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).