Kinetics of Germination of Individual Spores of Geobacillus stearothermophilus as Measured by Raman Spectroscopy and Differential Interference Contrast Microscopy

Tingting Zhou,Zhiyang Dong,Peter Setlow,Yong-Qing Li,Adam Driks

doi:10.1371/journal.pone.0074987

Abstract

Geobacillus stearothermophilus is a gram-positive, thermophilic bacterium, spores of which are very heat resistant. Raman spectroscopy and differential interference contrast microscopy were used to monitor the kinetics of germination of individual spores of G. stearothermophilus at different temperatures, and major conclusions from this work were as follows. 1) The CaDPA level of individual G. stearothermophilus spores was similar to that of Bacillus spores. However, the Raman spectra of protein amide bands suggested there are differences in protein structure in spores of G. stearothermophilus and Bacillus species. 2) During nutrient germination of G. stearothermophilus spores, CaDPA was released beginning after a lag time (T lag) between addition of nutrient germinants and initiation of CaDPA release. CaDPA release was complete at T release, and ΔT release (T release – T lag) was 1–2 min. 3) Activation by heat or sodium nitrite was essential for efficient nutrient germination of G. stearothermophilus spores, primarily by decreasing T lag values. 4) Values of T lag and T release were heterogeneous among individual spores, but ΔT release values were relatively constant. 5) Temperature had major effects on nutrient germination of G. stearothermophilus spores, as at temperatures below 65°C, average T lag values increased significantly. 6) G. stearothermophilus spore germination with exogenous CaDPA or dodecylamine was fastest at 65°C, with longer Tlag values at lower temperatures. 7) Decoating of G. stearothermophilus spores slowed nutrient germination slightly and CaDPA germination significantly, but increased dodecylamine germination markedly. These results indicate that the dynamics and heterogeneity of the germination of individual G. stearothermophilus spores are generally similar to that of Bacillus species.

Highlights

Many components of the spore germination machinery are conserved between spore forming members of the Bacillales [1]
7) Decoating of G. stearothermophilus spores slowed nutrient germination slightly and CaDPA germination significantly, but increased dodecylamine germination markedly. These results indicate that the dynamics and heterogeneity of the germination of individual G. stearothermophilus spores are generally similar to that of Bacillus species
The intensity of the CaDPA-specific 1,017 cm21 Raman band in the average spectrum from 30 individual spores indicated that the CaDPA level in the core of G. stearothermophilus spores was, 382 mM, and this value was only slightly higher than the values for B. subtilis and B. cereus spores (Table 1)

Summary

Introduction

Many components of the spore germination machinery are conserved between spore forming members of the Bacillales [1]. Bacillus subtilis spore germination can be initiated by a variety of chemicals, including nutrients, cationic surfactants, and enzymes, as well as by hydrostatic pressure [2]. Nutrient germinants for spore germination generally include amino acids, purine derivatives, and sugars, and are species and strain specific. These nutrient germinants interact with germination receptors (GRs) located in the inner spore membrane [2], stimulating the release of the spore core’s large (,10% of spore dry wt) depot of pyridine2,6-dicarboxylic acid (dipicolinic acid [DPA]) and divalent cations, predominantly Ca2+, which are likely present as a 1:1 chelate (CaDPA) [3]. CaDPA in the core is released and replaced by water in stage I of spore germination, and CaDPA release triggers stage II of germination, a major event which is the hydrolysis of spores’ peptidoglycan cortex by cortex lytic enzymes (CLEs) [2,4].

Methods

Results

Conclusion