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The Normalcy of Dormancy: Common Themes in Microbial Quiescence

2013; Cell Press; Volume: 13; Issue: 6 Linguagem: Inglês

10.1016/j.chom.2013.05.012

1934-6069

Emily S. C. Rittershaus, Seung-Hun Baek, Christopher M. Sassetti,

Fungal and yeast genetics research

All microorganisms are exposed to periodic stresses that inhibit growth. Many bacteria and fungi weather these periods by entering a hardy, nonreplicating state, often termed quiescence or dormancy. When this occurs during an infection, the resulting slowly growing pathogen is able to tolerate both immune insults and prolonged antibiotic exposure. While the stresses encountered in a free-living environment may differ from those imposed by host immunity, these growth-limiting conditions impose common pressures, and many of the corresponding microbial responses appear to be universal. In this review, we discuss the common features of these growth-limited states, which suggest new approaches for treating chronic infections such as tuberculosis. All microorganisms are exposed to periodic stresses that inhibit growth. Many bacteria and fungi weather these periods by entering a hardy, nonreplicating state, often termed quiescence or dormancy. When this occurs during an infection, the resulting slowly growing pathogen is able to tolerate both immune insults and prolonged antibiotic exposure. While the stresses encountered in a free-living environment may differ from those imposed by host immunity, these growth-limiting conditions impose common pressures, and many of the corresponding microbial responses appear to be universal. In this review, we discuss the common features of these growth-limited states, which suggest new approaches for treating chronic infections such as tuberculosis. A defining feature of Mycobacterium tuberculosis, the causative agent of tuberculosis, is its slow growth. The maximal doubling time of this bacterium is approximately 20 hr and is significantly slower when exposed to stresses such as those encountered in the host. Indeed, the bacterial population found in chronically infected animals replicates only once every 100 hr or more (Gill et al., 2009Gill W.P. Harik N.S. Whiddon M.R. Liao R.P. Mittler J.E. Sherman D.R. A replication clock for Mycobacterium tuberculosis.Nat. Med. 2009; 15: 211-214Crossref PubMed Scopus (203) Google Scholar; Muñoz-Elías et al., 2005Muñoz-Elías E.J. Timm J. Botha T. Chan W.T. Gomez J.E. McKinney J.D. Replication dynamics of Mycobacterium tuberculosis in chronically infected mice.Infect. Immun. 2005; 73: 546-551Crossref PubMed Scopus (148) Google Scholar), and subpopulations of bacteria are thought to cease growth entirely for significant periods. The importance of this relatively quiescent behavior is difficult to overstate, as it likely underlies the chronicity of the infection as well as the requirement for extended antibiotic therapy. Dormancy, latency, and persistence are conceptually related terms used to describe the propensity of M. tuberculosis to arrest its growth in response to host-imposed stress. Because this behavior is very different from well-studied model organisms or agents of acute infection, it is sometimes considered an unusual selective adaptation specific to the pathogenic mycobacteria. While this trait is likely adaptive, it is by no means unusual. In fact, slow to negligible replication is the norm in the microbial world, where organisms often inhabit environments that are incompatible with rapid growth. In this review, we will consider mycobacterial dormancy in this broader ecological context. All microbes are subjected to changing environments, and the basic requirements for growth (carbon, nitrogen, phosphorus, water, etc.) are not always available. The evolutionary success of virtually all microbial species requires the ability to weather these periods, and the spectrum of survival strategies used by different microbial species has been studied for decades (Steinhaus and Birkeland, 1939Steinhaus E.A. Birkeland J.M. Studies on the life and death of bacteria: I. The senescent phase in aging cultures and the probable mechanisms involved.J. Bacteriol. 1939; 38: 249-261PubMed Google Scholar). In general, these strategies can be described as variations of three general themes (Figure 1). The physiology of organisms that evolved in consistently nutrient-rich environments, such as the bacteria Escherichia coli, are tuned to maximize growth rate (Neidhardt, 1999Neidhardt F.C. Bacterial growth: constant obsession with dN/dt.J. Bacteriol. 1999; 181: 7405-7408Crossref PubMed Google Scholar). Under nutrient-replete conditions in which bacterial metabolism is often studied, these organisms maximize their growth at the expense of economy by using relatively inefficient fermentative pathways to generate energy (Wolfe, 2005Wolfe A.J. The acetate switch.Microbiol. Mol. Biol. Rev. 2005; 69: 12-50Crossref PubMed Scopus (734) Google Scholar). Upon nutrient exhaustion the majority of these bacterial populations die, leaving a few viable organisms that subsist on the corpses of their siblings. Slow growth and cell death are balanced during this period (Finkel, 2006Finkel S.E. Long-term survival during stationary phase: evolution and the GASP phenotype.Nat. Rev. Microbiol. 2006; 4: 113-120Crossref PubMed Scopus (294) Google Scholar). When environmental conditions become more favorable, the few survivors resume growth. The ability to replicate rapidly is likely to be an essential component of this strategy, as these organisms must outcompete neighboring microbes to consume the newly introduced nutrients. A distinct strategy for surviving periods of growth-limiting stress appears to be favored by both M. tuberculosis (Betts et al., 2002Betts J.C. Lukey P.T. Robb L.C. McAdam R.A. Duncan K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling.Mol. 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These “quiescent” cells can be differentiated from truly dormant spore-like forms because they display nominal metabolic capacity, maintain their membrane potential, and do not undergo obvious morphological differentiation (Gengenbacher et al., 2010Gengenbacher M. Rao S.P. Pethe K. Dick T. Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability.Microbiology. 2010; 156: 81-87Crossref PubMed Scopus (156) Google Scholar; Rao et al., 2008Rao S.P. Alonso S. Rand L. Dick T. Pethe K. The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic, nonreplicating Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. USA. 2008; 105: 11945-11950Crossref PubMed Scopus (305) Google Scholar). This strategy allows the viable bacterial population size to be maintained throughout the period of stress (Jones and Lennon, 2010Jones S.E. Lennon J.T. Dormancy contributes to the maintenance of microbial diversity.Proc. Natl. Acad. Sci. USA. 2010; 107: 5881-5886Crossref PubMed Scopus (451) Google Scholar), relieving the emphasis for rapid growth seen in the bust-and-boom model. Sporulation is the purest form of microbial dormancy. When exposed to growth-restricting stress, some bacteria undergo an asymmetric cell division to produce a hardy metabolically inactive daughter cell called a spore (Stragier and Losick, 1996Stragier P. Losick R. Molecular genetics of sporulation in Bacillus subtilis.Annu. Rev. Genet. 1996; 30 (297–41)Crossref PubMed Scopus (492) Google Scholar). Upon exposure to favorable environmental conditions, a fraction of spores germinate and initiate rapid growth to reestablish the population. This strategy could be viewed as a combination of the first two. The spore, while fundamentally distinct, shares many structural and biochemical features with quiescent cells, which promote long-term survival. Upon germination, however, rapid growth may be advantageous to repopulate the niche. Indeed, the 10 min replication time of some spore-forming species of the clostridia bacteria are among the fastest known (Kreidl et al., 2002Kreidl K.O. Green G.R. Wren S.M. Intravascular hemolysis from a Clostridium perfringens liver abscess.J. Am. Coll. Surg. 2002; 194: 387Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Historically, the strategies at either end of this spectrum have been most heavily studied. This is due to the experimental tractability of rapidly growing organisms, not because these strategies are more common or important. Indeed, it has been estimated that 60% of the microbial biomass on earth exists in a quiescent state (Cole, 1999Cole J.J. Aquatic microbiology for ecosystem scientists: new and recycled paradigms in ecological microbiology.Ecosystems (N.Y.). 1999; 2: 215-225Crossref Scopus (124) Google Scholar; Lewis and Gattie, 1991Lewis D.L. Gattie D.K. Ecology of quiescent microbes.ASM News. 1991; 57: 27-32Google Scholar). Despite its ubiquity, we still know relatively little about the regulatory mechanisms and physiological changes that define microbial quiescence. While these cellular adaptations are not exactly the same for all organisms or under all conditions, common themes can be defined (Figure 2). In this review, we will consider the general adaptations that are required for quiescence in diverse microorganisms and discuss how these insights might be used to develop more effective therapies for chronic infections such as tuberculosis. An almost universal property of quiescent cells is the accumulation of carbon stores, although the chemical structure of the storage form can differ. During low growth states, the yeast Saccharomyces cerevisiae accumulates glycogen, trehalose, and triglycerides as the main forms of metabolizable carbon (Gray et al., 2004Gray J.V. Petsko G.A. Johnston G.C. Ringe D. Singer R.A. Werner-Washburne M. “Sleeping beauty”: quiescence in Saccharomyces cerevisiae.Microbiol. Mol. Biol. Rev. 2004; 68: 187-206Crossref PubMed Scopus (401) Google Scholar). The bacterial pathogen Vibrio cholerae accumulates glycogen in preparation for survival in nutrient-poor environments (Bourassa and Camilli, 2009Bourassa L. Camilli A. Glycogen contributes to the environmental persistence and transmission of Vibrio cholerae.Mol. Microbiol. 2009; 72: 124-138Crossref PubMed Scopus (54) Google Scholar). Additionally, many bacteria store fatty acids in the form of triglycerides (Daniel et al., 2004Daniel J. Deb C. Dubey V.S. Sirakova T.D. Abomoelak B. Morbidoni H.R. Kolattukudy P.E. Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture.J. Bacteriol. 2004; 186: 5017-5030Crossref PubMed Scopus (238) Google Scholar; Kalscheuer et al., 2007Kalscheuer R. Stöveken T. Malkus U. Reichelt R. Golyshin P.N. Sabirova J.S. Ferrer M. Timmis K.N. Steinbüchel A. Analysis of storage lipid accumulation in Alcanivorax borkumensis: evidence for alternative triacylglycerol biosynthesis routes in bacteria.J. Bacteriol. 2007; 189: 918-928Crossref PubMed Scopus (85) Google Scholar) and wax esters (Sirakova et al., 2012Sirakova T.D. Deb C. Daniel J. Singh H.D. Maamar H. Dubey V.S. Kolattukudy P.E. Wax ester synthesis is required for Mycobacterium tuberculosis to enter in vitro dormancy.PLoS ONE. 2012; 7: e51641https://doi.org/10.1371/journal.pone.0051641Crossref PubMed Scopus (23) Google Scholar). Both triglycerides and wax esters also accumulate in plant seeds (Radunz and Schmid, 2000Radunz A. Schmid G.H. Wax esters and triglycerides as storage substances in seeds of Buxus sempervirens.Eur. J. Lipid Sci. Technol. 2000; 102: 734-738Crossref Google Scholar), indicating that this mode of storage is advantageous for organisms that represent vastly separated domains of life. In addition, linear plastic polymers like polyhydroxyalkanoates and poly-β-hydroxybutyric acid can serve as a carbon repository in a variety of bacteria living in the soil and the rhizosphere (Kadouri et al., 2005Kadouri D. Jurkevitch E. Okon Y. Castro-Sowinski S. Ecological and agricultural significance of bacterial polyhydroxyalkanoates.Crit. Rev. Microbiol. 2005; 31: 55-67Crossref PubMed Scopus (154) Google Scholar). What is the purpose of carbon storage? The most intuitive answer is that these cells are simply “storing nuts for winter,” and these nutritional stores can be rapidly mobilized to fuel growth when environmental conditions improve. This role has been most clearly demonstrated in the S. cerevisiae cell, where the trehalose stores that accumulate in stationary cultures are immediately consumed upon addition of fresh media to fuel rapid regrowth (Shi et al., 2010Shi L. Sutter B.M. Ye X. Tu B.P. Trehalose is a key determinant of the quiescent metabolic state that fuels cell cycle progression upon return to growth.Mol. Biol. Cell. 2010; 21: 1982-1990Crossref PubMed Scopus (79) Google Scholar). Glycogen may serve a similar role in V. cholerae, a bacterium whose life cycle relies on periodic switches from the nutrient-replete mammalian gut to nutrient-poor aquatic environments (Bourassa and Camilli, 2009Bourassa L. Camilli A. Glycogen contributes to the environmental persistence and transmission of Vibrio cholerae.Mol. Microbiol. 2009; 72: 124-138Crossref PubMed Scopus (54) Google Scholar). Carbon storage has also been found to play an important role in remodeling cellular carbon fluxes and facilitating entry into the quiescent state. Diverse stresses, such as low oxygen, low pH, or low iron, all induce a storage response in M. tuberculosis through the activation of a common sensor-kinase system, DosRST (Bacon et al., 2007Bacon J. Dover L.G. Hatch K.A. Zhang Y. Gomes J.M. Kendall S. Wernisch L. Stoker N.G. Butcher P.D. Besra G.S. Marsh P.D. Lipid composition and transcriptional response of Mycobacterium tuberculosis grown under iron-limitation in continuous culture: identification of a novel wax ester.Microbiology. 2007; 153: 1435-1444Crossref PubMed Scopus (58) Google Scholar; Baek et al., 2011Baek S.H. Li A.H. Sassetti C.M. Metabolic regulation of mycobacterial growth and antibiotic sensitivity.PLoS Biol. 2011; 9: e1001065https://doi.org/10.1371/journal.pbio.1001065Crossref PubMed Scopus (151) Google Scholar; Daniel et al., 2011Daniel J. Maamar H. Deb C. Sirakova T.D. Kolattukudy P.E. Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages.PLoS Pathog. 2011; 7: e1002093https://doi.org/10.1371/journal.ppat.1002093Crossref PubMed Scopus (298) Google Scholar). The DosS sensor likely responds to alterations in cellular redox state in these contexts (Honaker et al., 2010Honaker R.W. Dhiman R.K. Narayanasamy P. Crick D.C. Voskuil M.I. DosS responds to a reduced electron transport system to induce the Mycobacterium tuberculosis DosR regulon.J. Bacteriol. 2010; 192: 6447-6455Crossref PubMed Scopus (61) Google Scholar), and triggers the synthesis of triglycerides that are stored in large cytosolic inclusions (Garton et al., 2002Garton N.J. Christensen H. Minnikin D.E. Adegbola R.A. Barer M.R. Intracellular lipophilic inclusions of mycobacteria in vitro and in sputum.Microbiology. 2002; 148: 2951-2958Crossref PubMed Scopus (162) Google Scholar). The impact of this response appears to extend beyond the generation of nutrient stores. That is, disruption of the triglyceride biosynthesis pathway in M. tuberculosis reverses the growth arrest that is normally caused by these stresses, but has little effect on the subsequent recovery of growth when the stress is relieved (Baek et al., 2011Baek S.H. Li A.H. Sassetti C.M. Metabolic regulation of mycobacterial growth and antibiotic sensitivity.PLoS Biol. 2011; 9: e1001065https://doi.org/10.1371/journal.pbio.1001065Crossref PubMed Scopus (151) Google Scholar). This inverse relationship between growth and triglyceride production appears to result from the redirection of acetyl-CoA from the TCA cycle, where it is used to generate energy during aerobic respiration, into lipid synthesis, where acetyl CoA serves as a building block for fatty acids. The growth-limiting effect of carbon storage is unlikely to be restricted to mycobacteria. For example, S. cerevisiae mutants that are unable to produce glycogen or trehalose consume more CO2 than the wild-type strain during slow growth (Silljé et al., 1999Silljé H.H. Paalman J.W. ter Schure E.G. Olsthoorn S.Q. Verkleij A.J. Boonstra J. Verrips C.T. Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae.J. Bacteriol. 1999; 181: 396-400PubMed Google Scholar), indicating higher TCA flux in the absence of carbon storage. The almost universal propensity of microorganisms to accumulate acetyl CoA-derived carbon stores under growth-limiting stresses suggests that this may represent a common strategy for reducing growth and metabolic rate. Virtually all bacteria are surrounded by an elastic meshwork of peptidoglycan that maintains cellular integrity under changing environmental conditions. This structure is composed of glycan chains, consisting of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), crosslinked through short peptide moieties. Not surprisingly, the long-term survival of both spores and quiescent cells depends on specific alterations in the composition of this structure. For example, in stationary phase cultures, the Gram-positive bacteria Staphylococcus aureus generates a cell wall that is structurally different from the peptidoglycan found during exponential phase growth, in that it contains fewer pentaglycine bridges, which crosslink the glycan chains, and is significantly thicker (Zhou and Cegelski, 2012Zhou X. Cegelski L. Nutrient-dependent structural changes in S. aureus peptidoglycan revealed by solid-state NMR spectroscopy.Biochemistry. 2012; 51: 8143-8153Crossref PubMed Scopus (40) Google Scholar). Similarly, the level and gradient of crosslinking are important for the formation of bacterial spores. In the spore peptidoglycan layer of the soil-dwelling bacteria Bacillus subtilis, the peptide side chains serving as crosslinkers are completely or partially removed from the NAM residues and replaced by muramic-δ-lactam, a specificity determinant for germination autolytic enzymes, at every second NAM position in the cortex glycan strands. As a consequence, overall levels of crosslinking are markedly decreased in the spore cortex as compared to the vegetative cell wall (Atrih et al., 1996Atrih A. Zöllner P. Allmaier G. Foster S.J. Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.J. Bacteriol. 1996; 178: 6173-6183Crossref PubMed Scopus (118) Google Scholar). Thus, common features of the peptidoglycan in both quiescent cells and spores are reduced crosslinks and increased peptidoglycan mass. The regulation of these modifications is likely complex, but recent observations suggest that extracellular D-amino acids, such as D-methionine and D-leucine, could play an important role. D-amino acids accumulate to millimolar levels in the supernatants of stationary phase bacterial culture, where they regulate cell wall synthetic enzymes and are incorporated into the peptidoglycan polymer. The increased abundance of D-amino acids in cultures of nongrowing cells and their ability to alter the osmotic sensitivity of V. cholerae (Lam et al., 2009Lam H. Oh D.C. Cava F. Takacs C.N. Clardy J. de Pedro M.A. Waldor M.K. D-amino acids govern stationary phase cell wall remodeling in bacteria.Science. 2009; 325: 1552-1555Crossref PubMed Scopus (344) Google Scholar) suggests a likely role in remodeling the cell wall for quiescence. Like many other bacteria, M. tuberculosis may vary the crosslinking of its peptidoglycan in slow growth states (Lavollay et al., 2008Lavollay M. Arthur M. Fourgeaud M. Dubost L. Marie A. Veziris N. Blanot D. Gutmann L. Mainardi J.L. The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation.J. Bacteriol. 2008; 190: 4360-4366Crossref PubMed Scopus (216) Google Scholar). During exponential growth, M. tuberculosis peptidoglycan is crosslinked largely via linkages between the third and fourth amino acids in the stem peptide, the chain of amino acids in peptidoglycan that crosslinks adjacent strands (i.e., 4→3 linkages). However, in stationary phase the cell wall primarily consists of 3→3 crosslinks. While not all investigators have observed these changes (Kumar et al., 2012Kumar P. Arora K. Lloyd J.R. Lee I.Y. Nair V. Fischer E. Boshoff H.I. Barry 3rd, C.E. Meropenem inhibits D,D-carboxypeptidase activity in Mycobacterium tuberculosis.Mol. Microbiol. 2012; 86: 367-381Crossref PubMed Scopus (89) Google Scholar), altered crosslinking could significantly change the physical characteristics of the cell wall. In addition, 3→3 crosslinks are made by transpeptidases that are insensitive to β-lactam antibiotics that inhibit cell wall synthesis, suggesting the reduction in 4→3 linkages may reduce antibiotic susceptibility. Indeed, when the L,D-transpeptidase (MT2594/Rv2518c) responsible for making the 3→3 linkages in M. tuberculosis was inactivated, the bacteria became more susceptible to the β-lactam antibiotic amoxicillin, and persistence in animals was attenuated (Gupta et al., 2010Gupta R. Lavollay M. Mainardi J.L. Arthur M. Bishai W.R. Lamichhane G. The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin.Nat. Med. 2010; 16: 466-469Crossref PubMed Scopus (176) Google Scholar). The mycobacterial cell wall is much more complex than those surrounding the organisms discussed above, and the full complement of alterations that accompany quiescence have yet to be defined. Mycobacterial peptidoglycan is conjugated to an additional glycan layer and finally to a functional outer membrane composed of very long chain fatty acids called mycolic acids. Surrounding this hydrophobic layer is a capsule that is largely comprised of the polysaccharide α-glucan. Thickening of the mycobacterial cell wall upon hypoxia-induced stasis was first demonstrated more than 30 years ago (Wayne, 1976Wayne L.G. Dynamics of submerged growth of Mycobacterium tuberculosis under aerobic and microaerophilic conditions.Am. Rev. Respir. Dis. 1976; 114: 807-811PubMed Google Scholar). More recently, a computational model of the M. tuberculosis response to hypoxia was used to predict a large increase in production of cell wall components like mycolic acids and peptidoglycan (Fang et al., 2012Fang X. Wallqvist A. Reifman J. Modeling phenotypic metabolic adaptations of Mycobacterium tuberculosis H37Rv under hypoxia.PLoS Comput. Biol. 2012; 8: e1002688https://doi.org/10.1371/journal.pcbi.1002688Crossref PubMed Scopus (43) Google Scholar). This prediction is consistent with electron microscopy studies that demonstrate thickening of the outer mycolic acid and/or capsule layers of the cell wall (Cunningham and Spreadbury, 1998Cunningham A.F. Spreadbury C.L. Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton alpha-crystallin homolog.J. Bacteriol. 1998; 180: 801-808Crossref PubMed Google Scholar). A major physiological outcome of these changes is decreased permeability of the cell wall, and the uptake of several classes of antibiotics into quiescent M. tuberculosis is significantly decreased relative to replicating cells (Sarathy et al., 2013Sarathy J. Dartois V. Dick T. Gengenbacher M. Reduced drug uptake in phenotypically resistant nutrient-starved nonreplicating Mycobacterium tuberculosis.Antimicrob. Agents Chemother. 2013; 57: 1648-1653Crossref PubMed Scopus (75) Google Scholar). In addition to its structural roles, cell wall metabolism also appears to play an important role in generating signals that regulate the germination of spores and the exit from quiescence. In B. subtilis, the PrkC Ser/Thr kinase responds to the presence of extracellular peptidoglycan fragments and induces spore germination (Shah et al., 2008Shah I.M. Laaberki M.H. Popham D.L. Dworkin J. A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments.Cell. 2008; 135: 486-496Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). These fragments are released by growing cells, providing a mechanism by which the spore can sense the presence of a favorable growth environment using cues from neighboring bacteria. M. tuberculosis expresses a similar Ser/Thr kinase, PknB, which is also capable of binding extracellular peptidoglycan fragments (Mir et al., 2011Mir M. Asong J. Li X. Cardot J. Boons G.J. Husson R.N. The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr kinase PknB binds specific muropeptides and is required for PknB localization.PLoS Pathog. 2011; 7: e1002182https://doi.org/10.1371/journal.ppat.1002182Crossref PubMed Scopus (107) Google Scholar) and regulates cell wall synthesis and growth (Gee et al., 2012Gee C.L. Papavinasasundaram K.G. Blair S.R. Baer C.E. Falick A.M. King D.S. Griffin J.E. Venghatakrishnan H. Zukauskas A. Wei J.R. et al.A phosphorylated pseudokinase complex controls cell wall synthesis in mycobacteria.Sci. Signal. 2012; 5: ra7https://doi.org/10.1126/scisignal.2002525Crossref PubMed Scopus (101) Google Scholar). Activation of this kinase could explain the ability of spent culture medium to promote the regrowth of quiescent mycobacteria, as this activity depends on secreted lysozyme-like proteins (Mukamolova et al., 1998Mukamolova G.V. Kaprelyants A.S. Young D.I. Young M. Kell D.B. A bacterial cytokine.Proc. Natl. Acad. Sci. USA. 1998; 95: 8916-8921Crossref PubMed Scopus (314) Google Scholar) that could act by liberating peptidoglycan-derived PknB ligands. It may seem intuitive that RNA and protein synthesis will proceed at negligible rates in the quiescent cell. However, the dynamics of macromolecular synthesis are more complicated than they appear and vary during the entry, maintenance, and exit from quiescence. During entry and exit, protein synthesis accelerates. Protein turnover increases 5-fold in famished E. coli cells due to proteases that are produced in early stationary phase. This enhanced protein turnover during the transition to the growth-limited state facilitates de novo protein synthesis in the absence of an exogenous carbon source (Shaikh et al., 2010Shaikh A.S. Tang Y.J. Mukhopadhyay A. Martín H.G. Gin J. Benke P.I. Keasling J.D. Study of stationary phase metabolism via isotopomer analysis of amino acids from an isolated protein.Biotechnol. Prog. 2010; 26: 52-56PubMed Google Scholar), and the required amino acids are provided by peptidase-dependent autophagy, in which amino acids are produced via protein hydrolysis and degradation (Reeve et al., 1984Reeve C.A. Amy P.S. Matin A. Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12.J. Bacteriol. 1984; 160: 1041-1046Crossref PubMed Google Scholar). Similarly, increased protein turnover may also be required for exiting the quiescent state. Regrowth of M. tuberculosis from hypoxia-induced stasis is accompanied by an increase of the ClgR regulatory protein, which induces expression of the ClpXP protease that uses ATP hydrolysis to unfold proteins for subsequent degradation (Sherrid et al., 2010Sherrid A.M. Rustad T.R. Cangelosi G.A. Sherman D.R. Characterization of a Clp protease gene regulator and the reaeration response in Mycobacterium tuberculosis.PLoS ONE. 2010; 5: e11622https://doi.org/10.1371/journal.pone.0011622Crossref PubMed Scopus (55) Google Scholar). Here, increased protein turnover is a likely indicator of the wholesale metabolic remodeling necessary to shift between growth states. Once quiescence is established, however, it is reasonable to assume that the synthesis of RNA and protein will slow considerably. Indeed, quiescence in S. cerevisiae is accompanied by a 3- to 5-fold decrease in overall transcription rate (Choder, 1991Choder M. A general topoisomerase I-dependent transcriptional repression in the stationary phase in yeast.Genes Dev. 1991; 5: 2315-2326Crossref PubMed Scopus (94) Google Scholar), and a 20-fold decrease in protein synthesis (Fuge et al., 1994Fuge E.K. Braun E.L. Werner-Washburne M. Protein synthesis in long-term stationary-phase cultures of Saccharomyces cerevisiae.J. Bacteriol. 1994; 176: 5802-5813Crossref PubMed Scopus (127) Google Scholar). The mechanisms underlying the reduction of macromolecular synthesis in slowly growing E. coli have been explored in great detail. While the rate of nascent RNA and polypeptide chain elongation remains relatively constant, the number of synthetic sites decreases (Pedersen, 1986Pedersen S. The chain growth rate for protein synthesis varies in Escherichia coli.in: Schaechter M. Neidhardt F.C. Ingraham J.L. Kjeldgaard N.O. The Molecular Biology of Bacterial Growth. Jones and Bartlett, Inc., Boston, Mass1986: 13-20Google Scholar). As approximately half of the mass of the rap