Division of Mycobacterial Research, National Institute for Medical Research, London NW7 1AA, UK
Correspondence
Robert A. Cox
rcox{at}nimr.mrc.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Mycobacteria are aerobic, non-motile, rod-shaped bacteria that are Gram-positive and acid-fast. Mycobacterium leprae and M. tuberculosis are human pathogens; their slow growth [generation times of 12 days (Shepard, 1960) and approaching 1 day (Wayne, 1994
), respectively] is a notable property. The emergence of drug-resistant strains of M. tuberculosis has led to tuberculosis again becoming a threat to world health. Pathogenic mycobacteria have challenging properties such as an ability to survive within host cells. They are engulfed by host macrophages but survive and grow within phagosomes (Armstrong & D'Arcy-Hart, 1971
; Ferrari et al., 1999
). Treatment of tuberculosis is lengthy because bacilli persist despite chemotherapy, allowing the illness to resume its course if drug treatment is stopped prematurely. In the persistent state of the bacillus, drug resistance appears physiological rather than genetic in origin. It is known that, under hypoxic conditions, M. tuberculosis can exist in a dormant state that is resistant to standard antimycobacterial drugs. Tubercle bacilli can encounter hypoxic conditions in vivo (Weber et al., 2000
) and oxygen starvation is thought to halt growth and lead to dormancy (Wayne & Hayes, 1996
). Reactivation of dormant cells is thought to be the cause of the disease appearing many years after the exposure to the tubercle bacillus.
A knowledge of mycobacterial physiology during exponential growth in vitro and in vivo, within macrophages, adaptation to oxygen starvation and of the dormant state, would further our understanding of the course of tuberculosis. Current knowledge has been limited by technical problems such as slow growth and cell aggregation (for review see Ratledge, 1982; Wheeler & Ratledge, 1994
). For example, the macromolecular compositions of M. tuberculosis grown under different conditions have not been reported.
Proteins comprise approximately one half of the dry mass of a bacterial cell (Bremer & Dennis, 1996). Ninety-five per cent of the energy used by the cell to synthesize macromolecules is devoted to the synthesis of proteins and 5 % is used to synthesize DNA, RNA, peptidoglycan, phospholipid, lipopolysaccharides and polysaccharides (Ingraham et al., 1983
). The RNA content of a cell, of which approximately 83 % is rRNA (Bremer & Dennis, 1996
; Butcher et al., 1999
), reflects the number of ribosomes per cell. The central role played by ribosomes in protein biosynthesis suggests that there is a relationship between protein content, the rate of protein synthesis and RNA content.
The significance of the relationship between the specific growth rates and the protein and RNA contents of Mycobacterium bovis bacillus CalmetteGuérin (BCG), a close relative of M. tuberculosis (Brosch et al., 2000), is the subject of this study. Two questions were formulated. First, how is the average ribosome concentration of a bacterial culture related to µ, the specific growth rate? Second, how is the average ribosome concentration related to the average rate of protein synthesis?
Three concepts are implicit in the questions posed. First, the principal features of protein synthesis are thought to be common to all bacteria irrespective of growth rate (Maaløe & Kjelgaard, 1966). Second, the concentrations of reactants strongly influence the rate of a chemical reaction. Third, the ratio of RNA : protein is thought to be directly proportional to the concentration of ribosomes (Bremer & Dennis, 1996
).
Progress was made by first analysing definitive data for the macromolecular compositions of cells of Escherichia coli grown optimally in five media (doubling time, tD, 0·41·67 h). The macromolecular composition of M. bovis BCG was calculated from its chemical composition (Winder & Rooney, 1970). The relationships established for E. coli were then shown to apply to M. bovis BCG.
Theoretical section
Definition of slow growth.
Traditionally (see, for example, Wayne & Kubica, 1986) mycobacteria are classified as either fast-growing or slow-growing according to whether colonies appear on a solid medium within 5 days (fast-growers) or longer than 5 days (slow-growers). Bacteria growing optimally are defined according to µ, their specific growth rate (Fig. 1
); slow growth spans the range µ
0·14 h-1 (tD
5 h), fast growth span the range µ>0·14 h-1<0·7 h-1 (tD 15 h). Faster-growing bacteria (µ>0·7 h-1 tD<1 h) are classified as ultra-fast growers.
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Definitions, axioms and assumptions.
The analysis presented below concerns exponentially growing cell cultures of E. coli for which it is known that the conditions of growth govern both the specific growth rate µ and macromolecular composition (DNA : protein : RNA). Specifically, different growth rates correlate with different macromolecular compositions of cell cultures (see, for example, Bremer & Dennis, 1996). In contrast, when the specific growth rate was 0·2 h-1 or less, it was noted that the macromolecular content changed very little. Cultures had minimal contents of protein and RNA as judged by the DNA : protein and DNA : RNA ratios (Jacobsen, 1974
cited by Ingraham et al., 1983
). For cells of minimal protein and RNA contents, the growth rates are designated µmin; the subscript min denotes that protein and RNA contents are minimal. These cells are thought to have an excess of ribosomes over the apparent demand for protein synthesis, as was shown for a Vibrio sp. by Flärdh et al. (1992)
. Cultures characterized by µmin are not included in the analysis. However, the concept of minimal cells could be relevant to dormant mycobacterial cells. (Symbols are defined in Table 1
).
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A culture, which at time t comprises nt cells, accumulates protein at an instantaneous rate specified by equation (3), which is the differential of equation (1).
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It is thought that the amount of protein per cell increases exponentially over the range t=0 for a newborn cell to t=tD when the cells divide (Cooper, 1988). Hence, mp(t), the amount of protein per cell at time t, is given by equation (6) where mp(t=0) is the amount of protein per newborn cell.
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Over the same period (t=0 to t=tD) the specific protein synthesis rate [p(t)] at time t is given by equation (7).
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Thus, equations (3) and (8) are related through nt [equation (5)], which further illustrates the notion that the properties of a cell culture are determined by the properties of average cells. Thus, both mp(av) and p(av) are parameters that usefully describe the exponential growth of a cell culture.
Macromolecular composition of E. coli as a function of the specific growth rate.
For cells growing at a rate in the range 0·421·73 doublings h-1 (Table 2), the mass of protein [mp(av)] and dry cell mass [mdc(av)] increase concomitantly with growth rate; that is, mp(av) appears to be in constant proportion,
m, of mdc(av). The average cell volume [v(av)] will be directly proportional to mdc(av) and hence, to mp(av) if the fraction of cell mass due to water,
aq, and the buoyant density,
, are independent of growth rate. A value for
aq of 0·7 was established for E. coli growing optimally in glucose minimal medium (Ingraham et al., 1983
); this value appears to be accepted for E. coli cells in general. The buoyant density,
, of E. coli was found to lie within the range 1·09±0·015 g cell mass (ml cell volume)-1 [or pg fl-1 (or µm3)] for cells with growth rates of 0·43·0 h-1 and to remain constant during the growth cycle (Woldringh et al., 1981
); and was found to be independent of the composition of the growth medium below 1000 mosM (Baldwin et al., 1994
). Thus, as a first approximation,
may be regarded as a constant. The parameters
, mdc(av) (or mp(av)/
m) and v(av) are related by equations (9a) and (9b).
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Whereas the concentration of protein appears to be independent of the specific growth rate, the mass of protein per cell is not. The relationship between mp(av) and µ [equation (10)] is illustrated in Fig. 2.
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The macromolecular composition of stationary phase cells (day 8 of Table 3) provides a guide to the properties of minimal cells of the mycobacterium. When appropriate conversion factors (Table 3
) were applied to data for M. bovis BCG as to data for E. coli, the following picture emerged. Minimal cells of E. coli were estimated to have an average of 6200 ribosomes, a concentration of 8200 ribosomes per femtolitre of cell volume. In contrast, minimal cells of M. bovis BCG were estimated to have an average of 2200ribosomes per cell, a concentration of approx. 4500 ribosomes per femtolitre of cell volume, which is close to the limit inferred for viable cells from the intercept on the RNA : protein axis in Fig. 3
.
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DISCUSSION |
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The significance of the correlations specified in equations (11) and (12) lies in the relationship between the RNA : protein ratio and ribosome concentration. A simple proportionality between the RNA : protein ratio and ribosome concentration requires mRNA(av) to be directly proportional to the average number of ribosomes, which seems likely because rRNA and tRNA are thought to account for 98 % or so of the RNA fraction. Furthermore, mp(av) is required to be directly proportional to cell volume, or, more specifically, to the volume in which protein synthesis takes place. In other words, the concentration of protein is required to have a constant value that is independent of growth rate. The available data for E. coli (Table 2) indicate that protein constitutes a fixed proportion,
m, of dry cell mass that is independent of the specific growth rate; there are no data reported for M. tuberculosis.
On the basis of the assumption that the RNA : protein ratio is proportional to ribosome concentration, equation (12) may be revised to relate the specific protein synthesis rate directly to the third power of the ribosome concentration. This proposed relationship can be shown to be in accord with our knowledge of mRNA-directed protein synthesis. The exponential increases in mass, volume, RNA content and protein content during growth ensure that the concentrations of RNA and protein are kept constant and require that the amounts of substrates, enzymes and products increase concomitantly to maintain constant concentrations. The balance between components of the machinery for protein synthesis is maintained if the proportions of individual components such as tRNA and protein factors such as elongation factor EF-Tu, etc., are kept in balance with the number of ribosomes. For example, 9·269·29 tRNAmolecules per ribosome and five to seven copies of EF-Tu were reported for E. coli independently of growth rate (Table 2).
Protein synthesis involves many steps and many components (for review see Al-Karadagh et al., 2000). The rate-limiting step in peptide bond formation is the interaction of a ternary complex (tc) composed of aminoacyl tRNA (aatRNA), elongation factor EF-Tu and GTP with the A-site of the ribosome (Pape et al., 1998
). The overall reaction is given by equation (13).
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The reaction involves ribosomes, aatRNA and EF-Tu. The concentrations of the latter two components may be directly related to ribosome concentration (Table 2), suggesting that the rate of peptide bond formation is a function of the third power of the ribosome concentration. Two further factors apply to protein synthesis during cell growth; namely, the concentration of protein remains constant, whereas the cell volume increases exponentially.
The correlation observed between specific growth rates and macromolecular compositions of both an ultra-fast and a very-slow grower reflects the importance of both protein concentration and the rate of protein biosynthesis in cell growth. For example, the protein content reflects cell volume, the RNA : DNA ratio reflects the number of ribosomes per cell and the RNA : protein ratio reflects the concentration of ribosomes and hence, the specific protein synthesis rate. Finally, the ability to measure the ratios DNA : protein : RNA simply and accurately, using a minimal number of cells, would be advantageous; flow cytometry may offer this potential (Diaper & Edwards, 1994: Turner et al., 2000
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Armstrong, J. A. & D'Arcy-Hart, P. (1971). Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J Exp Med 134, 713740.
Arnstein, H. R. V. & Cox, R. A. (1992). Protein Biosynthesis: in Focus. Oxford: Oxford University Press.
Baldwin, W. W., Hirkish, M. A. & Koch, A. L. (1994). A change in a single gene of Salmonella typhimurium can dramatically change its buoyant density. J Bacteriol 176, 50015004.[Abstract]
Bremer, H. & Dennis, P. P. (1996). Modulation of chemical composition and other parameters of the cell growth rate. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 15531568. Editor by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Brosch, R., Gordon, S. V., Eiglmeier, K., Garnier, T., Tekaia, F., Yeramian, E. & Cole, S. T. (2000). Genomics, biology, and evolution of the Mycobacterium tuberculosis complex. In Molecular Genetics of Mycobacteria, pp. 1936. Edited by G. F. Hatfull & W. R. Jacobs, Jr. Washington, DC: American Society for Microbiology.
Butcher, P. O., Sole, K. M. & Mangan, J. A. (1999). RNA extraction. In Molecular Mycobacteriology: Techniques and Clinical Applications, pp. 385350. Edited by R. A. Ollar & N. O. Connell. New York: Marcel Dekker.
Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537544.[CrossRef][Medline]
Colston, M. J. & Cox, R. A. (1999). Mycobacterial growth and dormancy. In Mycobacteria: Molecular Biology and Virulence, pp. 198219. Edited by C. Ratledge & J. Dale. Oxford, UK: Blackwell Science.
Cooper, S. (1988). Rate and topography of cell wall synthesis during the division cycle of Salmonella typhimurium. J Bacteriol 170, 422430.[Medline]
Cooper, S. & Helmstetter, C. E. (1968). Chromosome replication and the division cycle of Escherichia coli B/r. J Mol Biol 31, 519540.[Medline]
Diaper, J. P. & Edwards, C. (1994). Survival of Staphylococcus aureus in lakewater monitored by flow cytometry. Microbiology 140, 3542.[Abstract]
Ferrari, G., Langen, H., Naito, M. & Pieters, J. (1999). A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell 97, 435447.[Medline]
Flärdh, K., Cohen, P. S. & Kellenberg, S. (1992). Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956. J Bacteriol 174, 67806788.[Abstract]
Gonzalez-y-Merchand, J. A., Colston, M. J. & Cox, R. A. (1999). Effects of growth conditions on expression of mycobacterial murA and tyrS genes and contributions of their transcripts to precursor rRNA synthesis. J Bacteriol 181, 46174627.
Helmstetter, C. E. & Cooper, S. (1968). DNA synthesis during the division cycle of rapidly growing E. coli B/r. J Mol Biol 31, 507518.[Medline]
Hiriyanna, K. T. & Ramakrishnan, T. (1986). Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv. Arch Microbiol 144, 105109.[Medline]
Ingraham, J. L., Maaløe, O. & Neidhardt, F. C. (1983). Growth of the Bacterial Cell. Sunderland, MA: Sinauer Associates.
Jacobs, W. R., Jr (2000). Mycobacterium tuberculosis: a once genetically intractable organism. In Molecular Genetics of Mycobacteria, pp. 116. Edited by G. F. Hatfull & W. R. Jacobs, Jr. Washington, DC: American Society for Microbiology.
Jacobsen, H. (1974). PhD thesis. University of Copenhagen.
Koch, A. L. (1979). Microbial growth in low concentrations of nutrients. In Strategies of Microbial Life in Extreme Environments, pp. 261269. Edited by M. Shilo. Weinheim: Verlag Chemie.
Maaløe, O. & Kjeldgaard, N. O. (1966). Control of Macromolecular Synthesis: a Study of DNA, RNA and Protein Synthesis in Bacteria. New York: W. A. Benjamin.
Pape, T., Wintermeyer, W. & Rodnina, M. V. (1998). Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A-site of the E. coli ribosome. EMBO J 17, 74907497.
Pedersen, S., Bloch, P. L., Reeh, S. & Neidhardt, F. C. (1978). Patterns of protein synthesis in E. coli: a catalog of the amount of 140 individual proteins at different growth rates. Cell 14, 179190.[Medline]
Powell, E. O. (1956). Growth rate and generation time of bacteria, with special reference to continuous culture. J Gen Microbiol 15, 492511.[Medline]
Ratledge, C. (1982). Nutrition, growth and metabolism. In Biology of the Mycobacteria, pp. 185271. Edited by C. Ratledge & J. L. Stanford. London: Academic Press.
Schaechter, M., Williamson, J. P., Hood, J. R., Jr & Koch, A. L. (1962). Growth, cell and nuclear divisions in some bacteria. J Gen Microbiol 29, 421434.[Medline]
Shepard, C. C. (1960). The experimental disease that follows the injection of human leprosy bacilli into the footpads of mice. J Exp Med 112, 445454.
Turner, K., Porter, J., Pickup, R. & Edwards, C. (2000). Changes in viability and macromolecular content of long-term batch cultures of Salmonella typhimurium measured by flow cytometry. J Appl Microbiol 89, 9099.[CrossRef][Medline]
Wayne, L. G. (1994). Cultivation of Mycobacterium tuberculosis for research purposes. In Tuberculosis: Pathogenesis, Protection and Control, pp. 7383. Edited by B. Bloom. Washington, DC: American Society for Microbiology.
Wayne, L. G. & Hayes, L. G. (1996). An in vitro model for sequential study of shift down of Mycobacterium tuberculosis through two stages of replicating persistence. Infect Immun 64, 20622069.[Abstract]
Wayne, L. G. & Kubica, G. P. (1986). The mycobacteria. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 14351457. Edited by P. H. A., Sneath, N. S., Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Weber, I., Fritz, C., Ruttkowski, S., Kreft, A. & Bange, F. C. (2000). Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice. Mol Microbiol 35, 10171025.[CrossRef][Medline]
Wheeler, P. R. & Ratledge, C. (1994). Metabolism of Mycobacterium tuberculosis. In Tuberculosis; Pathogenesis, Protection and Control, pp. 353385. Edited by B. R. Bloom. Washington, DC: American Society for Microbiology.
Winder, F. G. & Rooney, S. A. (1970). Effects of nitrogenous components of the medium on the carbohydrate and nucleic acid content of Mycobacterium tuberculosis BCG. J Gen Microbiol 63, 2939.[Medline]
Woldringh, C. L., Binnerts, J. S. & Mans, A. (1981). Variation in Escherichia coli buoyant density measured in percoll gradients. J Bacteriol 148, 5863.[Medline]
Yoshimura, F. & Nikaido, H. (1982). Permeability of Pseudomonas aeruginosa outer membrane to hydrophobic solutes. J Bacteriol 152, 636642.[Medline]
Received 3 April 2002;
revised 21 October 2002;
accepted 12 November 2002.