Raman Spectrum Of Spore Germination Of Bacillus Subtilis In Response To Formaldehyde Stress

The germination of Bacillus spores is the first step to return the vegetative growth, and is induced by nutrients and a variety of non-nutrient agents, and spores completely loss resistance to outer stress.As a characteristic composition of bacteria spores, pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plays an important role in spores resistance as well as spores stability to many environmental stresses. Following DPA-Ca2+ released entirely, spores lose their refraction and resistance completely. It is significant to explore the behavior of DPA-Ca2+ release and the rule of compositional changes in spores under environmental stresses for understanding the resistance mechanism of bacterial spores. We reported the application of laser tweezers Raman spectroscopy (LTRS) to research the character of DPA-Ca2+ release, the dynamics of an individual spore and the component difference combined with Principal Components Analysis (PCA) under formaldehyde stress. The results indicated that the toleration ability of spores to formaldehyde is 500 times stronger than germinated spores (DPA-Ca2+ released completely); Formaldehyde treatment didn't cause DPA-Ca2+ release of spores, which was different with germinants induction or chlorine/high temperature and wet heat stress:Formaldehyde was to penetrate multilayer barriers of the bacterial spore into the spores internal and interact with proteins and DNA directly. The PCA results showed that killing of spores by 1000mM of formaldehyde is due to damaging purine and G-C bases of nucleic acid, C-C backbone, and also harming to membrane lipids partly; oppositely, killing of germinated spores treated with 1.5mM of formaldehyde is due to C-C main chain and side chain damage, which adenine and membrane lipid have been damaged slightly, the maximum absolute value of PC1(principal component1) loading of 958cm-1 exhibited that the max damage to germinated spores by formaldehyde was spores proteins.the spores of bacillus subtitles in response to bleaching water and wet heat, the release of DPA-Ca+ from its core is not couple with spores'death, Ca2+-DPA retain when Spores were dead. bleaching water and wet heat make DPA-Ca2+ release; this process is very fast, generally in 1-2 minutes. after DPA-Ca2+ releasing, the band of 1655cm-1 shifts to 1665cm-1,α-helix of protein decreasing, no regular coil increasing, so space structure of Protein changes. Principal Components Analysis (PCA) analysis results indicate:bleaching mainly damage the spore's inner membrane, causing DPA-Ca2+ release, Chlorine damage C-C Lord chain of protein breaking into the spores, Damaging nucleic acid, making g.spores dead; high temperature damage the hydroxyl phenyl rings of Tyrosine,the indoles rings of tryptophan, damage side chain of Protein, even rupture, following accumulation of proline,suggesting that damage protein or make enzyme deactivation in wet heat stress.Bacillus subtilis is non-methylotrophic bacteria which can assimilate formaldehyde, whose vegetative cell can transform formaldehyde to one's own components by RuMP (Ribulose monophosphate) pathway. In order to explore germinated spores'(g.spore) of bacillus subtitles physical reaction and tolerance mechanism in response to formaldehyde, researching on the process of different concentrations formaldehyde stress g. spore 2 hours by laser tweezers Raman spectra system (LTRS).The result shows, g.spores contain the same genetic material and functional proteins with vegetative cell, which have the ability of tolerating formaldehyde, but the ability of vegetative cell is stronger than g.spore, and g.spore can't absorb and metabolize formaldehyde. The Raman spectrum band changes of g.spores in response to formaldehyde stress displays:g.spores in 0.4mmol/L formaldehyde medium, whose Change intensity of protein, Lipid and nucleic acid is stronger than g.spores in no formaldehyde medium, The trends reflect that g.spores makes a strong response pattern early in formaldehyde stress, many gene expression level rise, cells metabolism level increases, macromolecular substances accumulation also follow increase, After the 1.5 hours, various components in vivo decrease, showing cells are mild hurt; g.spores in 0.8mmol/L formaldehyde medium, various components in vivo decrease at 1.0 hour, the band of nucleic acid decreases slowly at the very start, indicating DNA can't duplicate in the whole process, the bands of membrane phospholipids and C-S(protein) stretching mode decline obviously after 0.5 hours, which suggests formaldehyde Causing the membrane lipid hydrocarbon chain breakedown;g.spores in 1.0mmol/L formaldehyde medium, the bands decrease at 0.5 hour, suggesting the concentration formaldehyde makes a severe stress effect on cells, the content of biological macromolecules drops significantly after 0.5 hour, making cell hurts gradually deteriorated.