- Email: [email protected]
Bacterial spore germination and protein mobility
Update
452
TRENDS in Microbiology
9 Rosengard, A.M. et al. (2002) Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement. Proc. Natl. Acad. Sci. U.S.A. 99, 8808– 8813 10 Rother, R.P. et al. (1994) Inhibition of complement-mediated cytolysis by the terminal complement inhibitor of herpesvirus saimiri. J. Virol. 68, 730 – 737 11 Albrecht, J.C. et al. (1992) Herpesvirus saimiri has a gene specifying a homologue of the cellular membrane glycoprotein CD59. Virology 190, 527 – 530 12 Fodor, W.L. et al. (1995) Primate terminal complement inhibitor homologues of human CD59. Immunogenetics 41, 51 13 Fodor, W.L. et al. (1995) The complement control protein homolog of herpesvirus saimiri regulates serum complement by inhibiting C3 convertase activity. J. Virol. 69, 3889 – 3892 14 Boshoff, C. et al. (1995) Kaposi’s sarcoma-associated herpesvirus infects endothelial and spindle cells. Nat. Med. 1, 1274– 1278 15 Chang, Y. et al. (1994) Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266, 1865– 1869 16 Cesarman, E. et al. (1995) Kaposi’s sarcoma-associated herpesviruslike DNA sequences in AIDS-related body-cavity-based lymphomas. N. Engl. J. Med. 332, 1186– 1191 17 Soulier, J. et al. (1995) Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman’s disease. Blood 86, 1276 – 1280
Vol.11 No.10 October 2003
18 Means, R.E. et al. (2002) Kaposi’s sarcoma associated herpesvirus immune evasion strategies. Front. Biosci. 7, e185 – e203 19 Neipel, F. et al. (1997) Cell-homologous genes in the Kaposi’s sarcomaassociated rhadinovirus human herpesvirus 8: determinants of its pathogenicity? J. Virol. 71, 4187 – 4192 20 Russo, J.J. et al. (1996) Nucleotide sequence of the Kaposi sarcomaassociated herpesvirus (HHV8). Proc. Natl. Acad. Sci. U.S.A. 93, 14862 – 14867 21 Kapadia, S.B. et al. (2002) Critical role of complement and viral evasion of complement in acute, persistent, and latent gammaherpesvirus infection. Immunity 17, 143– 155 22 Kapadia, S.B. et al. (1999) Murine gammaherpesvirus 68 encodes a functional regulator of complement activation. J. Virol. 73, 7658– 7670 23 Krych, M. et al. (1992) Complement receptors. Curr. Opin. Immunol. 4, 8 – 13 24 Nemerow, G.R. et al. (1987) Identification of gp350 as the viral glycoprotein mediating attachment of Epstein – Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d. J. Virol. 61, 1416 – 1420
0966-842X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2003.08.004
Bacterial spore germination and protein mobility Anne Moir Department of Molecular Biology & Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK
Fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) has been used to report on protein mobility in single spores. Proteins found in dormant Bacillus spores are not mobile; however, mobility is restored when germination occurs and the core rehydrates. Spores of a cwlD mutant, in which the cortex is resistant to hydrolysis, are able to complete the earliest stages of germination in response to a specific germinant stimulus; in these circumstances, the protein in the spore remains immobile. Therefore, the earliest stages of spore germination, including loss of resistance to extreme heat and the complete release of the spore component dipicolinic acid, are achieved without the restoration of protein mobility.
Spore resistance and dormancy Bacterial endospores, formed by bacilli and clostridia, are commonly found in any soil or water environment [1], and are the most resistant form of life on Earth. The complex structure of the spore ensures the maintenance of a dormant state and protects the cellular compartment from environmental insult [2]. Cowan et al. [3] provide visual evidence that proteins in the core of the dormant spore are not mobile. The core of a dormant spore contains normal cellular cytoplasmic components, from enzymes and ribosomes to DNA, but all are in an inactive state. Even for spores suspended in water, the core has a low water content (estimated at 28 – 55% for different species [4]); the state of Corresponding author: Anne Moir ([email protected]). http://www.trends.com
the water molecules is not understood – they may not be freely mobile, but they can be exchanged with external water [5]. The wet-heat resistance of dormant spores correlates with the level of dehydration [6]. The DNA in the core is complexed with small acid-soluble proteins (SASPs) [7] that protect it from heat and UV-damage. Dipicolinic acid (DPA) accumulates in the core during sporulation, mainly as a calcium chelate [8]. The inner membrane of the dormant spore is in an unusual environment and has been described as ‘semicrystalline’, only regaining the fluidity characteristics of a normal cell membrane when the spore germinates [9]. It is surrounded by a thin peptidoglycan germ-cell wall that is retained on spore germination, and a very thick cortex consisting of a modified peptidoglycan that is important in the maintenance of the spore’s dehydrated state and which is degraded during germination. This modified peptidoglycan is synthesized across a mother-cell-derived membrane, which remains in a residual, but probably incomplete, form under the spore coat. The coat is composed of multiple layers of protein arrays and is impermeable to enzymes (Figure 1). Spore germination The spore germinates in response to an environmental signal; the most common germinants are amino acids, sugars or ribosides. This germination process uses components already established in the spore during its formation, and is essentially a biophysical and degradative one [10,11]. The germinant penetrates the coat and cortex and interacts with a receptor complex [12] located in the
Update
TRENDS in Microbiology
453
the two germination-specific lytic enzymes (CwlJ) is localized at the coat– cortex interface, and can be activated artificially by high concentrations of external CaDPA, which might also be the natural activating signal for this enzyme [21]. The other major lytic enzyme, SleB, is present in the spore inner membrane and in the outer layers [16]; how it is activated, and whether it is necessary at both locations, remains unclear. There is no information about the enzymes that destroy the integrity of the protein coat.
Spore core Inner membrane Peptidoglycan cortex Multilayered protein coats
TRENDS in Microbiology
Figure 1. A representation of spore and germination events. Blue arrow: the germinant interacts with the receptor at the inner membrane; the membrane becomes transiently permeable – ions move out of the core. Yellow arrow: cortex lytic enzymes are activated – the cortex degrades, the core rehydrates and proteins become mobile – metabolism resumes.
inner spore membrane [13] (Figure 2). Multiple operons that encode such receptors are annotated in the genome sequences of all spore-formers. However, the precise mode of action of this integral membrane receptor remains elusive. Another germination protein in B. cereus [14] acts as an ion antiporter and is required for early germination events. A homologue to this antiporter has also been found in B. megaterium [15]. This suggests that the local movement of ions is probably involved in the membrane receptor function. The downstream consequences of germinant– receptor interaction include a rapid efflux of monovalent cations (Hþ, Naþ and Kþ) from the core. Release of DPA and calcium ions follows, and the core becomes slightly more hydrated. Lytic enzymes that hydrolyse the specialized peptidoglycan of the cortex are activated (Figure 1), leading to full rehydration [16–19]. The internal pH of the spore core (as low as pH6.5 late in sporulation) increases [11]. Enzymes are reactivated and ATP is synthesized. The SASPs are degraded by a specific protease, releasing the DNA from its complexed state [11]. The metabolizing and energized germinated spore can then resume RNA, protein and DNA synthesis in the outgrowth phase [20]. There might be alternative modes of cortex-lytic enzyme activation in different species [19,21]. In B. subtilis, one of
(a)
(b)
1 2 3 4
Figure 2. The structure of dormant and germinated spores. The scale bar represents 0.4 mm. Horizontal lines end in 1, core; 2, inner membrane; 3, cortex; 4, coat layers. (a) A dormant spore of Bacillus subtilis, showing the dark central core, surrounded by the electron-transparent peptidoglycan cortex and the thick, multilayered coats. (b) A germinating spore of B. subtilis. The core has swelled and is rehydrated; substructures, such as transparent nucleoid and granular ribosomes, are visible. The inner membrane, around the core, is now clearly defined. The cortex is partially degraded and coat layers are thinner. http://www.trends.com
Vol.11 No.10 October 2003
Protein mobility measurement It is hard to study dormancy because any biochemical approach that breaks the spore structure also destroys its crucial characteristics. In addition, biophysical measurements on intact spores, although informative [6], are often difficult to interpret in functional terms because the spore is composed of heterogeneous layers. Cowan et al. [3] exploited a B. subtilis strain in which green fluorescent protein (GFP) is produced exclusively in the forespore compartment during sporulation, a region destined to become the core of the dormant spore. FRAP (fluorescence recovery after photobleaching, a technology reviewed in [22]), was used to report on protein mobility in single spores [3]. Using a confocal laser-scanning microscope, fluorescent images of the spore were collected. A region corresponding to one-half of the spore was then exposed to unattenuated laser light, irreversibly photobleaching the GFP and destroying the fluorescence locally. Fluorescence is restored as nonbleached GFP from the untreated half diffuses into the bleached region. Successive images were collected and processed to estimate the kinetics of this process [3]. Comparing dormant, germinated and part-germinated spores In fully germinated spores, the photobleached GFP was quickly replaced by mobile protein from the remainder of the germinated spore core, as would be expected in normal cell cytoplasm. Dramatically, this was not the case in dormant spores; the GFP did not move through the cytoplasm at a significant rate. With the use of controls, the diffusion rate of GFP in the dormant spore was estimated to be at least 104-fold lower than in normal cytoplasm, demonstrating that essentially all the GFP is immobile [3]. Spores will germinate, lose their heat-resistance and rehydrate if lysozyme gains access to the cortex through artificially permeabilised coats. Paradoxically, the loss of the spore’s extreme heat resistance during normal germination does not require cortex hydrolysis! In cwlD mutant spores of B. subtilis the cortex peptidoglycan lacks the muramic lactam that identifies the cortex as a substrate for lytic enzymes. In this mutant, the spore core remains imprisoned inside the cortex [23], even though many of the early events in spore germination including cation release, DPA release, and a small measure of rehydration (from 40% to , 60% water [3]) are completed [24]. Full rehydration is dependent on cortex hydrolysis, which does not occur. Therefore, enzymic processes such as ATP synthesis do not resume and proteolysis of SASPs is minimal [24]. FRAP analysis of these ‘stage I germinated’ cwlD mutant spores revealed that the GFP was still immobile [3],
Update
454
TRENDS in Microbiology
despite the small increase in water content. Therefore, protein immobility correlates with the failure of spores to resume metabolism. These spores are no longer as heatresistant as dormant spores (although they are more heat-resistant than fully germinated spores). The extreme heat-resistance in dormant spores is lost, therefore, following a relatively small degree of rehydration, without the cytoplasmic proteins becoming mobile. Which characteristic of the partial germinated state is responsible for this dramatic reduction in heat-resistance is not clear; changes in the spore inner membrane properties represent an attractive candidate. The loss of DPA from the core cannot be responsible because a DPA-minus B. subtilis mutant, stabilized by defects in all three major germinant receptors, was shown to be relatively heat resistant [8]. Conclusions Cowan et al. [3] have demonstrated the immobility of protein in dormant and part-germinated spores, and the restoration of mobility following cortex hydrolysis. Studies such as these help to define and separate events in the molecular physiology of germination, none of which are fully understood. A better appreciation of the biophysical properties of core and inner spore membrane in the partgerminated mutant spore might be the key to understanding the role of the germinant receptors and other membrane-active proteins in early stages of germination. References 1 Slepecky, R.A. and Leadbetter, E.R. (1984) On the prevalence and roles of spore-forming bacteria and their spores in nature. In The Bacterial Spore (Vol. 2) (Hurst, A. and Gould, G., eds), pp. 79–99, Academic Press 2 Nicholson, W.L. et al. (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol. Mol. Biol. Rev. 64, 548– 572 3 Cowan, A.E. et al. (2003) A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy. Proc. Natl. Acad. Sci. U. S. A. 100, 4209–4214 4 Beaman, T.C. and Gerhardt, P. (1986) Heat resistance of bacterial spores correlated with protoplast dehydration, mineralisation, and thermal adaptation. Appl. Environ. Microbiol. 52, 1242 – 1246 5 Leuschner, R.G.K. and Lillford, P.J. (2000) Effects of hydration on molecular mobility in phase-bright Bacillus subtilis spores. Microbiol. 146, 49 – 55 6 Gerhardt, P. and Marquis, R.E. (1989) Spore thermoresistance mechanisms. In Regulation of Prokaryotic Development (Smith, I. et al., eds), pp. 43 – 63, American Society for Microbiology
Vol.11 No.10 October 2003
7 Setlow, P. (1995) Mechanisms for the prevention of damage to DNA in spores of Bacillus species. Annu. Rev. Microbiology. 49, 29– 54 8 Paidhungat, M. et al. (2000) Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J. Bacteriol. 182, 5505– 5512 9 Stewart, G.S.A.B. et al. (1980) An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium KM measured by electron spin and nuclear magnetic resonance spectroscopy. Biochim. Biophys. Acta 600, 270 – 290 10 Moir, A. et al. (2002) Spore germination. Cell. Mol. Life Sci. 59, 403–409 11 Paidhungat, M. and Setlow, P. (2002) Spore germination and outgrowth. In Bacillus Subtilis and its Closest Relatives: From Genes to Cells (Sonenshein, A.L. et al., eds), pp. 537 – 548, ASM Press 12 Paidhungat, M. and Setlow, P. (1999) Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant D- alanine. J. Bacteriol. 181, 3341 – 3350 13 Hudson, K.D. et al. (2001) Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore. J. Bacteriol. 183, 4317–4322 14 Southworth, T.W. et al. (2001) GerN, an endospore germination protein of Bacillus cereus, is an Naþ/Hþ-Kþ antiporter. J. Bacteriol. 183, 5896–5903 15 Tani, K. et al. (1996) Cloning and sequencing of the spore germination gene of Bacillus megaterium ATCC 12872: Similarities to the NaHantiporter gene of Enterococcus hirae. Microbiol. Immunol. 40, 99 – 105 16 Chirakkal, H. et al. (2002) Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination. Microbiol. 148, 2383– 2392 17 Bagyan, I. and Setlow, P. (2002) Localization of the cortex lytic enzyme CwlJ in spores of Bacillus subtilis. J. Bacteriol. 184, 1219– 1224 18 Boland, F.M. et al. (2000) Complete spore-cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB. Microbiol. 146, 57–64 19 Shimamoto, S. et al. (2001) Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity. J. Bacteriol. 183, 3742 – 3751 20 Horsburgh, M.J. et al. (2001) Transcriptional responses during outgrowth of Bacillus subtilis endospores. Microbiol. 147, 2933– 2941 21 Paidhungat, M. et al. (2001) Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2 þ -dipicolinate. J. Bacteriol. 183, 4886– 4893 22 Reits, E.A.J. and Neefjes, J.J. (2001) From fixed to FRAP: measuring protein mobility and activity in living cells. Nat. Cell Biol. 3, E145–E147 23 Popham, D.L. et al. (1996) Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc. Natl. Acad. Sci. U. S. A. 93, 15405 – 15410 24 Setlow, B. et al. (2001) Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination. J. Bacteriol. 183, 4894–4899 0966-842X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2003.08.001
| Letter
Searching for keys under the devil’s lamppost Eric W. Brown, J. Eugene LeClerc and Thomas A. Cebula Division of Molecular Biology, Center for Food Safety & Applied Nutrition, Food and Drug Administration, Laurel, MD 20708, USA
In a recent Trends in Microbiology article, ‘The devil is in the detail’, Bentley et al. [1] comment on recent reports regarding the whole-genome sequencing of Salmonella enterica serovar Typhi (S. Typhi) and several other enteric Corresponding author: Thomas A. Cebula ([email protected]). http://www.trends.com
and soil-dwelling microbial species. Having enjoyed this well-written synopsis, we nevertheless felt compelled to discuss some of their S. Typhi conclusions further. The authors highlight a recent manuscript by Deng and colleagues [2] that compares the complete sequence of S. Typhi strain Ty2 with the previously
452
TRENDS in Microbiology
9 Rosengard, A.M. et al. (2002) Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement. Proc. Natl. Acad. Sci. U.S.A. 99, 8808– 8813 10 Rother, R.P. et al. (1994) Inhibition of complement-mediated cytolysis by the terminal complement inhibitor of herpesvirus saimiri. J. Virol. 68, 730 – 737 11 Albrecht, J.C. et al. (1992) Herpesvirus saimiri has a gene specifying a homologue of the cellular membrane glycoprotein CD59. Virology 190, 527 – 530 12 Fodor, W.L. et al. (1995) Primate terminal complement inhibitor homologues of human CD59. Immunogenetics 41, 51 13 Fodor, W.L. et al. (1995) The complement control protein homolog of herpesvirus saimiri regulates serum complement by inhibiting C3 convertase activity. J. Virol. 69, 3889 – 3892 14 Boshoff, C. et al. (1995) Kaposi’s sarcoma-associated herpesvirus infects endothelial and spindle cells. Nat. Med. 1, 1274– 1278 15 Chang, Y. et al. (1994) Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266, 1865– 1869 16 Cesarman, E. et al. (1995) Kaposi’s sarcoma-associated herpesviruslike DNA sequences in AIDS-related body-cavity-based lymphomas. N. Engl. J. Med. 332, 1186– 1191 17 Soulier, J. et al. (1995) Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman’s disease. Blood 86, 1276 – 1280
Vol.11 No.10 October 2003
18 Means, R.E. et al. (2002) Kaposi’s sarcoma associated herpesvirus immune evasion strategies. Front. Biosci. 7, e185 – e203 19 Neipel, F. et al. (1997) Cell-homologous genes in the Kaposi’s sarcomaassociated rhadinovirus human herpesvirus 8: determinants of its pathogenicity? J. Virol. 71, 4187 – 4192 20 Russo, J.J. et al. (1996) Nucleotide sequence of the Kaposi sarcomaassociated herpesvirus (HHV8). Proc. Natl. Acad. Sci. U.S.A. 93, 14862 – 14867 21 Kapadia, S.B. et al. (2002) Critical role of complement and viral evasion of complement in acute, persistent, and latent gammaherpesvirus infection. Immunity 17, 143– 155 22 Kapadia, S.B. et al. (1999) Murine gammaherpesvirus 68 encodes a functional regulator of complement activation. J. Virol. 73, 7658– 7670 23 Krych, M. et al. (1992) Complement receptors. Curr. Opin. Immunol. 4, 8 – 13 24 Nemerow, G.R. et al. (1987) Identification of gp350 as the viral glycoprotein mediating attachment of Epstein – Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d. J. Virol. 61, 1416 – 1420
0966-842X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2003.08.004
Bacterial spore germination and protein mobility Anne Moir Department of Molecular Biology & Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK
Fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) has been used to report on protein mobility in single spores. Proteins found in dormant Bacillus spores are not mobile; however, mobility is restored when germination occurs and the core rehydrates. Spores of a cwlD mutant, in which the cortex is resistant to hydrolysis, are able to complete the earliest stages of germination in response to a specific germinant stimulus; in these circumstances, the protein in the spore remains immobile. Therefore, the earliest stages of spore germination, including loss of resistance to extreme heat and the complete release of the spore component dipicolinic acid, are achieved without the restoration of protein mobility.
Spore resistance and dormancy Bacterial endospores, formed by bacilli and clostridia, are commonly found in any soil or water environment [1], and are the most resistant form of life on Earth. The complex structure of the spore ensures the maintenance of a dormant state and protects the cellular compartment from environmental insult [2]. Cowan et al. [3] provide visual evidence that proteins in the core of the dormant spore are not mobile. The core of a dormant spore contains normal cellular cytoplasmic components, from enzymes and ribosomes to DNA, but all are in an inactive state. Even for spores suspended in water, the core has a low water content (estimated at 28 – 55% for different species [4]); the state of Corresponding author: Anne Moir ([email protected]). http://www.trends.com
the water molecules is not understood – they may not be freely mobile, but they can be exchanged with external water [5]. The wet-heat resistance of dormant spores correlates with the level of dehydration [6]. The DNA in the core is complexed with small acid-soluble proteins (SASPs) [7] that protect it from heat and UV-damage. Dipicolinic acid (DPA) accumulates in the core during sporulation, mainly as a calcium chelate [8]. The inner membrane of the dormant spore is in an unusual environment and has been described as ‘semicrystalline’, only regaining the fluidity characteristics of a normal cell membrane when the spore germinates [9]. It is surrounded by a thin peptidoglycan germ-cell wall that is retained on spore germination, and a very thick cortex consisting of a modified peptidoglycan that is important in the maintenance of the spore’s dehydrated state and which is degraded during germination. This modified peptidoglycan is synthesized across a mother-cell-derived membrane, which remains in a residual, but probably incomplete, form under the spore coat. The coat is composed of multiple layers of protein arrays and is impermeable to enzymes (Figure 1). Spore germination The spore germinates in response to an environmental signal; the most common germinants are amino acids, sugars or ribosides. This germination process uses components already established in the spore during its formation, and is essentially a biophysical and degradative one [10,11]. The germinant penetrates the coat and cortex and interacts with a receptor complex [12] located in the
Update
TRENDS in Microbiology
453
the two germination-specific lytic enzymes (CwlJ) is localized at the coat– cortex interface, and can be activated artificially by high concentrations of external CaDPA, which might also be the natural activating signal for this enzyme [21]. The other major lytic enzyme, SleB, is present in the spore inner membrane and in the outer layers [16]; how it is activated, and whether it is necessary at both locations, remains unclear. There is no information about the enzymes that destroy the integrity of the protein coat.
Spore core Inner membrane Peptidoglycan cortex Multilayered protein coats
TRENDS in Microbiology
Figure 1. A representation of spore and germination events. Blue arrow: the germinant interacts with the receptor at the inner membrane; the membrane becomes transiently permeable – ions move out of the core. Yellow arrow: cortex lytic enzymes are activated – the cortex degrades, the core rehydrates and proteins become mobile – metabolism resumes.
inner spore membrane [13] (Figure 2). Multiple operons that encode such receptors are annotated in the genome sequences of all spore-formers. However, the precise mode of action of this integral membrane receptor remains elusive. Another germination protein in B. cereus [14] acts as an ion antiporter and is required for early germination events. A homologue to this antiporter has also been found in B. megaterium [15]. This suggests that the local movement of ions is probably involved in the membrane receptor function. The downstream consequences of germinant– receptor interaction include a rapid efflux of monovalent cations (Hþ, Naþ and Kþ) from the core. Release of DPA and calcium ions follows, and the core becomes slightly more hydrated. Lytic enzymes that hydrolyse the specialized peptidoglycan of the cortex are activated (Figure 1), leading to full rehydration [16–19]. The internal pH of the spore core (as low as pH6.5 late in sporulation) increases [11]. Enzymes are reactivated and ATP is synthesized. The SASPs are degraded by a specific protease, releasing the DNA from its complexed state [11]. The metabolizing and energized germinated spore can then resume RNA, protein and DNA synthesis in the outgrowth phase [20]. There might be alternative modes of cortex-lytic enzyme activation in different species [19,21]. In B. subtilis, one of
(a)
(b)
1 2 3 4
Figure 2. The structure of dormant and germinated spores. The scale bar represents 0.4 mm. Horizontal lines end in 1, core; 2, inner membrane; 3, cortex; 4, coat layers. (a) A dormant spore of Bacillus subtilis, showing the dark central core, surrounded by the electron-transparent peptidoglycan cortex and the thick, multilayered coats. (b) A germinating spore of B. subtilis. The core has swelled and is rehydrated; substructures, such as transparent nucleoid and granular ribosomes, are visible. The inner membrane, around the core, is now clearly defined. The cortex is partially degraded and coat layers are thinner. http://www.trends.com
Vol.11 No.10 October 2003
Protein mobility measurement It is hard to study dormancy because any biochemical approach that breaks the spore structure also destroys its crucial characteristics. In addition, biophysical measurements on intact spores, although informative [6], are often difficult to interpret in functional terms because the spore is composed of heterogeneous layers. Cowan et al. [3] exploited a B. subtilis strain in which green fluorescent protein (GFP) is produced exclusively in the forespore compartment during sporulation, a region destined to become the core of the dormant spore. FRAP (fluorescence recovery after photobleaching, a technology reviewed in [22]), was used to report on protein mobility in single spores [3]. Using a confocal laser-scanning microscope, fluorescent images of the spore were collected. A region corresponding to one-half of the spore was then exposed to unattenuated laser light, irreversibly photobleaching the GFP and destroying the fluorescence locally. Fluorescence is restored as nonbleached GFP from the untreated half diffuses into the bleached region. Successive images were collected and processed to estimate the kinetics of this process [3]. Comparing dormant, germinated and part-germinated spores In fully germinated spores, the photobleached GFP was quickly replaced by mobile protein from the remainder of the germinated spore core, as would be expected in normal cell cytoplasm. Dramatically, this was not the case in dormant spores; the GFP did not move through the cytoplasm at a significant rate. With the use of controls, the diffusion rate of GFP in the dormant spore was estimated to be at least 104-fold lower than in normal cytoplasm, demonstrating that essentially all the GFP is immobile [3]. Spores will germinate, lose their heat-resistance and rehydrate if lysozyme gains access to the cortex through artificially permeabilised coats. Paradoxically, the loss of the spore’s extreme heat resistance during normal germination does not require cortex hydrolysis! In cwlD mutant spores of B. subtilis the cortex peptidoglycan lacks the muramic lactam that identifies the cortex as a substrate for lytic enzymes. In this mutant, the spore core remains imprisoned inside the cortex [23], even though many of the early events in spore germination including cation release, DPA release, and a small measure of rehydration (from 40% to , 60% water [3]) are completed [24]. Full rehydration is dependent on cortex hydrolysis, which does not occur. Therefore, enzymic processes such as ATP synthesis do not resume and proteolysis of SASPs is minimal [24]. FRAP analysis of these ‘stage I germinated’ cwlD mutant spores revealed that the GFP was still immobile [3],
Update
454
TRENDS in Microbiology
despite the small increase in water content. Therefore, protein immobility correlates with the failure of spores to resume metabolism. These spores are no longer as heatresistant as dormant spores (although they are more heat-resistant than fully germinated spores). The extreme heat-resistance in dormant spores is lost, therefore, following a relatively small degree of rehydration, without the cytoplasmic proteins becoming mobile. Which characteristic of the partial germinated state is responsible for this dramatic reduction in heat-resistance is not clear; changes in the spore inner membrane properties represent an attractive candidate. The loss of DPA from the core cannot be responsible because a DPA-minus B. subtilis mutant, stabilized by defects in all three major germinant receptors, was shown to be relatively heat resistant [8]. Conclusions Cowan et al. [3] have demonstrated the immobility of protein in dormant and part-germinated spores, and the restoration of mobility following cortex hydrolysis. Studies such as these help to define and separate events in the molecular physiology of germination, none of which are fully understood. A better appreciation of the biophysical properties of core and inner spore membrane in the partgerminated mutant spore might be the key to understanding the role of the germinant receptors and other membrane-active proteins in early stages of germination. References 1 Slepecky, R.A. and Leadbetter, E.R. (1984) On the prevalence and roles of spore-forming bacteria and their spores in nature. In The Bacterial Spore (Vol. 2) (Hurst, A. and Gould, G., eds), pp. 79–99, Academic Press 2 Nicholson, W.L. et al. (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol. Mol. Biol. Rev. 64, 548– 572 3 Cowan, A.E. et al. (2003) A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy. Proc. Natl. Acad. Sci. U. S. A. 100, 4209–4214 4 Beaman, T.C. and Gerhardt, P. (1986) Heat resistance of bacterial spores correlated with protoplast dehydration, mineralisation, and thermal adaptation. Appl. Environ. Microbiol. 52, 1242 – 1246 5 Leuschner, R.G.K. and Lillford, P.J. (2000) Effects of hydration on molecular mobility in phase-bright Bacillus subtilis spores. Microbiol. 146, 49 – 55 6 Gerhardt, P. and Marquis, R.E. (1989) Spore thermoresistance mechanisms. In Regulation of Prokaryotic Development (Smith, I. et al., eds), pp. 43 – 63, American Society for Microbiology
Vol.11 No.10 October 2003
7 Setlow, P. (1995) Mechanisms for the prevention of damage to DNA in spores of Bacillus species. Annu. Rev. Microbiology. 49, 29– 54 8 Paidhungat, M. et al. (2000) Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J. Bacteriol. 182, 5505– 5512 9 Stewart, G.S.A.B. et al. (1980) An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium KM measured by electron spin and nuclear magnetic resonance spectroscopy. Biochim. Biophys. Acta 600, 270 – 290 10 Moir, A. et al. (2002) Spore germination. Cell. Mol. Life Sci. 59, 403–409 11 Paidhungat, M. and Setlow, P. (2002) Spore germination and outgrowth. In Bacillus Subtilis and its Closest Relatives: From Genes to Cells (Sonenshein, A.L. et al., eds), pp. 537 – 548, ASM Press 12 Paidhungat, M. and Setlow, P. (1999) Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant D- alanine. J. Bacteriol. 181, 3341 – 3350 13 Hudson, K.D. et al. (2001) Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore. J. Bacteriol. 183, 4317–4322 14 Southworth, T.W. et al. (2001) GerN, an endospore germination protein of Bacillus cereus, is an Naþ/Hþ-Kþ antiporter. J. Bacteriol. 183, 5896–5903 15 Tani, K. et al. (1996) Cloning and sequencing of the spore germination gene of Bacillus megaterium ATCC 12872: Similarities to the NaHantiporter gene of Enterococcus hirae. Microbiol. Immunol. 40, 99 – 105 16 Chirakkal, H. et al. (2002) Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination. Microbiol. 148, 2383– 2392 17 Bagyan, I. and Setlow, P. (2002) Localization of the cortex lytic enzyme CwlJ in spores of Bacillus subtilis. J. Bacteriol. 184, 1219– 1224 18 Boland, F.M. et al. (2000) Complete spore-cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB. Microbiol. 146, 57–64 19 Shimamoto, S. et al. (2001) Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity. J. Bacteriol. 183, 3742 – 3751 20 Horsburgh, M.J. et al. (2001) Transcriptional responses during outgrowth of Bacillus subtilis endospores. Microbiol. 147, 2933– 2941 21 Paidhungat, M. et al. (2001) Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2 þ -dipicolinate. J. Bacteriol. 183, 4886– 4893 22 Reits, E.A.J. and Neefjes, J.J. (2001) From fixed to FRAP: measuring protein mobility and activity in living cells. Nat. Cell Biol. 3, E145–E147 23 Popham, D.L. et al. (1996) Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc. Natl. Acad. Sci. U. S. A. 93, 15405 – 15410 24 Setlow, B. et al. (2001) Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination. J. Bacteriol. 183, 4894–4899 0966-842X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2003.08.001
| Letter
Searching for keys under the devil’s lamppost Eric W. Brown, J. Eugene LeClerc and Thomas A. Cebula Division of Molecular Biology, Center for Food Safety & Applied Nutrition, Food and Drug Administration, Laurel, MD 20708, USA
In a recent Trends in Microbiology article, ‘The devil is in the detail’, Bentley et al. [1] comment on recent reports regarding the whole-genome sequencing of Salmonella enterica serovar Typhi (S. Typhi) and several other enteric Corresponding author: Thomas A. Cebula ([email protected]). http://www.trends.com
and soil-dwelling microbial species. Having enjoyed this well-written synopsis, we nevertheless felt compelled to discuss some of their S. Typhi conclusions further. The authors highlight a recent manuscript by Deng and colleagues [2] that compares the complete sequence of S. Typhi strain Ty2 with the previously