Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering
Zhang, Pengfei; Kong, Lingbo; Setlow, Peter; Li, Yong-qing
Dual-trap laser tweezers Raman spectroscopy (LTRS) and elastic light scattering (ELS) were used to investigate dynamic processes during high-temperature treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis in water. Major conclusions from these studies included the following. (i) After spores of all three species were added to water at 80 to 90°C, the level of the 1:1 complex of Ca2+ and dipicolinic acid (CaDPA; ∼25% of the dry weight of the spore core) in individual spores remained relatively constant during a highly variable lag time (Tlag), and then CaDPA was released within 1 to 2 min. (ii) The Tlag values prior to rapid CaDPA release and thus the times for wet-heat killing of individual spores of all three species were very heterogeneous. (iii) The heterogeneity in kinetics of wet-heat killing of individual spores was not due to differences in the microscopic physical environments during heat treatment. (iv) During the wet-heat treatment of spores of all three species, spore protein denaturation largely but not completely accompanied rapid CaDPA release, as some changes in protein structure preceded rapid CaDPA release. (v) Changes in the ELS from individual spores of all three species were strongly correlated with the release of CaDPA. The ELS intensities of B. cereus and B. megaterium spores decreased gradually and reached minima at T1 when ∼80% of spore CaDPA was released, then increased rapidly until T2 when full CaDPA release was complete, and then remained nearly constant. The ELS intensity of B. subtilis spores showed similar features, although the intensity changed minimally, if at all, prior to T1. (vi) Carotenoids in B. megaterium spores' inner membranes exhibited two changes during heat treatment. First, the carotenoid's two Raman bands at 1,155 and 1,516 cm−1 decreased rapidly to a low value and to zero, respectively, well before Tlag, and then the residual 1,155-cm−1 band disappeared, in parallel with the rapid CaDPA release beginning at Tlag. Bacterial spores of Bacillus species are formed in sporulation and are metabolically dormant and extremely resistant to a variety of harsh conditions, including heat, radiation, and many toxic chemicals (37). Since spores of these species are generally present in foodstuffs and cause food spoilage and food-borne disease (37, 38), there has long been interest in the mechanisms of both spore resistance and spore killing, especially for wet heat, the agent most commonly used to kill spores. The killing of dormant spores by wet heat generally requires temperatures about 40°C higher than those for the killing of growing cells of the same strain (37, 43). A number of factors influence spore wet-heat resistance, with a major factor being the spore core's water content, as spores with higher core water content are less wet-heat resistant than are spores with lower core water (15, 25). The high level of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) and the types of its associated divalent cations, predominantly Ca2+, that comprise ∼25% of the dry weight of the core also contribute to spore wet-heat resistance, although how low core water content and CaDPA protect spores against wet heat is not known. The protection of spore DNA against depurination by its saturation with a group of α/β-type small, acid-soluble spore proteins also contributes to spore wet-heat resistance (14, 23, 33, 37). Despite knowledge of a number of factors important in spore wet-heat resistance, the mechanism for wet-heat killing of spores is not known. Wet heat does not kill spores by DNA damage or oxidative damage (35, 37). Instead, spore killing by this agent is associated with protein denaturation and enzyme inactivation (2, 7, 44), although specific proteins for which damage causes spore death have not been identified. Wet-heat treatment also often results in the release of the spore core's large depot of CaDPA. The mechanism for this CaDPA release is not known but is presumably associated with the rupture of the spore's inner membrane (7). In addition, the relationship between protein denaturation and CaDPA release is not clear, although recent work suggests that significant protein denaturation can occur prior to CaDPA release (7). Almost all information on spore killing by moist heat has been obtained with spore populations, and essentially nothing is known about the behavior of individual spores exposed to potentially lethal temperatures in water. Given the likely heterogeneity of spores in populations, in particular in their wet-heat resistances (16, 18, 39, 40), it could be most informative to analyze the behavior of individual spores exposed to high temperatures in water. Raman spectroscopy is widely used in biochemical studies, as this technique has high sensitivity and responds rapidly to subtle changes in molecule structure (1, 22, 31). In addition, when Raman spectroscopy is combined with confocal microscopy and optical tweezers, the resultant laser tweezers Raman spectroscopy (LTRS) allows the nondestructive, noninvasive detection of biochemical processes at the single-cell level (9, 10, 19, 46). Indeed, LTRS has been used to analyze the DPA level and the germination of individual Bacillus spores (5, 19, 30). In order to obtain information more rapidly, dual- and multitrap laser tweezers have been developed to allow multiple individual cells or particles to be analyzed simultaneously (11, 13, 24, 27), and the dual trap has been used to measure the hydrodynamic cross-correlations of two particles (24). In addition to Raman scattering, the elastic light scattering (ELS) from trapped individual cells also provides valuable information on cell shape, orientation, refractive index, and morphology (12, 45) and has been used to monitor spore germination dynamics as well (30). In this work, we report studies of wet-heat treatment of individual spores of three different Bacillus species by dual-trap LTRS and ELS. A number of important processes related to wet-heat inactivation of spores, including CaDPA release and protein denaturation, and the correlation between these processes were investigated by monitoring changes in Raman scattering at CaDPA-, protein structure-, and phenylalanine-specific bands and changes in ELS intensity.
Zhang, Pengfei, & Kong, Lingbo, & Setlow, Peter, & Li, Yong-qing. (March 2010). Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering. Applied and Environmental Microbiology, (76:6), p.1796-1805. Retrieved from http://hdl.handle.net/10342/8872
Zhang, Pengfei, and Kong, Lingbo, and Setlow, Peter, and Li, Yong-qing. "Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering". Applied and Environmental Microbiology. 76:6. (1796-1805.), March 2010. April 21, 2021. http://hdl.handle.net/10342/8872.
Zhang, Pengfei and Kong, Lingbo and Setlow, Peter and Li, Yong-qing, "Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering," Applied and Environmental Microbiology 76, no. 6 (March 2010), http://hdl.handle.net/10342/8872 (accessed April 21, 2021).
Zhang, Pengfei, Kong, Lingbo, Setlow, Peter, Li, Yong-qing. Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering. Applied and Environmental Microbiology. March 2010; 76(6) 1796-1805. http://hdl.handle.net/10342/8872. Accessed April 21, 2021.