Comparative analysis of thioflavin T and other fluorescent dyes for fluorescent staining of Bacillus subtilis vegetative cell, sporulating cell, and mature spore.

Thioflavin T, a cationic benzothiazole dye, is typically used to detect amyloid fibrils. In this study, we analyzed the staining properties of Bacillus subtilis cells using several fluorescent dyes, including thioflavin T analogs, 2-(4'-methylaminophenyl) benzothiazole (BTA-1), and 2-(4-aminophenyl) benzothiazole (APBT). Thioflavin T stained vegetative cells in the early log phase and outer layer structures of forespores and mature spores. The inner parts of forespores and heat-killed mature spores were also stained with thioflavin T. Congo red, auramine O, and rhodamine B stained forespores and mature spores similar to thioflavin T. In contrast, APBT and BTA-1 fluorescence was detected in the outer layers of vegetative cells, mother cells, forespores, and mature spores, indicating that they bind to the cell membrane and/or cell wall. The combination of the fluorescent dyes used in this study will help analyze morphogenetic processes during the sporulation and the damage mechanisms of vegetative cells and spores.

Transcriptional Profile during Deoxycholate-Induced Sporulation in a Clostridium perfringens Isolate Causing Foodborne Illness.

UNLABELLED: Clostridium perfringens type A is a common source of foodborne illness (FBI) in humans. Vegetative cells sporulate in the small intestinal tract and produce the major pathogenic factor C. perfringens enterotoxin. Although sporulation plays a critical role in the pathogenesis of FBI, the mechanisms inducing sporulation remain unclear. Bile salts were shown previously to induce sporulation, and we confirmed deoxycholate (DCA)-induced sporulation in C. perfringens strain NCTC8239 cocultured with human intestinal epithelial Caco-2 cells. In the present study, we performed transcriptome analyses of strain NCTC8239 in order to elucidate the mechanism underlying DCA-induced sporulation. Of the 2,761 genes analyzed, 333 were up- or downregulated during DCA-induced sporulation and included genes for cell division, nutrient metabolism, signal transduction, and defense mechanisms. In contrast, the virulence-associated transcriptional regulators (the VirR/VirS system, the agr system, codY, and abrB) were not activated by DCA. DCA markedly increased the expression of signaling molecules controlled by Spo0A, the master regulator of the sporulation process, whereas the expression of spo0A itself was not altered in the presence or absence of DCA. The phosphorylation of Spo0A was enhanced in the presence of DCA. Collectively, these results demonstrated that DCA induced sporulation, at least partially, by facilitating the phosphorylation of Spo0A and activating Spo0A-regulated genes in strain NCTC8239 while altering the expression of various genes. IMPORTANCE: Disease caused by Clostridium perfringens type A consistently ranks among the most common bacterial foodborne illnesses in humans in developed countries. The sporulation of C. perfringens in the small intestinal tract is a key event for its pathogenesis, but the factors and underlying mechanisms by which C. perfringens sporulates in vivo currently remain unclear. Bile salts, major components of bile, which is secreted from the liver for the emulsification of lipids, were shown to induce sporulation. However, the mechanisms underlying bile salt-induced sporulation have not yet been clarified. In the present study, we demonstrate that deoxycholate (one of the bile salts) induces sporulation by facilitating the phosphorylation of Spo0A and activating Spo0A-regulated genes using a transcriptome analysis. Thus, this study enhances our understanding of the mechanisms underlying sporulation, particularly that of bile salt-induced sporulation, in C. perfringens.

Morphological and genetic characterization of group I Clostridium botulinum type B strain 111 and the transcriptional regulator spoIIID gene knockout mutant in sporulation.

Clostridium botulinum is a heat-resistant spore-forming bacterium that causes the serious paralytic illness botulism. Heat-resistant spores may cause food sanitation hazards and sporulation plays a central role in the survival of C. botulinum. We observed morphological changes and investigated the role of the transcriptional regulator SpoIIID in the sporulation of C. botulinum type B strain 111 in order to elucidate the molecular mechanism in C. botulinum. C. botulinum type B formed heat-resistant spores through successive morphological changes corresponding to those of Bacillus subtilis, a spore-forming model organism. An analysis of the spoIIID gene knockout mutant revealed that the transcriptional regulator SpoIIID contributed to heat-resistant spore formation by C. botulinum type B and activated the transcription of the sigK gene later during sporulation. Transcription of the spoIIID gene, which differed from that in B. subtilis and Clostridium difficile, was observed in the sigE gene knockout mutant of C. botulinum type B. An analysis of the sigF gene knockout mutant showed that the sporulation-specific sigma factor SigF was essential for transcription of the spoIIID gene in C. botulinum type B. These results suggest that the regulation of sporulation in C. botulinum is not similar to that in B. subtilis and other clostridia.

SpoIVA is an essential morphogenetic protein for the formation of heat- and lysozyme-resistant spores in Clostridium sporogenes NBRC 14293.

Clostridium sporogenes is an anaerobic spore-forming bacterium genetically related to Clostridium botulinum but lacks toxin genes. The sporulation mechanism and spore structures of anaerobic bacteria, including C. sporogenes, have not been comprehensively analyzed. Based on 16S rRNA gene analysis, it has been determined that C. sporogenes NBRC 14293 belongs to C. botulinum Group I. Moreover, SpoIVA is highly conserved in Bacillus and Clostridium species. Therefore, the aim of the present study is to investigate the mechanism of spore formation in C. sporogenes by performing a functional analysis of spoIVA encoding SpoIVA, a protein involved in the early development of the spore coat and cortex in Bacillus subtilis. Inactivation of spoIVA in C. sporogenes resulted in the loss of resistance of sporulating cells to lysozyme and heat treatments. Phase-contrast microscopy indicated that the inactivation of spoIVA caused the development of abnormal forespores and production of only a few immature spores. In the spoIVA mutant, abnormal swirl structures were detected in the mother cell using both phase-contrast and transmission electron microscopy. These swirls were stained with auramine O, pararosaniline hydrochloride, and 2-(4-aminophenyl)benzothiazole to examine the surface of mature spores of the wild-type strain. We found that the spore coat and exosporium proteins were misassembled and that they accumulated in the mother cells of the mutant. The results of this study indicate that SpoIVA is a spore morphogenetic protein, providing novel insights into spore morphogenesis in C. sporogenes.

Ionic liquid-assisted sample preparation mediates sensitive proteomic analysis of Bacillus subtilis spores.

Endospore-forming bacteria are ubiquitous. Bacterial endospores are multilayered proteinaceous structures that protects the bacterial genome during stress conditions. They are also responsible for a wide range of critical clinical infections in humans. Precise analysis of spore-forming pathogens remains a major challenge in the field of proteomics because spore structures are highly resistant to conventional solubilizers and denaturing agents, such as sodium dodecyl sulfate and urea. We present an ionic liquid-assisted (i-soln) technique of sample preparation, called pTRUST, which enables shotgun analysis of Bacillus subtilis spores even when the starting materials are in the sub-microgram range. In proteomic analysis, this technique shows 50-2000-fold higher sensitivity than other conventional gel-based or gel-free methods (including one-pot sample processing). Using this technique, we identified 445 proteins with high confidence from trace amounts of highly pure spore preparations, including 52 of the 79 proteins (approximately 70%) previously demonstrated to be localized in spores in the SubtiWiki database and detected through direct protein analysis. Consequently, 393 additional proteins were identified as candidates for spore constitutive proteins. Twenty of these newly identified candidates were produced as green fluorescent protein fusion proteins, and each was evaluated for authenticity as a spore constituent using fluorescence microscopy analysis. The pTRUST method's sensitivity and reliability using the i-soln system, together with hitherto unreported proteins in spores, will enable an array of spore research for biological and clinical applications.

With respect to coefficient of linear thermal expansion, bacterial vegetative cells and spores resemble plastics and metals, respectively.

BACKGROUND: If a fixed stress is applied to the three-dimensional z-axis of a solid material, followed by heating, the amount of thermal expansion increases according to a fixed coefficient of thermal expansion. When expansion is plotted against temperature, the transition temperature at which the physical properties of the material change is at the apex of the curve. The composition of a microbial cell depends on the species and condition of the cell; consequently, the rate of thermal expansion and the transition temperature also depend on the species and condition of the cell. We have developed a method for measuring the coefficient of thermal expansion and the transition temperature of cells using a nano thermal analysis system in order to study the physical nature of the cells. RESULTS: The tendency was seen that among vegetative cells, the Gram-negative Escherichia coli and Pseudomonas aeruginosa have higher coefficients of linear expansion and lower transition temperatures than the Gram-positive Staphylococcus aureus and Bacillus subtilis. On the other hand, spores, which have low water content, overall showed lower coefficients of linear expansion and higher transition temperatures than vegetative cells. Comparing these trends to non-microbial materials, vegetative cells showed phenomenon similar to plastics and spores showed behaviour similar to metals with regards to the coefficient of liner thermal expansion. CONCLUSIONS: We show that vegetative cells occur phenomenon of similar to plastics and spores to metals with regard to the coefficient of liner thermal expansion. Cells may be characterized by the coefficient of linear expansion as a physical index; the coefficient of linear expansion may also characterize cells structurally since it relates to volumetric changes, surface area changes, the degree of expansion of water contained within the cell, and the intensity of the internal stress on the cellular membrane. The coefficient of linear expansion holds promise as a new index for furthering the understanding of the characteristics of cells. It is likely to be a powerful tool for investigating changes in the rate of expansion and also in understanding the physical properties of cells.

Excessive ultraviolet C irradiation causes spore protein denaturation and prohibits the initiation of spore germination in Bacillus subtilis.

Ultraviolet (UV) -C is widely used to kill bacteria as it damages chromosomal DNA. We analyzed the denaturation of the protein function of Bacillus subtilis spores after UV-C irradiation. Almost all of the B. subtilis spores germinated in Luria-Bertani (LB) liquid medium, but the colony-forming unit (CFU) of the spores on LB agar plates decreased to approximately 1/103 by 100 mJ/cm2 of UV-C irradiation. Some of the spores germinated in LB liquid medium under phase-contrast microscopy, but almost no colonies formed on the LB agar plates after 1 J/cm2 of UV-C irradiation. The fluorescence of the green fluorescent protein (GFP) -fused spore proteins, YeeK-GFP, YeeK is a coat protein, decreased following UV-C irradiation of over 1 J/cm2, while that of SspA-GFP, SspA is a core protein, decreased following UV-C irradiation of over 2 J/ cm2, respectively. These results revealed that UV-C affected on coat proteins more than core proteins. We conclude that 25 to 100 mJ/cm2 of UV-C irradiation can cause DNA damage, and more than 1 J/cm2 of UV-C irradiation can cause the denaturation of spore proteins involved in germination. Our study would contribute to improve the technology to detect the bacterial spores, especially after UV sterilization.

Discrimination of the Bacillus cereus group members by pattern analysis of random amplified polymorphic DNA-PCR.

We tried to discriminate 16 strains of the Bacillus cereus group including B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis strains by the pattern analysis of Random Amplified Polymorphic DNA (RAPD) -PCR. Eight oligonucleotides primers were prepared and the polymorphic patterns of the DNA of each strain were compared with those of others. The primers E and F gave different patterns of RAPD-PCR products in all strains of the B. cereus group, so these primers are effective tools for the discrimination of closely related strains. All eight primers showed different polymorphic patterns of DNA for the four strains of B. cereus isolated from the kitchen of a private home, which verifies the advantage of the RAPD-PCR analysis for the discrimination of isolated strains of B. cereus from the environment.

The Study of Diversity in Sporulation among Closely Genetically Related Bacillus cereus Strains.

Bacillus cereus is an important foodborne pathogenic bacterium. Although several B. cereus strains have been isolated from the environment, the differences among these strains with respect to spore formation ability and cell morphology need clarification. In this study, a phylogenetic tree was constructed based on the 16S rRNA gene sequences of nine strains of B. cereus. Spore formation and morphology of these nine strains were compared using both phase-contrast and fluorescence microscopy to create an index of the designated sporulation stages. Additionally, to investigate the efficiency of heat-resistant spore formation. Phylogenetic analysis revealed that five strains (ATCC 14579T, NBRC 3457, NBRC 3514, NBRC 3836, and NBRC 13597) clustered together and the remaining four (ATCC 10987, NBRC 3003, NBRC 13494, and NBRC 13690) were genetically distinct from each other. Phase-contrast microscopy revealed significant differences in the sporulation stages among the nine strains. Furthermore, the efficiency of heat-resistant spore formation also differed, even among genetically related strains. In conclusion, a variety of cell morphologies during sporulation were observed among the nine B. cereus strains. We propose a designation of sporulation stages in B. cereus ATCC 14579T, which may be used as an index for evaluating the sporulation progress of B. cereus.

Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis.

In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) inEscherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulphhydryl groups exerted significant effects strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibitedGraphical Abstract the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the MEROPS peptidase database.

Proteins involved in formation of the outermost layer of Bacillus subtilis spores.

To investigate the outermost structure of the Bacillus subtilis spore, we analyzed the accessibility of antibodies to proteins on spores of B. subtilis. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not cgeA mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the B. subtilis spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of cotW affected the assembly of CotV-GFP, and a disruption of cotX affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that cgeA and genes in the cotVWXYZ cluster are involved in spore crust formation.

A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly.

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.

Localization of proteins to different layers and regions of Bacillus subtilis spore coats.

Bacterial spores are encased in a multilayered proteinaceous shell known as the coat. In Bacillus subtilis, over 50 proteins are involved in spore coat assembly but the locations of these proteins in the spore coat are poorly understood. Here, we describe methods to estimate the positions of protein fusions to fluorescent proteins in the spore coat by using fluorescence microscopy. Our investigation suggested that CotD, CotF, CotT, GerQ, YaaH, YeeK, YmaG, YsnD, and YxeE are present in the inner coat and that CotA, CotB, CotC, and YtxO reside in the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat and were more abundant at the mother cell proximal pole of the forespore, whereas CotA and CotC were more abundant at the mother cell distal pole of the forespore. These polar localizations were observed both in sporangia prior to the release of the forespore from the mother cell and in mature spores after release. Moreover, CotB was observed at the middle of the spore as a ring- or spiral-like structure. Formation of this structure required cotG expression. Thus, we conclude not only that the spore coat is a multilayered assembly but also that it exhibits uneven spatial distribution of particular proteins.

Expression of yeeK during Bacillus subtilis sporulation and localization of YeeK to the inner spore coat using fluorescence microscopy.

The yeeK gene of Bacillus subtilis is predicted to encode a protein of 145 amino acids composed of 28% glycine, 23% histidine, and 12% tyrosine residues. Previous studies were unable to detect YeeK in wild-type spores; however, the 18-kDa YeeK polypeptide has been identified in yabG mutant spores. In this study, we analyze the expression and localization of YeeK to explore the relationship between YeeK and YabG. Northern hybridization analysis of wild-type RNA indicated that transcription of the yeeK gene, which was initiated 5 h after the onset of sporulation, was dependent on a SigK-containing RNA polymerase and the GerE protein. Genetic disruption of yeeK did not impair vegetative growth, development of resistant spores, or germination. Fluorescent microscopy of in-frame fusions of YeeK with green fluorescent protein (YeeK-GFP) and red fluorescent protein (YeeK-RFP) confirmed that YeeK assembles into the spore integument. CotE, SafA, and SpoVID were required for the proper localization of YeeK-GFP. Comparative analysis of YeeK-RFP and an in-frame GFP fusion of YabG indicated that YeeK colocalized with YabG in the spore coat. This is the first use of fluorescent proteins to show localization to different layers of the spore coat. Immunoblotting with anti-GFP antiserum indicated that YeeK-GFP was primarily synthesized as a 44-kDa molecule, which was then digested into a 29-kDa fragment that corresponded to the molecular size of GFP in wild-type spores. In contrast, a minimal amount of 44-kDa YeeK-GFP was digested in yabG mutant spores. Our findings demonstrate that YeeK is guided into the spore coat by CotE, SafA, and SpoVID. We conclude that YabG is directly or indirectly involved in the digestion of YeeK.

[Protein modification system in dormant cells].

Dormancy in an organism is an adaptive response to environmental stress. The initiation, maintenance, and breaking of dormancy are adaptations to environmental signals. In active cells, an environmental response is genetically controlled general phenomenon. However, no such system is available in dormant cells, in which almost all gene expression is repressed. Bacterial spores are dormant and highly resistant to many environmental stresses. I analyzed the protein profile of Bacillus subtilis spores by a combination of SDS-PAGE and LC-MS/MS to reveal protein modification in dormant cells. I found that protein modification was mediated by spore built-in enzymes YabG (a protease) and Tgl (a transglutaminase) in the spores of B. subtilis. The rearrangement of spore coat proteins caused by the activities of these built-in enzymes proceeded independently of gene expression or de novo protein synthesis in dormant cells. The results suggest that some built-in enzymes are activated under certain conditions and thereafter become involved in the modification of proteins and other cellular materials in dormant cells. I propose the idea that "Active" adaptation in active cells is dependent on gene expression, and that "Quiet" adaptation in dormant cells is dependent on the activity of some built-in enzymes independently of gene expression or de novo protein synthesis. Other enzymes are involved in restoration of dormancy in response to signals such as the nutrition.

Expression, localization and modification of YxeE spore coat protein in Bacillus subtilis.

YxeE is a spore coat protein of Bacillus subtilis. We have previously reported that YxeE can be extracted from yabG mutant spores. In the present study, we analysed the expression and localization of YxeE. Northern analysis detected the transcript of yxeE from sporulating cells 4 h after the onset of sporulation, and revealed that the synthesis of yxeE mRNA was dependent on expression of the SigK RNA polymerase and the GerE regulator. Immunoblotting with anti-YxeE antiserum detected YxeE from sporulating cells 4 h after the onset of sporulation. YxeE was detected in the extracts from mature spores of yabG mutant strain but not in those from wild-type spores. On the other hand, fluorescence microscope observations showed that YxeE-green fluorescent protein (GFP) is located at the surface of both wild-type and yabG spores. Immunoblot analysis with anti-GFP antiserum showed that YxeE-GFP was not digested in yabG-disrupted strain and that only the GFP portion remained in the wild-type yabG background. We conclude that YxeE is a substrate of YabG protease.

Development of a Novel Scanning Thermal Microscopy (SThM) Method to Measure the Thermal Conductivity of Biological Cells.

　Differences in the physical properties of individual cells cannot be evaluated with conventional experimental methods that are used to study groups of cells obtained from pure cultures. To examine the differences in the thermal tolerance of individual cells that are genetically identical, a method is needed to measure the thermal energy required to kill single cells. We developed a scanning thermal microscopy (SThM) system and measured the thermal conductivity of various bacterial cells, for example, spore formeing Bacillus genus and non spore-forming bacteria such as Escherichia coli. The thermal conductivity of vegetative cells (0.61 to 0.75 W/m・K) was found to be higher than that of spores (0.29 to 0.45 W/m・K). Furthermore the newly developed method enables us to estimate the thermal energy needed to kill individual cells or spores. We believe that this method can estimate the thermal energy required to achieve the cell for sterilization by heating.

Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores.

Here we describe the functional relationship between YabG and transglutaminase (Tgl), enzymes that modify the spore coat proteins of Bacillus subtilis. In wild-type spores at 37 degrees C, Tgl mediates the crosslinking of GerQ into higher molecular mass forms; however, some GerQ multimers are found in tgl mutant spores, indicating that Tgl is not essential. Immunoblotting showed that spores isolated from a yabG mutant after sporulation at 37 degrees C contain only very low levels of GerQ multimers. Heat treatment for 20 min at 60 degrees C, which maximally activates the enzymatic activity of Tgl, caused crosslinking of GerQ in isolated yabG spores but not in tgl/yabG double-mutant spores. In addition, the germination frequency of the tgl/yabG spores in the presence of l-alanine with or without heat activation at 60 degrees C was lower than that of wild-type spores. These findings suggest that Tgl cooperates with YabG to mediate the temperature-dependent modification of the coat proteins, a process associated with spore germination in B. subtilis.

The ylbO gene product of Bacillus subtilis is involved in the coat development and lysozyme resistance of spore.

The Bacillus subtilis YlbO protein is a Myb-like DNA binding domain-containing protein that is expressed under the control of SigE. Here, we analyzed gene expression and protein composition in ylbO-negative cells. SDS-PAGE analysis revealed that the protein profile of ylbO- negative spores differed from that of wild-type. Specifically, the expression of coat proteins CgeA, CotG, and CotY, which are controlled by SigK and GerE, was reduced in ylbO -negative cells. Northern blot analysis revealed that YlbO regulated the transcription of cgeA, cotG, and cotY. These results suggest that YlbO regulates the expression of some coat proteins during sporulation in B. subtilis directly or indirectly.

The Heat Resistance of Microbial Cells Represented by D Values Can be Estimated by the Transition Temperature and the Coefficient of Linear Expansion.

We previously developed a method for evaluating the heat resistance of microorganisms by measuring the transition temperature at which the coefficient of linear expansion of a cell changes. Here, we performed heat resistance measurements using a scanning probe microscope with a nano thermal analysis system. The microorganisms studied included six strains of the genus Bacillus or related genera, one strain each of the thermophilic obligate anaerobic bacterial genera Thermoanaerobacter and Moorella, two strains of heat-resistant mold, two strains of non-sporulating bacteria, and one strain of yeast. Both vegetative cells and spores were evaluated. The transition temperature at which the coefficient of linear expansion due to heating changed from a positive value to a negative value correlated strongly with the heat resistance of the microorganism as estimated from the D value. The microorganisms with greater heat resistance exhibited higher transition temperatures. There was also a strong negative correlation between the coefficient of linear expansion and heat resistance in bacteria and yeast, such that microorganisms with greater heat resistance showed lower coefficients of linear expansion. These findings suggest that our method could be useful for evaluating the heat resistance of microorganisms.