The Mechanism Of Endospores Of Bacillus Subtilis Stressed By Alkali And Formaldehyde

Spores (endospores) are resting states of some microorganisms and are formed under unfavorable conditions for the survival of growing cells. The dormant spore is hypersensitive to environment, and monitors the environment all time; the spore will germinate when conditions are again favorable for its growth, and go through outgrowth, ultimately being converted back into a vegetative cell. The dipicolinic acid (DPA) is the special components of spores which is chelated with divalent cations (generally Ca2+) in the spore’s core,and it plays important role in the stability and resistance of spores.In this study, laser tweezers Raman spectroscopy (LTRS), combined with Principal Components Analysis (PCA), was used to investigate the physiological processand the lethal mechanism of endospores, germinated spores and vegetative cells while stressed by alkali and formaldehyde.The Ca2+-DPA content of spores was linearly correlated with the intensity of its Raman specific band at1017cm-1. Spores’ Ca2+-DPA content was determined by Raman spectroscopy using LTRS as described previously.The behavior of Ca2+-DPA release from the spores was different after excitated with different germinants, in which the behavior was divided into two phase, Tiag and Trelease. The process of Ca2+-DPA release was a gradual slope and about10-30%of Ca2+-DPA was released to the outside in Tlag phase. Trelease phase was a steep slope which about70%Ca2+-DPA was released to the outside in several minutes (<5minutes generally). It meant that all channels of Ca2+-DPA release were open at short time in the Trelease phase..As of now we don’t know much about the mechanism how the channels open in a very short time.The behavior of Ca2+-DPA release was different with those of different chemical reagents treatment obviously. Only a little of Ca2+-DPA was found releasing from inside spores under alkali stress after LTRS was used to as a tool to study the behavior of Ca2+-DPA release, but the Ca2+-DPA did not release while stressed with formaldehyde. However, the behavior of Ca2+-DPA release was similar the behavior of germinants treatment after stressed with the chlorine and wet heat, both two the mechanism of Ca2+-DPA release were quite different. We presumed besides partial channels of Ca2+-DPA release were open, the membrane of spores was injured and leaked after stressed by chlorine and wet heat, so Ca2+-DPA could release from both channels and slits simultaneously. This release pattern shorten the time of T-release phase (finished in1-3minutes).To study the lethal mechanism of endospores stressed by alkali, the peak and intensity of laser tweezers Raman spectroscopy (LTRS) was used to investigate the physiological process of single spores under alkali stress. The result showed that both endspores and germinated spores (g.spores) had a range of tolerance to alkali, but the ability of g.spores to resist alkali was obviously lower than that of endospores due to the release of their Ca2+-DPA which plays an role in spores resistance as well as spores stability to many stresses; A small quantity of Ca2+-DPA of endospores was observed to release after alkali stress, however, the behavior of release was different with the normal behavior of Ca2+-DPA release induced by L-alanine; The data before and after alkali stress of the endospores and g.spores with PCA displayed that alkali mainly injured the member of spores, and alkali was easily entered into the internal structure of spores to destroy the structure of protein backbone and injure the nucleic acid of spores. We show that the alkali could result the small quantities of Ca2+-DPA release by damaging the member channel of spores.Both germinated spores (g.spores) and vegetative cells had a range of tolerance to formaldehyde, but the g.spores had a lower tolerance than those of vegetative cells and g.spores couldn’t absorb and metabolize formaldehyde as well. The time point of absorbing formaldehyde of the growth spores could be found after g.spores were cultured in2×SG medium,The data of Raman spectra of endospores, g.spores, growthing cells and vegetative cells was analysed statistically with PCA after stressed by formaldehyde. The results displayed that formaldehyde mainly injured the membrane of spores, and the structure of protein backbone and the nucleic acid of spores were injured.