Levels of Glycine Betaine in Growing Cells and Spores of Bacillus Species and Lack of Effect of Glycine Betaine on Dormant Spore Resistance
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《细菌学杂志》
Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3305
ABSTRACT
Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and 20-fold lower in B. cereus and B. megaterium spores. Athough GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.
TEXT
Cells of free-living bacteria can face drastic changes in the osmotic strength of their environment. This is particularly true for members of Bacillus species, some of which can grow in media whose salt concentration can be close to zero or as high as 1 to 2 M (4). Bacillus species respond to elevated-ionic-strength media by synthesizing or accumulating any of a variety of osmolytes, including amino acids such as proline and glutamic acid, various ectoines, and glycine betaine (GB) (2, 4, 13). While cells of almost all Bacillus species can synthesize glutamic acid and proline, synthesis of other osmolytes, in particular GB, is often minimal. This is especially the case for the well-studied soil bacterium Bacillus subtilis (4). This organism can synthesize GB from exogenous choline but makes little GB upon growth in minimal media (3, 4). However, B. subtilis has a number of systems for GB uptake, and it is likely that GB and other osmolytes derived from root or animal exudates and dying cells of both eukaryotes and prokaryotes are present in soils and other environmental niches (4, 12, 26). GB appears to be the most effective osmolyte in growing B. subtilis cells, and it can accumulate to 500 mM (12, 27). In addition to providing osmoprotection to growing B. subtilis cells, GB can also provide significant thermal protection (12) and has been reported to provide desiccation resistance to bacteria of a number of genera (3, 5).
Bacillus species are capable of undergoing sporulation, producing a dormant endospore. These spores are resistant to environmental stresses, including heat, desiccation, toxic chemicals, and extremes of ionic strength (22). Given the effects of GB on resistance properties of growing cells, it is possible that GB might play a similar role in spore resistance. However, GB levels in spores have never been quantitated, although GB has been detected (25). In this communication, we report the levels of GB in growing cells and spores of B. subtilis and several other Bacillus species prepared in rich and minimal media and with and without high salt and GB. The heat and desiccation resistance of B. subtilis spores with very different GB levels and with or without other molecules involved in protecting spores from heat and desiccation are also reported.
Strains used and preparation of growing cells and spores. The strains used in this work were B. cereus T (originally obtained from H. O. Halvorson); B. megaterium QMB1551 (ATCC 12872; originally obtained from H. S. Levinson); B. subtilis PS533 (wild type) (19), a prototrophic derivative of strain 168 carrying plasmid pUB110 conferring resistance to kanamycin (10 μg/ml); B. subtilis PS482 (–––) (11), isogenic with PS533 but lacking plasmid pUB110 and the sspA and sspB genes that encode the majority of the DNA-protective, /-type small, acid-soluble spore proteins (SASP) and with the sspE gene encoding the single -type SASP replaced with a chloramphenicol resistance (3 μg/ml) marker; and B. subtilis strain FB122 (16), also isogenic with PS533 but lacking pUB110 as well as having the spoVF operon that encodes the subunits of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) synthetase replaced with a tetracycline resistance (10 μg/ml) marker and the sleB gene encoding a spore cortex lytic enzyme replaced with a spectinomycin resistance (100 μg/ml) marker. Strain FB122 makes stable spores that lack the large depot of DPA that makes up 20% of the spore core's dry weight (9, 16). Cells were grown in SNB medium (10), 2x SG medium (15), LB medium (17), or Spizizen's medium without Casamino Acids (24) and with additions as noted for individual experiments but without antibiotics. Growing cells (10 to 30 ml) were harvested at an optical density at 600 nm (OD600) of 0.3 to 0.5 (Spizizen's medium) or 1 to 2 (all other media) and washed twice by centrifugation with 2 to 5 ml of 25 mM KPO4 buffer (pH 7.4) plus NaCl to give the ionic strength of the growth medium used. Final cell pellets were frozen and lyophilized prior to GB analyses.
Spores of various strains were prepared at 37°C on 2x SG or Spizizen's medium agar plates (B. subtilis) or at 30°C on SNB medium plates (B. cereus and B. megaterium) (15, 17). Spores were purified by repeated washing with water over a period of 1 to 2 weeks until the spores were free (>98%) of growing or sporulating cells and cell debris as observed by phase-contrast microscopy and were stored in water at 4°C protected from light (15).
Measurement of GB levels. Dried growing cells or spores (3 to 14 mg [dry weight]) were boiled in water (1 ml) for 30 min, a procedure that has been used previously for GB extraction from growing cells and to extract all small molecules from dormant spores (12, 23, 26). The extracts were cooled on ice and centrifuged, and the pellet was washed twice with 1 ml of water. The pooled supernatant fluids were passed through a 1-ml Chelex column in water at 23°C to remove divalent cations, the column was rinsed with 2 ml water, and the runthrough and column washes were pooled and lyophilized. The dry residue was dissolved in 700 μl of D2O (99.96%; Cambridge Isotope Laboratories, Andover, MA), and t-butanol (anhydrous; Aldrich, Milwaukee, WI) was added to 1 mM to serve as an internal standard. The solution was then subjected to nuclear magnetic resonance (NMR) spectroscopy with either 500 MHz or 600 MHz Varian Inova NMR spectrometers. VNMR software supported data acquisition, processing, and display. The pulse program used was the water sequence from a Biopack (Varian), with weak presaturation (0.03-kHz field strength). All proton NMR spectra were processed and analyzed using Felix 95 software from Biosym/MSI, San Diego, CA. GB levels were calculated by comparison of the peak height of the signal from GB's nine methyl protons relative to the peak height of the signal from t-butanol's nine methyl protons. The peak height of GB's methyl protons was linear over the concentration range measured in this work. All GB levels presented are averages of determinations from at least two growing cell or spore preparations and are expressed relative to the dry weight of the spores or growing cells.
Growing B. subtilis cells have 4 g of cytoplasmic water/g (dry weight) (12, 26). However, the amount of water in the spore core, the equivalent of a growing cell protoplast, is much less (9). Indeed, when B. subtilis spores are made at 37°C, only 35% of the core's wet weight is water (16). Using this value and the fact that the core contributes 50% of a spore's dry weight gives 0.3 g of core water/g dry spores. Consequently, when GB levels are expressed on the basis of dry weight, a value for spores that is 10-fold lower than that in growing cells translates into almost identical GB concentrations in these two cell types, since GB is undoubtedly present only in the spore core, where other spore small molecules have been found (9, 18, 21).
The nine methyl protons of GB and the position of their signal made it relatively easy to observe the GB signal among those from other small molecules in extracts from spores (Fig. 1A) or growing cells (data not shown), as noted previously (14, 26). GB extracts were routinely passed through a Chelex column to remove divalent cations prior to NMR analysis. Levels of divalent cations are extremely high in spores due to their chelation by the spore core's large DPA depot (9). These divalent cations include much Mn2+, and we feared that high levels of Mn2+ in spore extracts might decrease the resolution of GB peaks in NMR spectra because of peak broadening. Indeed, peaks in the NMR spectrum of a spore extract not treated with Chelex were much broader than those in a Chelex-treated extract, and the methyl proton peak from GB was not well resolved (Fig. 1B). Using the procedure for preparation and Chelex treatment of extracts described above, >90% of low levels (180 nmol) of GB added before boiling to samples of spores or growing cells of B. subtilis strain PS533 prepared in 2x SG medium were recovered in the final solutions analyzed by NMR (data not shown). Thus, the method for quantitation of GB in growing cells and spores was both sensitive and accurate.
Levels of GB in growing cells and spores of Bacillus species prepared in rich media. Growing cells and spores of B. subtilis prepared in a rich medium had significant levels of GB, although levels in spores were almost 10-fold lower (Table 1). Note, however, that as discussed above these low levels in spores translate into GB concentrations that are similar to those in comparable growing cells. GB levels were 8- to 16-fold higher in growing cells and spores of B. subtilis prepared in rich medium containing high salt. Levels of GB in B. subtilis spores prepared in rich medium without salt were not increased by addition of GB. However, GB levels in spores were increased 2-fold by GB addition if the rich medium contained high salt. Spores of B. subtilis strain FB122 lack the large depot of DPA that provides resistance to wet heat, as well as a variety of other stresses (1, 17). These DPA-less spores had a GB level much lower than that in wild-type B. subtilis spores (Table 1; also see below).
Levels of GB in cells of B. cereus and B. megaterium grown in rich medium with or without high salt were comparable to those in B. subtilis cells grown similarly (Table 1). However, levels of GB in spores of B. cereus and B. megaterium were low, even if the spores were prepared with high salt.
Levels of GB in growing cells and spores of B. subtilis prepared in minimal medium. Although GB was present in growing cells and spores of various Bacillus species prepared in rich media, GB levels were much lower than values in the literature, as GB concentrations in growing cells are reported to be 100 to 175 mM in low salt and 500 to 700 mM in high salt (12, 26). However, the latter values have generally been determined by use of minimal media to which GB was added. Consequently, we also measured GB levels in growing cells and spores of B. subtilis prepared in Spizizen's minimal medium without Casamino Acids and with or without high salt and GB (Table 2). As expected given the poor synthesis of GB by B. subtilis unless it is supplied with choline, growing cells had very little GB, although the GB level was increased 3-fold by high salt. Addition of GB increased GB levels in growing cells even more, and the concentration of GB in cells grown in high salt plus GB is calculated to be 200 mM. While this value is lower than values in the literature, it is still a very high internal GB concentration.
GB was present at only low levels in wild-type spores prepared in minimal medium with or without salt or GB alone (Table 2). However, addition of GB and high salt to minimal medium resulted in higher levels of GB in spores, although the levels were lower than those in spores prepared in rich medium (Tables 1 and 2). GB levels in spores of strain PS482 prepared in minimal medium with or without high salt and GB were similar to those in wild-type spores, while GB levels in spores of strain FB122 were much lower, as they were in spores prepared in rich medium (Tables 1 and 2). Thus, the absence of neither SASP nor DPA resulted in an increased accumulation of GB.
Effect of GB levels on cell growth and spore resistance. Cells of B. subtilis strain PS533 growing in Spizizen's medium at 37°C had a doubling time of 64 ± 3 min, and this value was essentially identical (62 ± 3 min) with 1 mM GB. The presence of 0.4 M NaCl increased the population doubling time to 135 ± 7 min. However, the efficacy of GB as an osmolyte in B. subtilis was shown by the reduction in the latter doubling time to 76 ± 4 min in the minimal medium containing 0.4 M NaCl plus 1 mM GB.
GB is one of the most common and effective osmolytes found in bacteria and has strong stabilizing effects on macromolecules (4, 5, 8). Indeed, elevated GB levels increase the tolerance of growing B. subtilis cells to elevated temperatures (12). Thus, it was of obvious interest to examine the heat resistance of spores with very different GB levels. Spore resistance to wet heat was determined essentially as described previously (7, 17). Spores at an OD600 of 1 in water were incubated at elevated temperatures; at various times aliquots were diluted 1/100 in water at 23°C, diluted further in water, and spotted on LB medium plates (17) containing the appropriate antibiotic. Then, the plates were incubated for 24 h at 37°C and colonies were counted. Longer incubation gave no increase in colony numbers. Wild-type spores (PS533) prepared in minimal medium that had GB levels that varied by more than 15-fold exhibited identical wet heat resistance (Fig. 2A; Table 2). Similar results were obtained with wild-type spores prepared in rich medium with or without high salt plus GB (data not shown). We also examined the wet heat resistance of spores of strain PS482 that lack all major SASP, including the DNA-protective /-type SASP (6, 11). Spores that lack /-type SASP, including PS482 spores, are significantly less heat resistant than wild-type spores, due to DNA damage generated by wet heat in the absence of /-type SASP (7) (Fig. 2A and B). However, PS482 spores with very different GB levels exhibited essentially identical wet heat resistance (Fig. 2B).
Spores of strain FB122 lack DPA and are significantly less heat resistant than are wild-type spores, due to the lower core water content in wild-type spores than in DPA-less FB122 spores (9, 16, 17). DPA-less FB122 spores also germinate extremely poorly in response to nutrients, although they germinate well with high concentrations of a 1:1 complex of Ca2+ and DPA (Ca-DPA) (16). Consequently, survival of FB122 spores upon wet heat treatment was assessed by incubating these spores at an OD600 of 1 in water, and at various times aliquots were diluted 1/100 in 60 mM Ca-DPA at pH 7.5 (16). After incubation of the latter dilutions for 60 min at 23°C, aliquots of serial dilutions were spotted on LB medium plates and the colonies formed were determined as described above. Again, DPA-less FB122 spores prepared with and without salt and GB had identical resistance to wet heat (Fig. 2C), although none of these FB122 spores had very high levels of GB (Table 2).
Wild-type B. subtilis spores are also resistant to multiple cycles of desiccation and rehydration, but spores lacking most /-type SASP are desiccation sensitive (22). GB has also been reported to provide desiccation resistance to bacteria (5), so it was of interest to examine the desiccation resistance of spores with different GB levels. One-milliliter samples of PS482 spores at an OD600 of 10 were centrifuged, freeze-dried, and rehydrated in 1 ml water multiple times, and spore viability was determined after various numbers of freeze-drying cycles by plating on LB medium plates as described above. As expected based on previous work (1, 22), PS482 spores that lack the majority of their /-type SASP were sensitive to desiccation (Fig. 2D). PS482 spores prepared with high salt were slightly more resistant to desiccation than were spores prepared without high salt. However, spore sensitivity to desiccation was unaffected by variations in GB levels (Fig. 2D; Table 2).
In addition to the tests of the resistance properties noted above, we found that the viability of dormant spores with various levels of GB was not affected by incubation for 90 min at 23°C in either very high salt (1.5 M NaCl) or distilled water (data not shown). This was not surprising, since (i) the spore's peptidoglycan cortex restricts swelling of the spore core, (ii) the spore core already has high concentrations of osmolytes (see below), and (iii) the spore's inner membrane restricts passage of small charged compounds (9, 22).
Conclusions. The work reported in this communication leads to a number of conclusions. First, GB levels can vary over 10-fold in growing cells, with GB and high salt in growth media leading to increases in GB levels. The latter results confirm what has been found previously (2, 4, 12, 27), although levels of GB found in growing B. subtilis cells in the current work were lower than levels reported previously. We have no explanation for this discrepancy, although there are certainly differences in the genetic backgrounds of the B. subtilis strains used in the current and previous works.
Further conclusions are new and concern levels of GB in spores. First, GB can indeed be present in spores, as was reported previously (25). Spore GB levels also responded in the same way to high salt and GB in sporulation media as did GB levels in growing cells. As a consequence, GB levels in spores can vary 15- to 30-fold depending on the additions to sporulation media. Second, GB levels in spores are much lower than levels in growing cells prepared in the same medium, from 25- to 90-fold lower for spores prepared in minimal medium (Table 2). Some of this difference may be due to the lower amount of core water in spores relative to dry weight, compared to that in growing cells. However, this is at most a 10-fold effect, as noted above. In addition, during sporulation, the developing spore initially has much more core water, and the low core water content of the dormant spore is attained only very late in sporulation. Another factor that may explain the extremely low GB levels in dormant spores is that spores have a number of additional osmolytes that may replace GB. One obvious additional osmolyte is DPA, a molecule that comprises 20% of the core dry weight. However, the solubility of a 1:1 chelate of Ca2+ and DPA, the likely form of most DPA in spores (9), is 70 mM at 23°C, and DPA-less spores have less GB, not more, than do wild-type spores. The spore also has high levels of two other osmolytes, glutamate and 3-phosphoglyceric acid (3PGA), which are present in the core at 50 (3PGA) to 100 (glutamate) mM (21), and glutamate is an important osmoprotectant in growing cells of a number of Bacillus species (2, 4). However, this does not explain the decreased GB levels in DPA-less spores, and we have no good explanation for the latter observation. However, it should be possible to test whether levels of glutamate or 3PGA or both compounds increase in DPA-less spores and thus substitute for GB and perhaps also for DPA. We also note that the pH in the core of DPA-less spores is 7.7, while this value is only 6.5 in wild-type spores (18, 20). Perhaps core pH also plays a role in determining spore GB levels.
The final conclusion from this work comes from analyses of the wet heat and desiccation resistance of spores with very different GB levels. Wild-type B. subtilis spores prepared in rich medium and with and without high salt and GB had identical wet heat resistance and were also insensitive to extremes of ionic strength. This was also the case for wild-type or ––– spores prepared in minimal medium. Spores of these strains prepared with high salt and GB had 20- to 30-fold- higher GB levels than spores prepared without salt or GB. PS482 spores with very different GB levels also had identical resistance to desiccation. Thus, GB and other osmolytes appear to play no role in spore resistance to wet heat and desiccation, and GB cannot substitute for /-type SASP in conferring spore resistance to the latter treatments.
ACKNOWLEDGMENTS
We are grateful for the assistance of Swaroopa Atluri in some aspects of this work.
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ABSTRACT
Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and 20-fold lower in B. cereus and B. megaterium spores. Athough GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.
TEXT
Cells of free-living bacteria can face drastic changes in the osmotic strength of their environment. This is particularly true for members of Bacillus species, some of which can grow in media whose salt concentration can be close to zero or as high as 1 to 2 M (4). Bacillus species respond to elevated-ionic-strength media by synthesizing or accumulating any of a variety of osmolytes, including amino acids such as proline and glutamic acid, various ectoines, and glycine betaine (GB) (2, 4, 13). While cells of almost all Bacillus species can synthesize glutamic acid and proline, synthesis of other osmolytes, in particular GB, is often minimal. This is especially the case for the well-studied soil bacterium Bacillus subtilis (4). This organism can synthesize GB from exogenous choline but makes little GB upon growth in minimal media (3, 4). However, B. subtilis has a number of systems for GB uptake, and it is likely that GB and other osmolytes derived from root or animal exudates and dying cells of both eukaryotes and prokaryotes are present in soils and other environmental niches (4, 12, 26). GB appears to be the most effective osmolyte in growing B. subtilis cells, and it can accumulate to 500 mM (12, 27). In addition to providing osmoprotection to growing B. subtilis cells, GB can also provide significant thermal protection (12) and has been reported to provide desiccation resistance to bacteria of a number of genera (3, 5).
Bacillus species are capable of undergoing sporulation, producing a dormant endospore. These spores are resistant to environmental stresses, including heat, desiccation, toxic chemicals, and extremes of ionic strength (22). Given the effects of GB on resistance properties of growing cells, it is possible that GB might play a similar role in spore resistance. However, GB levels in spores have never been quantitated, although GB has been detected (25). In this communication, we report the levels of GB in growing cells and spores of B. subtilis and several other Bacillus species prepared in rich and minimal media and with and without high salt and GB. The heat and desiccation resistance of B. subtilis spores with very different GB levels and with or without other molecules involved in protecting spores from heat and desiccation are also reported.
Strains used and preparation of growing cells and spores. The strains used in this work were B. cereus T (originally obtained from H. O. Halvorson); B. megaterium QMB1551 (ATCC 12872; originally obtained from H. S. Levinson); B. subtilis PS533 (wild type) (19), a prototrophic derivative of strain 168 carrying plasmid pUB110 conferring resistance to kanamycin (10 μg/ml); B. subtilis PS482 (–––) (11), isogenic with PS533 but lacking plasmid pUB110 and the sspA and sspB genes that encode the majority of the DNA-protective, /-type small, acid-soluble spore proteins (SASP) and with the sspE gene encoding the single -type SASP replaced with a chloramphenicol resistance (3 μg/ml) marker; and B. subtilis strain FB122 (16), also isogenic with PS533 but lacking pUB110 as well as having the spoVF operon that encodes the subunits of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) synthetase replaced with a tetracycline resistance (10 μg/ml) marker and the sleB gene encoding a spore cortex lytic enzyme replaced with a spectinomycin resistance (100 μg/ml) marker. Strain FB122 makes stable spores that lack the large depot of DPA that makes up 20% of the spore core's dry weight (9, 16). Cells were grown in SNB medium (10), 2x SG medium (15), LB medium (17), or Spizizen's medium without Casamino Acids (24) and with additions as noted for individual experiments but without antibiotics. Growing cells (10 to 30 ml) were harvested at an optical density at 600 nm (OD600) of 0.3 to 0.5 (Spizizen's medium) or 1 to 2 (all other media) and washed twice by centrifugation with 2 to 5 ml of 25 mM KPO4 buffer (pH 7.4) plus NaCl to give the ionic strength of the growth medium used. Final cell pellets were frozen and lyophilized prior to GB analyses.
Spores of various strains were prepared at 37°C on 2x SG or Spizizen's medium agar plates (B. subtilis) or at 30°C on SNB medium plates (B. cereus and B. megaterium) (15, 17). Spores were purified by repeated washing with water over a period of 1 to 2 weeks until the spores were free (>98%) of growing or sporulating cells and cell debris as observed by phase-contrast microscopy and were stored in water at 4°C protected from light (15).
Measurement of GB levels. Dried growing cells or spores (3 to 14 mg [dry weight]) were boiled in water (1 ml) for 30 min, a procedure that has been used previously for GB extraction from growing cells and to extract all small molecules from dormant spores (12, 23, 26). The extracts were cooled on ice and centrifuged, and the pellet was washed twice with 1 ml of water. The pooled supernatant fluids were passed through a 1-ml Chelex column in water at 23°C to remove divalent cations, the column was rinsed with 2 ml water, and the runthrough and column washes were pooled and lyophilized. The dry residue was dissolved in 700 μl of D2O (99.96%; Cambridge Isotope Laboratories, Andover, MA), and t-butanol (anhydrous; Aldrich, Milwaukee, WI) was added to 1 mM to serve as an internal standard. The solution was then subjected to nuclear magnetic resonance (NMR) spectroscopy with either 500 MHz or 600 MHz Varian Inova NMR spectrometers. VNMR software supported data acquisition, processing, and display. The pulse program used was the water sequence from a Biopack (Varian), with weak presaturation (0.03-kHz field strength). All proton NMR spectra were processed and analyzed using Felix 95 software from Biosym/MSI, San Diego, CA. GB levels were calculated by comparison of the peak height of the signal from GB's nine methyl protons relative to the peak height of the signal from t-butanol's nine methyl protons. The peak height of GB's methyl protons was linear over the concentration range measured in this work. All GB levels presented are averages of determinations from at least two growing cell or spore preparations and are expressed relative to the dry weight of the spores or growing cells.
Growing B. subtilis cells have 4 g of cytoplasmic water/g (dry weight) (12, 26). However, the amount of water in the spore core, the equivalent of a growing cell protoplast, is much less (9). Indeed, when B. subtilis spores are made at 37°C, only 35% of the core's wet weight is water (16). Using this value and the fact that the core contributes 50% of a spore's dry weight gives 0.3 g of core water/g dry spores. Consequently, when GB levels are expressed on the basis of dry weight, a value for spores that is 10-fold lower than that in growing cells translates into almost identical GB concentrations in these two cell types, since GB is undoubtedly present only in the spore core, where other spore small molecules have been found (9, 18, 21).
The nine methyl protons of GB and the position of their signal made it relatively easy to observe the GB signal among those from other small molecules in extracts from spores (Fig. 1A) or growing cells (data not shown), as noted previously (14, 26). GB extracts were routinely passed through a Chelex column to remove divalent cations prior to NMR analysis. Levels of divalent cations are extremely high in spores due to their chelation by the spore core's large DPA depot (9). These divalent cations include much Mn2+, and we feared that high levels of Mn2+ in spore extracts might decrease the resolution of GB peaks in NMR spectra because of peak broadening. Indeed, peaks in the NMR spectrum of a spore extract not treated with Chelex were much broader than those in a Chelex-treated extract, and the methyl proton peak from GB was not well resolved (Fig. 1B). Using the procedure for preparation and Chelex treatment of extracts described above, >90% of low levels (180 nmol) of GB added before boiling to samples of spores or growing cells of B. subtilis strain PS533 prepared in 2x SG medium were recovered in the final solutions analyzed by NMR (data not shown). Thus, the method for quantitation of GB in growing cells and spores was both sensitive and accurate.
Levels of GB in growing cells and spores of Bacillus species prepared in rich media. Growing cells and spores of B. subtilis prepared in a rich medium had significant levels of GB, although levels in spores were almost 10-fold lower (Table 1). Note, however, that as discussed above these low levels in spores translate into GB concentrations that are similar to those in comparable growing cells. GB levels were 8- to 16-fold higher in growing cells and spores of B. subtilis prepared in rich medium containing high salt. Levels of GB in B. subtilis spores prepared in rich medium without salt were not increased by addition of GB. However, GB levels in spores were increased 2-fold by GB addition if the rich medium contained high salt. Spores of B. subtilis strain FB122 lack the large depot of DPA that provides resistance to wet heat, as well as a variety of other stresses (1, 17). These DPA-less spores had a GB level much lower than that in wild-type B. subtilis spores (Table 1; also see below).
Levels of GB in cells of B. cereus and B. megaterium grown in rich medium with or without high salt were comparable to those in B. subtilis cells grown similarly (Table 1). However, levels of GB in spores of B. cereus and B. megaterium were low, even if the spores were prepared with high salt.
Levels of GB in growing cells and spores of B. subtilis prepared in minimal medium. Although GB was present in growing cells and spores of various Bacillus species prepared in rich media, GB levels were much lower than values in the literature, as GB concentrations in growing cells are reported to be 100 to 175 mM in low salt and 500 to 700 mM in high salt (12, 26). However, the latter values have generally been determined by use of minimal media to which GB was added. Consequently, we also measured GB levels in growing cells and spores of B. subtilis prepared in Spizizen's minimal medium without Casamino Acids and with or without high salt and GB (Table 2). As expected given the poor synthesis of GB by B. subtilis unless it is supplied with choline, growing cells had very little GB, although the GB level was increased 3-fold by high salt. Addition of GB increased GB levels in growing cells even more, and the concentration of GB in cells grown in high salt plus GB is calculated to be 200 mM. While this value is lower than values in the literature, it is still a very high internal GB concentration.
GB was present at only low levels in wild-type spores prepared in minimal medium with or without salt or GB alone (Table 2). However, addition of GB and high salt to minimal medium resulted in higher levels of GB in spores, although the levels were lower than those in spores prepared in rich medium (Tables 1 and 2). GB levels in spores of strain PS482 prepared in minimal medium with or without high salt and GB were similar to those in wild-type spores, while GB levels in spores of strain FB122 were much lower, as they were in spores prepared in rich medium (Tables 1 and 2). Thus, the absence of neither SASP nor DPA resulted in an increased accumulation of GB.
Effect of GB levels on cell growth and spore resistance. Cells of B. subtilis strain PS533 growing in Spizizen's medium at 37°C had a doubling time of 64 ± 3 min, and this value was essentially identical (62 ± 3 min) with 1 mM GB. The presence of 0.4 M NaCl increased the population doubling time to 135 ± 7 min. However, the efficacy of GB as an osmolyte in B. subtilis was shown by the reduction in the latter doubling time to 76 ± 4 min in the minimal medium containing 0.4 M NaCl plus 1 mM GB.
GB is one of the most common and effective osmolytes found in bacteria and has strong stabilizing effects on macromolecules (4, 5, 8). Indeed, elevated GB levels increase the tolerance of growing B. subtilis cells to elevated temperatures (12). Thus, it was of obvious interest to examine the heat resistance of spores with very different GB levels. Spore resistance to wet heat was determined essentially as described previously (7, 17). Spores at an OD600 of 1 in water were incubated at elevated temperatures; at various times aliquots were diluted 1/100 in water at 23°C, diluted further in water, and spotted on LB medium plates (17) containing the appropriate antibiotic. Then, the plates were incubated for 24 h at 37°C and colonies were counted. Longer incubation gave no increase in colony numbers. Wild-type spores (PS533) prepared in minimal medium that had GB levels that varied by more than 15-fold exhibited identical wet heat resistance (Fig. 2A; Table 2). Similar results were obtained with wild-type spores prepared in rich medium with or without high salt plus GB (data not shown). We also examined the wet heat resistance of spores of strain PS482 that lack all major SASP, including the DNA-protective /-type SASP (6, 11). Spores that lack /-type SASP, including PS482 spores, are significantly less heat resistant than wild-type spores, due to DNA damage generated by wet heat in the absence of /-type SASP (7) (Fig. 2A and B). However, PS482 spores with very different GB levels exhibited essentially identical wet heat resistance (Fig. 2B).
Spores of strain FB122 lack DPA and are significantly less heat resistant than are wild-type spores, due to the lower core water content in wild-type spores than in DPA-less FB122 spores (9, 16, 17). DPA-less FB122 spores also germinate extremely poorly in response to nutrients, although they germinate well with high concentrations of a 1:1 complex of Ca2+ and DPA (Ca-DPA) (16). Consequently, survival of FB122 spores upon wet heat treatment was assessed by incubating these spores at an OD600 of 1 in water, and at various times aliquots were diluted 1/100 in 60 mM Ca-DPA at pH 7.5 (16). After incubation of the latter dilutions for 60 min at 23°C, aliquots of serial dilutions were spotted on LB medium plates and the colonies formed were determined as described above. Again, DPA-less FB122 spores prepared with and without salt and GB had identical resistance to wet heat (Fig. 2C), although none of these FB122 spores had very high levels of GB (Table 2).
Wild-type B. subtilis spores are also resistant to multiple cycles of desiccation and rehydration, but spores lacking most /-type SASP are desiccation sensitive (22). GB has also been reported to provide desiccation resistance to bacteria (5), so it was of interest to examine the desiccation resistance of spores with different GB levels. One-milliliter samples of PS482 spores at an OD600 of 10 were centrifuged, freeze-dried, and rehydrated in 1 ml water multiple times, and spore viability was determined after various numbers of freeze-drying cycles by plating on LB medium plates as described above. As expected based on previous work (1, 22), PS482 spores that lack the majority of their /-type SASP were sensitive to desiccation (Fig. 2D). PS482 spores prepared with high salt were slightly more resistant to desiccation than were spores prepared without high salt. However, spore sensitivity to desiccation was unaffected by variations in GB levels (Fig. 2D; Table 2).
In addition to the tests of the resistance properties noted above, we found that the viability of dormant spores with various levels of GB was not affected by incubation for 90 min at 23°C in either very high salt (1.5 M NaCl) or distilled water (data not shown). This was not surprising, since (i) the spore's peptidoglycan cortex restricts swelling of the spore core, (ii) the spore core already has high concentrations of osmolytes (see below), and (iii) the spore's inner membrane restricts passage of small charged compounds (9, 22).
Conclusions. The work reported in this communication leads to a number of conclusions. First, GB levels can vary over 10-fold in growing cells, with GB and high salt in growth media leading to increases in GB levels. The latter results confirm what has been found previously (2, 4, 12, 27), although levels of GB found in growing B. subtilis cells in the current work were lower than levels reported previously. We have no explanation for this discrepancy, although there are certainly differences in the genetic backgrounds of the B. subtilis strains used in the current and previous works.
Further conclusions are new and concern levels of GB in spores. First, GB can indeed be present in spores, as was reported previously (25). Spore GB levels also responded in the same way to high salt and GB in sporulation media as did GB levels in growing cells. As a consequence, GB levels in spores can vary 15- to 30-fold depending on the additions to sporulation media. Second, GB levels in spores are much lower than levels in growing cells prepared in the same medium, from 25- to 90-fold lower for spores prepared in minimal medium (Table 2). Some of this difference may be due to the lower amount of core water in spores relative to dry weight, compared to that in growing cells. However, this is at most a 10-fold effect, as noted above. In addition, during sporulation, the developing spore initially has much more core water, and the low core water content of the dormant spore is attained only very late in sporulation. Another factor that may explain the extremely low GB levels in dormant spores is that spores have a number of additional osmolytes that may replace GB. One obvious additional osmolyte is DPA, a molecule that comprises 20% of the core dry weight. However, the solubility of a 1:1 chelate of Ca2+ and DPA, the likely form of most DPA in spores (9), is 70 mM at 23°C, and DPA-less spores have less GB, not more, than do wild-type spores. The spore also has high levels of two other osmolytes, glutamate and 3-phosphoglyceric acid (3PGA), which are present in the core at 50 (3PGA) to 100 (glutamate) mM (21), and glutamate is an important osmoprotectant in growing cells of a number of Bacillus species (2, 4). However, this does not explain the decreased GB levels in DPA-less spores, and we have no good explanation for the latter observation. However, it should be possible to test whether levels of glutamate or 3PGA or both compounds increase in DPA-less spores and thus substitute for GB and perhaps also for DPA. We also note that the pH in the core of DPA-less spores is 7.7, while this value is only 6.5 in wild-type spores (18, 20). Perhaps core pH also plays a role in determining spore GB levels.
The final conclusion from this work comes from analyses of the wet heat and desiccation resistance of spores with very different GB levels. Wild-type B. subtilis spores prepared in rich medium and with and without high salt and GB had identical wet heat resistance and were also insensitive to extremes of ionic strength. This was also the case for wild-type or ––– spores prepared in minimal medium. Spores of these strains prepared with high salt and GB had 20- to 30-fold- higher GB levels than spores prepared without salt or GB. PS482 spores with very different GB levels also had identical resistance to desiccation. Thus, GB and other osmolytes appear to play no role in spore resistance to wet heat and desiccation, and GB cannot substitute for /-type SASP in conferring spore resistance to the latter treatments.
ACKNOWLEDGMENTS
We are grateful for the assistance of Swaroopa Atluri in some aspects of this work.
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