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Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination

2009; Oxford University Press; Volume: 49; Issue: 5 Linguagem: Inglês

10.1111/j.1472-765x.2009.02721.x

1472-765X

Jie Wei, Peter Setlow, Dallas G. Hoover,

Seed Germination and Physiology

Letters in Applied MicrobiologyVolume 49, Issue 5 p. 646-651 Free Access Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination J. Wei, J. Wei Department of Animal & Food Sciences, University of Delaware, Newark, DE, USASearch for more papers by this authorP. Setlow, P. Setlow Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorD.G. Hoover, D.G. Hoover Department of Animal & Food Sciences, University of Delaware, Newark, DE, USASearch for more papers by this author J. Wei, J. Wei Department of Animal & Food Sciences, University of Delaware, Newark, DE, USASearch for more papers by this authorP. Setlow, P. Setlow Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorD.G. Hoover, D.G. Hoover Department of Animal & Food Sciences, University of Delaware, Newark, DE, USASearch for more papers by this author First published: 12 October 2009 https://doi.org/10.1111/j.1472-765X.2009.02721.xCitations: 11 Dallas G. Hoover, Department of Animal & Food Sciences, University of Delaware, Newark, DE 19716-2150, USA. E-mail: dgh@udel.edu AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat. Methods: The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined. Results: Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50°C. Conclusions: Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed. Significance and Impact of the Study: The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus. Introduction Germination of spores of Bacillus species can be initiated by a variety of agents including specific nutrients, enzymes such as lysozyme, a 1 : 1 chelate of Ca2+ and pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA)] and cationic detergents such as dodecylamine (Setlow 2003). The molecular genetics of spore germination has been most studied in Bacillus subtilis 168 whose spores have three major nutrient germinant receptors of the GerA family encoded by the homologous tricistronic gerA, gerB and gerK operons (Setlow 2003). Stimulation of the germinant receptors triggers the release of the spore's large depot of Ca2+-DPA, and this release activates enzymes that initiate hydrolysis of the spore's peptidoglycan cortex, allowing completion of spore germination (Setlow 2003). Spores of a number of Bacillus species are germinated by pressures of 100–1000 MPa (Gould and Sale 1970; Heinz and Knorr 2001). At moderately high pressures (MHP; 50–300 MPa), germination of B. subtilis spores is triggered through activation of the germinant receptors (Wuytack et al. 2000; Black et al. 2005). Individual B. subtilis germinant receptors appear to exhibit different effectiveness in responding to MHP, the GerA receptor being more effective than the GerB receptor and even more effective than the GerK receptor (Black et al. 2005); however, it is not clear whether the responsiveness of different germinant receptors to MHP is because of intrinsic differences between individual receptors or to differences in their levels with receptors present at highest levels being most effective. Indeed, an increase in levels of individual germinant receptors results in more rapid spore germination with MHP (Black et al. 2005). Very high pressures (VHP; 400–1000 MPa) also germinate spores of various Bacillus species, but induction of B. subtilis spore germination by VHP does not require the germinant receptors (Black et al. 2007). Rather VHP may trigger spore germination by causing changes in the spore's inner membrane that trigger DPA release. Bacillus cereus spores are found at significant levels in foods and can be an important cause of spoilage and food-borne diseases (Setlow and Johnson 2007). Bacillus cereus spores germinate best with inosine or l-alanine, with a combination of these germinants eliciting the most rapid response, although other l-amino acids, can also trigger germination but less efficiently (Hornstra et al. 2006a). Seven operons encoding germinant receptors have been identified in B. cereus, all members of the gerA family identified in B. subtilis (Clements and Moir 1998; Barlass et al. 2002; Hornstra et al. 2005, 2006a). In B. cereus ATCC 14579, the GerR receptor is crucial for germination with both l-alanine and inosine but not with l-glutamine, the GerG receptor for germination with l-glutamine and the GerI and Q receptors for germination with inosine (Hornstra et al. 2006a). The situation in B. cereus 569 is similar, as GerI is the major germinant receptor involved in inosine germination with GerQ playing a minor role, while the GerL receptor is the major one involved in l-alanine germination (Clements and Moir 1998; Barlass et al. 2002); however, with the exception of loss of the GerR receptor in strain ATCC 14579 (Hornstra et al. 2005, 2006a), loss of an individual germinant receptor essential for normal germination with inosine does not have a major effect on germination with l-alanine and vice versa. In addition to germinant receptors, another group of proteins important in B. cereus spore germination is Na+/H+-K+ antiporters with two such proteins, GerN and GerT, in B. cereus 569 (Southworth et al. 2001; Thackray et al. 2001; Senior and Moir 2008). Loss of GerN greatly decreases spore germination with inosine, and loss of GerT in a gerN mutant background eliminates the residual inosine germination of gerN mutant spores, in particular at high inosine concentrations (Senior and Moir 2008). GerT also plays a significant role in spore outgrowth under some conditions; however, to date, no such antiporters have been identified as involved in spore germination with l-alanine. In this study, we have examined spores of B. cereus wild type and gerI, gerL, gerN, gerQ, gerR, gerT and gerN gerT mutant strains for their germination and inactivation by MHP at various temperatures in buffer. Materials and methods Bacillus cereus strains and spore preparation Bacillus cereus 569 and its isogenic gerI (AM1314), gerL (AM1316), gerQ (AM1311), gerN (AM1419), gerT (AM1631) and gerN gerT (AM1632) mutant derivatives (Clements and Moir 1998; Thackray et al. 2001; Barlass et al. 2002; Senior and Moir 2008) were obtained from Anne Moir (University of Sheffield, UK). Bacillus cereus ATCC 14579 and its isogenic gerR mutant derivative (LH1219) were obtained from Roy Moezelaar (Waginengen Centre for Food Science, the Netherlands). For spore preparation, B. cereus 569 was grown in 10 ml of nutrient broth (NB; Sigma Chemical Company, St Louis, MO, USA) for 12 h at 37°C with agitation. Twenty millilitres of the NB culture was transferred into 1 l of casein containing yeast extract (CCY) broth (Stewart et al. 1981) and incubated with shaking at 37°C for 7 days until >90% free spores were present as determined by phase-contrast microscopy. Bacillus cereus ATCC 14579 and its gerR mutant derivative were sporulated at 30°C in Schaeffer's sporulation medium (Nicholson and Setlow 1990). Spores were harvested and washed eight times by repeated centrifugation at 4000–9000 g for c. 25 min with distilled water, discarding the upper layer of cell debris in the pellet in early washing steps. Spores were stored at an optical density of 600 nm (OD600 nm) of 5·0 in distilled water at −80°C. All spore preparations used in this work were free (>98%) from sporulating cells, germinated spores and cell debris as determined by phase-contrast microscopy. Pressure treatment For pressure treatment, spores were diluted with Tris–HCl buffer (50 mmol l−1, pH 7·5) (TB) to an OD600 nm of 1·0 (c. 1 × 108 spores per ml). Diluted spore suspensions (1·5 ml) were aseptically transferred to sterile pouches (VWR International, Mississauga, ON, Canada), and the pouches were heat-sealed after expelling as much air as possible. MHP treatments were carried out in a PT-1 Research System pressure unit (Avure Technologies, Kent, WA, USA), using water as the hydrostatic medium. Pressurization was conducted at 150 MPa and various temperatures by thermostatically controlling the pressure chamber of the PT-1 unit for various times. For each experiment, an untreated similarly prepared sample was used to determine the initial spore number. Control experiments showed that by itself, the heat treatments had no effect on spore viability (data not shown). The pressure and temperature data were recorded every 2 s (DASUTEC USA, Bedford, NH, USA), and the temperature of the water bath was monitored using a K-type thermocouple. The pressure come-up rate was c. 22 MPa s−1, and the pressure release time was <4 s. The reported pressurization time did not include the pressure come-up or release times. Adiabatic heating generated by compression increased the temperature of water bath to c. 4·5°C for 150 MPa, and most of the heat dissipated after c. 3·5 min after application of pressure. Measurement of spore germination and inactivation Following pressure treatment, 1 ml of sample was transferred into 9 ml of TB, diluted with TB, pour-plated with Nutrient Agar (Sigma), and plates were incubated at 37°C for 24 h to enumerate survivors. The extent of inactivation was calculated as N0/N, with N the number of survivors after pressure treatment and N0 the number of initial spores. To determine the degree of spore germination, pressure-treated and control spore suspensions were heated at 70°C for 10 min to inactivate germinated spores. Control experiments showed that dormant spores were unaffected by this heat treatment (data not shown). Pressure-induced germination was also calculated as N0/N, with N0 and N the numbers of survivors in the control (nonpressurized and unheated) sample and in the pressurized and subsequently heat-treated samples, respectively. In experiments, analysing the germination and inactivation of gerR mutant spores and spores of the latter's wild-type parent, the extent of spore germination was also assessed by phase-contrast microscopy. Reproducibility and statistical analyses All experiments were carried out at least in duplicate, and averages of all data sets were shown in figures. The standard deviations for all values reported were always ≤40%. Significant differences between treatments were determined by a single factor test with significance levels of 1%, using the anova test tool within the Microsoft Excel spreadsheet program (Microsoft Excel professional edition, 2003). Results Pressure treatment of wild-type spores in TB To study the effects of pressure and temperature on the germination and inactivation of spores of wild-type B. cereus, suspensions of strain 569 spores were exposed to an MHP of 150 MPa at various temperatures. As expected, the degree of spore inactivation and germination rose with increases in temperature and pressure-exposure time (Fig. 1a,b); however, semi-log plots of MHP inactivation and germination as a function of time were nonlinear, with the greatest effects seen in the early minutes of treatment (Fig. 1a,b). The highest levels of spore germination and inactivation were at 65°C, where there were 5–6 logs of spore germination and c. 4 logs of spore inactivation. Figure 1Open in figure viewerPowerPoint Effect of moderately high pressure (MHP) at various temperatures on inactivation (a) and germination (b) of wild-type Bacillus cereus spores. Spores of B. cereus 569 (wild type) in Tris–HCl buffer were treated with MHP at various temperatures, and the degree of spore germination and inactivation were determined. The symbols used to denote the pressure treatment temperature are (○) 20°C; (•) 30°C; (△) 40°C; () 50°C and (□) 65°C. As noted previously, temperature plays an important role in MHP-induced germination and inactivation of B. subtilis spores (Black et al. 2007), and germination and inactivation of B. cereus spores by MHP also increased with increases in treatment temperature (Fig. 1a,b). The degree of spore germination was also higher than the degree of spore inactivation for all time/temperature combinations. This observation is consistent with the premise that MHP and heat cause spore inactivation by first triggering spore germination and only then inactivating the germinated spores (Sale et al. 1970). In contrast to results with MHP, completion of B. subtilis spore germination with VHP is maximal at 60°C (Black et al. 2005, 2007), as was also the case with B. cereus spores (data not shown). Pressure treatment in TB of spores lacking nutrient germinant receptors With B. subtilis spores, germination by MHP requires the presence of at least one of the spore's germinant receptors, and either different germinant receptors exhibit different intrinsic responsiveness to MHP or receptor levels determine effectiveness of MHP (Black et al. 2005); however, B. cereus 569 spores lacking either the GerL or GerQ receptors exhibited no significant differences in their germination and inactivation by MHP at various temperatures from that of wild-type spores (Fig. 2a,b). The inactivation of gerI mutant spores by MHP was also relatively similar to that of wild-type spores (Fig. 2a), and the MHP germination of gerI mutant spores was lower, but not significantly different than that of wild-type spores, in particular at higher temperatures (Fig. 2b) (P < 0·01). MHP treatment of wild-type and gerR mutant spores of B. cereus ATCC 14579 also showed that the absence of the GerR receptor resulted in only small decreases in rates of spore inactivation and germination, and the latter decreases were not significant (Fig. 3a,b). In this latter experiment, examination of spores by phase-contrast microscopy showed that levels of phase dark, and thus fully germinated spores, after various treatment times were again consistent with spore germination being required for spore inactivation (data not shown). Figure 2Open in figure viewerPowerPoint Effect of moderately high pressure (MHP) at various temperatures on inactivation (a) and germination (b) of Bacillus cereus spores with and without different nutrient germinant receptors. Spores of B. cereus 569 (wild type) and its isogenic gerI, gerL and gerQ mutant strains were treated with MHP at various temperatures, and after 15 min, the degree of spore germination and inactivation were determined. The spores represented by the different bars are open bars, wild-type spores; stippled bars, gerL mutant spores; solid bars, gerI mutant spores; and horizontally striped bars, gerQ mutant spores. Figure 3Open in figure viewerPowerPoint Effect of moderately high pressure (MHP) on inactivation (a) and germination (b) of Bacillus cereus spores with and without GerR. Spores of B. cereus ATCC 14579 (wild type) and its isogenic gerR mutant strain were treated at 40°C with MHP, and at various times the degree of spore germination and inactivation were determined. The spores represented by the different symbols are (○) wild-type spores and (•) gerR mutant spores. Pressure treatment of spores lacking cation antiporters that affect spore germination In addition to nutrient germinant receptors, two monovalent cation antiporters, GerN and GerT, also appear to be involved in inosine germination of B. cereus 569 spores (Southworth et al. 2001; Thackray et al. 2001; Senior and Moir 2008). Loss of GerN greatly decreases inosine germination, while loss of GerT alone has very little effect; however, loss of GerT eliminates the residual inosine germination of gerN mutant spores, in particular at high inosine concentrations (Senior and Moir 2008). The gerN, gerT and gerN gerT mutant spores were treated with MHP at 20–65°C, and the inactivation and germination of these spores were compared to that of wild-type spores (Fig. 4a,b). Loss of GerT alone did not reduce either the inactivation or the germination of B. cereus spores by MHP; indeed, at 20 and 30°C gerT mutant spores had significantly higher germination than wild-type spores (P < 0·01) (Fig. 4a,b). Loss of GerN or both GerN and GerT had very little effect on spore inactivation by MHP at all temperatures tested (Fig. 4a) and little effect on spore germination at 20, 30 and 65°C (Fig. 4b); however, loss of GerN and especially both GerN and GerT significantly decreased MHP germination of spores at 40 and 50°C (Fig. 4b) (P < 0·01). Figure 4Open in figure viewerPowerPoint Effect of moderately high pressure (MHP) at various temperatures on inactivation (a) and germination (b) of Bacillus cereus spores with and without germination-associated antiporters. Spores of B. cereus 569 (wild-type) and its isogenic gerN, gerT and gerN gerT mutant strains were treated with MHP at various temperatures, and after 15 min, the degree of spore germination and inactivation were determined. The spores represented by the different bars are open bars, wild-type spores; black bars with horizontal white lines, gerT mutant spores; bars with diagonal lines, gerN mutant spores; and solid bars, gerN gerT mutant spores. Discussion The results from this work indicated that both inactivation and germination of B. cereus spores by MHP increased as treatment temperature increased; however, the degree of B. cereus spore inactivation and germination by MHP increased up to 65°C, which was different from results with B. subtilis spores in which the degree of completion of spore germination triggered by MHP is maximal at 40°C (Black et al. 2007). Possibly, some protein essential for spore germination is more temperature sensitive in B. subtilis spores exposed to MHP than in B. cereus spores. The finding that under all conditions tested the degree of B. cereus spore germination triggered by MHP was greater than the degree of spore inactivation was consistent with MHP causing spore inactivation by initially triggering spore germination with the now more sensitive germinated spores then inactivated by the combination of MHP and heat; however, the precise mechanism for the inactivation of the MHP-germinated spores is not clear and is certainly a matter for further work. Another notable finding in the current work was that the degree of MHP germination of spores of B. cereus strains lacking individual germinant receptors was largely similar to that of wild-type spores. One possible conclusion from this observation is that germinant receptors are not involved in MHP germination of B. cereus spores; however, given results with B. subtilis spores (Black et al. 2005), this seems unlikely. In addition, the MHP germination of spores of B. cereus gerI and gerR strains was consistently lower, albeit only slightly, than that of wild-type spores. It thus seems more likely that with B. cereus spores, the seven different germinant receptors, each of which appears to be present in spores (Hornstra et al. 2006b), all trigger germination with MHP. In addition, levels of at least the GerI, L, R and S receptors are probably similar in spores prepared in rich media, albeit higher than levels of the GerG, K and Q receptors (Hornstra et al. 2006b). Thus, if the degree of MHP germination because of any one germinant receptor is dependent primarily on that receptor's level, then loss of any individual receptor would be expected not to have a large effect on the degree of B. cereus spore germination with MHP. Indeed, loss of the GerB or K receptors from B. subtilis spores has only a minimal effect on the MHP germination (Black et al. 2005). Consequently, what is likely needed for a definitive assessment of the role of germinant receptors in MHP germination of B. cereus spores is a strain lacking a large number of germinant receptors, but such a strain is not currently available. The final notable finding in the current work was that the loss of the GerN and especially the GerN and T monovalent cation antiporters had a significant effect on the MHP germination of B. cereus spores, especially at 40 and 50°C. Loss of GerN and T eliminates all germination with inosine and thus appears to have the effect on spore germination of loss of multiple germinant receptors. Consequently, one conclusion from these two findings is that these monovalent cation antiporters play a significant role in MHP germination as well; however, it may be advisable to be cautious in drawing this conclusion, as it is not clear that these antiporters play a direct role in spore germination. Antiporters do appear to play some role in inosine germination of B. cereus spores and in germination of spores of one Bacillus megaterium strain (Tani et al. 1996; Senior and Moir 2008); however, l-alanine germination of B. cereus spores is independent of the GerN and T antiporters (Senior and Moir 2008), and germination of spores of another B. megaterium strain is also independent of antiporters (Christie and Lowe 2007). In addition, while the gerN and gerT genes and their homologs are expressed only during sporulation in B. cereus and Bacillus anthracis as well as in Clostridium perfringens, at least in the latter organism, the genes encoding the antiporters whose absence reduces spore germination are expressed only in the mother cell compartment of the sporulating cell and thus likely are not present in the spore's inner membrane (Liu et al. 2004; Senior and Moir 2008; Paredes-Sabja et al. 2009). Consequently, it is not clear how these antiporters can be involved in ion movement across the spore's inner membrane during germination, and thus in the germination process itself. Indeed, there is some evidence with Cl. perfringens spores that antiporters do not function in the process of spore germination itself (Paredes-Sabja et al. 2009). Acknowledgements This work was supported by a grant from the US Department of Agriculture to P.S. and D.G.H. (grant no. 2003-35201-13553). 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