HU-like capacity, condensing the chromosome and maintaining DNA structure during the cold shock. It may also act as an RNA-associated protein, preventing the formation of secondary structures in the mRNA transcripts and aiding translation during cold shock. Hlp may therefore be similar to CspA of E. coli, which regulates cold-shock gene expression by binding directly to gene promoters (Jones et al., 1992b; Brandi et al., 1994), as well as by binding to mRNA transcripts (Jones and Inouye, 1994; Yamanaka et al., 1998).

Only the N-terminus of CipMa shows homology to HU. The C-terminus bears no resemblance to HUβ or HUα, the other subunit of HU. This C-terminus, which consists of degenerate repeats of proline, lysine and alanine residues, is similar in composition, but not in amino acid sequence, to several eukaryotic H1 histones (PAKK and KAAK repeats) (Prabhakar et al., 1998). It is also similar to the C-terminus (40 amino acids) of another mycobacterial protein, the heparin-binding haemagglutinin protein (HBHA), which is involved in mycobacterial adhesion (Menozzi et al., 1998) (accession number AF074390). According to the analysis of Menozzi et al. (1998), the lysine/proline-rich C-terminus of HBHA is responsible for the cellular adhesion properties exhibited by this protein. It has been shown that a number of respiratory pathogens produce heparin-binding adhesins that interact with sulphated carbohydrates on the surface of epithelial cells (Rostand and Esko, 1997) as an initial step in a bacterial infection. The finding that the HBHA C-terminus is involved in binding to sulphated polysaccharides and, by implication, in binding to epithelial cells suggests an important role for HBHA and possibly the mycobacterial histone-like DNA-binding proteins (CipMa) in the initial stages of a mycobacterial infection.

Hlp of M. smegmatis, as well as the M. tuberculosis homologue, have been associated with two interesting aspects of mycobacterial pathogenicity. First, in M. tuberculosis, Hlp was found to be highly immunogenic, inducing lymphocyte proliferation (Prabhakar et al., 1998). These authors were also able to demonstrate Hlp binding to different DNA templates, confirming the DNA-binding properties of this protein (Prabhakar et al., 1998). If Hlp is involved in DNA ‘packaging’, then its expression may be cell cycle dependant, as is the case with histones. Our studies in M. smegmatis have shown that the expression of CipMa/Hlp is indeed linked to the growth phase of the bacterium. Several histone-like proteins play a role in transcriptional regulation through changes in DNA topology, and Prabhakar et al. (1998) suggested that this mycobacterial equivalent may also have a regulatory function. Secondly, Hlp is the major protein expressed during anaerobically induced dormancy in M. smegmatis (Dick et al., 1998; Lee et al., 1998). However, gene disruption experiments have demonstrated that this protein is not essential for viability during anaerobic stress. Lee et al. (1998) conclude that the apparent redundancy of this protein may result from the presence of other ‘histone-like’ proteins in the genome, which compensate for the loss of Hlp. The results from these studies as well as our own suggest that Hlp may be a key regulatory protein and, therefore, a potential antimycobacterial target. The expression of Hlp in both an anaerobic/dormant and cold shock environments, as shown in our studies, adds to the evidence that dormancy (oxygen-starved stationary phase) involves a general stress response mechanism (Hu et al., 1998; Murugasu-Cei et al., 1999).

Experimental procedures

Culture conditions and cold shock

M. smegmatis strain LR222 (Beggs et al., 1995) was used throughout this study. MC2155 (Snapper et al., 1990) and hlp::km1 (Lee et al., 1998) were used for the mutant analysis. Mycobacterial cultures were seeded (1:1000 dilution) from a frozen, stationary phase (14 days) stock culture and grown in 200 ml of Middlebrook 7H9 medium (Difco Laboratories), supplemented with 10% (v/v) ADC (5% albumin, 2% dextrose) and 0.05% (v/v) Tween 80 (polyoxyethylene-sorbitan monooleate), unless otherwise indicated. The cultures were incubated at 37°C with aeration for 20–24 h (mid-exponential phase, OD600 = 0.1). After this period, the mycobacterial cultures were split into two separate culture bottles. One was retained at 37°C (control), while the other was transferred to an enclosed shaking water bath (180 r.p.m.) that was set to the appropriate cold-shock temperature (20°C, 15°C or 10°C). During growth at 37°C and at the cold-shock temperatures, cultures were continually assessed for contamination using the Ziehl–Nielsen mycobacterial stain (Heifets and Good, 1994). It must be noted that, when cultures of M. smegmatis were cold shocked to temperatures below 25°C, the bacteria were found to aggregate together forming clumps as a result of Tween additive. The formation of these clumps was found to affect the growth assessment studies and, consequently, all growth measurements were performed on cultures grown in the absence of Tween 80. This omission did not affect normal growth at 37°C.

Experimental design

For all the metabolic experiments described, three separate measurements were made at each time point, and each experiment was repeated three times. The data from only one of the experiments are presented, with the error bars (calculated for each time point) indicating intraexperimental error. These curves are, however, representative of the trend shown in all the experiments.

Growth assessment

The single-point bioluminescent assay for ATP was modified from that recommended by the manufacturers of the Bio-Orbit 1250 luminometer using the Promega Enliten luciferase/luciferin bioluminescence detection reagent (B.A. Ntolosi, personal communication). Culture turbidity was measured at 600 nm (OD600), whereas the number of cfus was calculated from serial dilutions of 100 µl aliquots of cells taken at each time point. These dilutions were plated, in duplicate, on Middlebrook 7H9 agar plates and incubated at 37°C for 5 days. To determine the generation time and growth rate (division rate) of the bacterium, the following equations were used (Schlegel, 1986):

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where g = generation time; V = division rate; and x = growth measurement at time t.

Metabolic assessment

Quantification of RNA synthesis. [5,6-3H]-uracil (49 Ci mmol−1; Amersham) and a solution of 20 mM non-radioactive uracil (Sigma) were mixed at a ratio of 1:1 ([3H]-uracil mix) to give a specific activity of 50 mCi mmol−1 and a final uracil concentration of 200 µM when added to the culture samples. At each time point, 1 ml samples of culture were removed and incubated with 20 µl of the [3H]-uracil mix (10 µCi) for 10 min at the appropriate temperature. After this labelling period, an equal volume (1 ml) of ice-cold 10% (w/v) trichloroacetic acid (TCA) was added, and the mixture was placed on ice for 30 min. The acid-precipitable material was collected by centrifugation (4000 r.p.m. for 10 min) and washed three times with cold 5% (w/v) TCA (4000 r.p.m. for 5 min). The final pellet was dissolved in 1 ml of 0.1 M NaOH and added to 9 ml of scintillation fluid (Insta-gel II Plus; Packard). The radioactivity content (c.p.m.) of the acid-precipitable material (bulk nucleic acids) was determined using a liquid scintillation counter (Beckman LS 7800). Nucleic acid synthesis was expressed as c.p.m. ml−1 cell culture. Under the conditions of this assay, only the RNA portion of the bulk nucleic acid was radiolabelled with the [3H]-uracil (as tested by KOH treatment; Wayne, 1977).

Quantification of protein synthesis. At each time point, 1 ml samples of culture were removed and incubated with 10 µl (0.5 µCi) of l-[U-14C]-leucine (304 mCi mmol−1; Amersham) for 10 min at the appropriate temperature. After this labelling period, the proteins were precipitated by adding an equal volume (1 ml) of 10% TCA and placing on ice for 30 min. The unincorporated radioactivity was removed, and the radioactivity content of the final acid-precipitable material was determined as described above. Protein synthesis was expressed as c.p.m. ml−1 cell culture. The acid precipitation of macromolecules and subsequent washing with 5% TCA was found to be more effective than filtration (Wayne, 1977; Granozzi et al., 1990) in removing unincorporated radioisotope in these studies. This method resulted in background levels (no control cells) of less than 5% when labelling at 37°C.

Protein analysis

One-dimensional SDS–PAGE. Culture samples (2 ml), taken at each time point, were incubated with 1 µl (15 µCi) of l-[35S]-methionine (1000 Ci mmol−1; Amersham) at 10°C or 37°C for 60 min. The labelled cells were collected by centrifugation (13 000 r.p.m. for 10 min), washed with 0.5 ml of PBS (pH 7.2) and resuspended in 100 µl of SDS loading buffer [3% (w/v) SDS, 10% (v/v) glycerol, 0.625 M Tris-Cl, pH 6.8]. Cell lysis and protein denaturation were achieved by boiling for 30 min before the determination of the protein concentration using the Bio-Rad DC protein assay kit. The tricine buffer system of Schagger and von Jagow, 1987) was used to separate the radiolabelled proteins on 16.5% polyacrylamide gels (13 × 16 × 0.2 cm). Before loading, 0.5% (v/v) β-mercaptoethanol and 0.01% (w/v) bromophenol blue were added to the samples. After electrophoresis, the gels were left to soak in 6% glycerol−20% methanol overnight, dried under vacuum and then exposed to Hyperfilm βMax X-ray film (Amersham) for 3–7 days (−70°C). For protein sequencing purposes, the proteins were transferred immediately after electrophoresis to a polyvinylidene difluoride (PVDF) nitrocellulose membrane (Amersham) using a semi-dry electroblotter (JDI) (200 mA for 1–2 h).

Two-dimensional protein electrophoresis. Volumes (20 ml) of culture were removed at each time point and incubated with 10 µl (150 µCi) of l-[35S]-methionine at 10°C or 37°C for 60 min. The cell pellets were washed in 1 ml of PBS (pH 7.2) and then resuspended in 1 ml of PBS (pH 7.2). Cell lysis was achieved using a Fast-Prep FR120 apparatus (Bio 101, Savant Instruments) (2 cycles for 40 s at 6000 r.p.m.). Cell debris was removed by centrifugation (13 000 r.p.m. for 10 min), and the cellular proteins were concentrated with the aid of a Centricon-3 filtration unit (Amicon), according to the manufacturer's specifications. An aliquot (5 µl) of the concentrated protein sample was added to 5 ml of scintillation fluid, and the radioactivity content (c.p.m.) was determined by scintillation counting. Two-dimensional protein separation was performed according to the methods developed by Görg et al. (1988; 1991). Immobiline Drystrips (18 cm), pH gradient 3–10 (non-linear) (Pharmacia), were used as the gel medium for the isoelectric focusing step (first dimension), using samples containing 2 × 106 c.p.m. (≈ 50 µg of protein). The electrophoresis conditions were as follows: 500 V for 5 h, followed by 3200 V for a further 13–14 h, using a Multiphor II electrophoresis unit (Pharmacia) set to 20°C. Proteins contained within the Immobiline Drystrip were separated according to molecular weight on 10% tricine SDS–polyacrylamide gels (25 × 20 × 0.1 cm) (Schagger and von Jagow, 1987). The SDS–polyacrylamide gels were silver stained according to the method of Morrissey (1981), with glutaraldehyde being omitted to enable mass spectrometric analysis. For sequencing purposes, silver-stained spots were cut out and freeze dried in 2% acetic acid. To visualize the radioactive proteins, the gels were also soaked in 6% glycerol−20% methanol overnight, dried under vacuum at 50°C and exposed to Hyperfilm βMax X-ray film (Amersham) for 3–7 days at −70°C.

Southern hybridization

For Southern hybridization analysis, genomic DNA was extracted from turbid bacterial cultures, according to the method of Sambrook et al. (1989) for E. coli and Jacobs et al. (1991) for the mycobacterial strains. DNA (10 µg) was digested with 10 units of restriction endonuclease for 12–16 h at 37°C (Sambrook et al., 1989), and the fragments were separated by electrophoresis [1.5% (w/v) agarose gels with 1× TAE running buffer]. The DNA was transferred to a Hybond N+ nylon membrane (Amersham) according to the methods of Southern, 1975) and Sambrook et al. (1989) and then cross-linked to the membrane with UV light (120 J). DNA double-stranded probes (100 ng) were randomly labelled using the ‘Ready-to-go’ dCTP DNA labelling beads (Pharmacia Biotech) according to the manufacturer's specifications. After denaturation, these probes were added to the prehybridization buffer (50% formamide, 5× SSPE, 0.1% SDS, 5× Denhardt's solution, 100 µg ml−1 herring sperm DNA) and incubated at 42°C for 16–14 h with the membranes. The membranes were treated with two washes of 2× SSC−0.1%SDS for 20 min at 42°C and then two washes of 1× SSC−0.1%SDS for 20 min at 55°C before being exposed to autoradiography film (Agfa CP-BU) for 1–2 days at −70°C.

Screening of the M. smegmatis genomic library

The M. smegmatis (LR222) genomic library was prepared by Dr M. Everett at Glaxo Wellcome (Stevenage, UK) and consisted of E. coli (XL1-Blue) containing partially digested Sau3A M. smegmatis genomic fragments ligated to BamHI-restricted pBluescript-II SK+. These colonies were imprinted upon a Hybond N+ membrane. The single-stranded DNA primer probe was end labelled with [γ-32P]-dATP as follows: 100 ng (10 pmol) of DNA primer, 10 units of polynucleotide kinase, 1× polynucleotide kinase buffer and 4 µl (40 µCi) of [γ-32P]-dATP (3000 Ci mmol−1) in a 20 µl reaction volume for 60 min at 37°C. The unincorporated, radioactive nucleotides were removed using a Microspin G25 Sephadex column (Pharmacia Biotech). Hybridization and membrane washing was performed as above.

Northern hybridization

RNA was extracted from cultures (1000 ml) of M. smegmatis grown at 37°C or cold shocked at 10°C. The RNA was extracted from 50 ml culture volumes at each time point (Fast-RNA kit-Blue; Bio 101), using the Fast-Prep FR120 apparatus (Bio 101, Savant Instruments) to disrupt the cells (2 × 40 s cycles at 6000 r.p.m. with cooling on ice between cycles). The method used for Northern transfer was that described by Fourney et al. (1988). Total RNA (40 µg) was electrophoresed on 1.5% denaturing agarose–formaldehyde gels, photographed to check for comparable levels of total RNA and then transferred to nylon membrane (Hybond N+ Amersham). Membranes were prehybridized (the same prehybridization buffer as for Southern hybridization) for at least 4 h at 42°C before the addition of the radiolabelled probe. Linear DNA (100 ng) generated in a cipMa-specific polymerase chain reaction (PCR) reaction (primers CTAGTCGGTTCTGGAGC and CAGGGCAAGGTGATTCC; 1100 bp product) was labelled with [α-32P]-dCTP (3000 Ci mmol−1; Amersham) via the process of random priming, using the ‘Ready-to-go’ dCTP DNA labelling beads (Pharmacia Biotech). DNA–RNA hybridization was allowed to proceed for 16–24 h at 42°C, after which the membranes were washed as follows: two washes with 2× SSC−0.1%SDS for 20 min at 23°C, followed by two higher stringency washes with 1× SSC−0.1%SDS for 20 min at 42°C. After exposure to autoradiography film (Agfa CP-BU), the resulting bands were scanned using a densitometer (Shimadzu CS-9000).


We would like to thank Dr T. Dick (The Institute of Molecular and Cell Biology, Singapore) for kindly donating the hlp mutant strain.