Properties Of Spores Research Articles

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

Termination factor Rho, responsible for the main factor-dependent pathway of transcription termination and the major inhibitor of antisense transcription, is an emerging regulator of various physiological processes in microorganisms. In Gram-positive bacterium Bacillus subtilis, Rho is involved in the control of cell adaptation to starvation and, in particular, in the control of sporulation, a complex differentiation program leading to formation of a highly resistant dormant spore. While the initiation of sporulation requires a decrease in Rho protein levels during the transition to stationary phase, the mechanisms regulating the expression of rho gene throughout the cell cycle remain largely unknown. Here we show that a drop in the activity of the vegetative SigA-dependent rho promoter causes the inhibition of rho expression in stationary phase. However, after the initiation of sporulation, rho gene is specifically reactivated in two compartments of the sporulating cell using distinct mechanisms. In the mother cell, rho expression occurs by read-through transcription initiated at the SigH-dependent promoter of the distal spo0F gene. In the forespore, rho gene is transcribed from the intrinsic promoter recognized by the alternative sigma factor SigF. These regulatory elements ensure the activity of Rho during sporulation, which appears important for the proper formation of spores. We provide experimental evidence that disruption of the spatiotemporal expression of rho during sporulation affects the resistance properties of spores, their morphology, and the ability to return to vegetative growth under favorable growth conditions.

Isolation and characterization of superdormant Bacillus subtilis spores under high hydrostatic pressure at 200 MPa and 500 MPa

High hydrostatic pressure (HHP) (200 MPa/500 MPa) superdormant (SD) spores were isolated and characterized for germination and resistance properties. Compared with untreated spores, two-hundred MPa-SD spores showed germination defect with L-alanine, AGFK, dodecylamine, 200 MPa or 500 MPa. Meanwhile, they exhibited elevated resistance to 93 °C and UV254 nm with decimal reduction time (D-value) increased by 2.7 and 1.5 fold respectively, reduced resistance to 0.22 M HCl and 3.2 M H2O2 with D-value decreased by 1.4 and 1.3 fold respectively, and no change in resistance to 0.83 M formaldehyde and 0.34 M NaClO. Five-hundred MPa-SD spores showed germination defect with 500 MPa, no change in germination with L-alanine, AGFK or 200 MPa, but increased germination efficiency with dodecylamine. Accordingly, their resistance to 0.34 M NaClO and UV254 nm elevated with D-value increased by 1.8 and 1.3 fold respectively, while resistance to 93 °C, 0.22 M HCl, 0.83 M formaldehyde and 3.2 M H2O2 reduced with D-value decreased by 2, 1.4, 1.4 and 2.3 fold respectively. These properties were noninheritable as re-sporulated spores from SD spores showed identical germination behavior compared to initial untreated spores. Proteomics analysis revealed that the mechanism of SD spores' superdomancy was likely attributed to the variation of coat proteins and SASPs, and inner membrane modifications. Industrial relevanceIt has long been acknowledged that the existence of SD spores greatly limits the successful application of HHP technology in low-acid foods. The data in this work provide valuable knowledge to understand the properties of SD spores under HHP, and thus hopefully can promote developing new strategies to eliminate bacterial spores in low-acid foods during industrial HHP processing.

Single-cell analysis reveals microbial spore responses to sodium hypochlorite.

Pollution from toxic spores has caused us a lot of problems because spores are extremely resistant and can survive most disinfectants. Therefore, the detection of spore response to disinfectant is of great significance for the development of effective decontamination strategies. In this work, we investigated the effect of 0.5% sodium hypochlorite on the molecular and morphological properties of single spores of Bacillus subtilis using single-cell techniques. Laser tweezers Raman spectroscopy showed that sodium hypochlorite resulted in Ca2+-dipicolinic acid release and nucleic acid denaturation. Atomic force microscopy showed that the surface of treated spores changed from rough to smooth, protein shells were degraded at 10 min, and the permeability barrier was destroyed at 15 min. The spore volume decreased gradually over time. Live-cell imaging showed that the germination and growth rates decreased with increasing treatment time. These results provide new insight into the response of spores to sodium hypochlorite.

Development and production of antibodies against gamma inactivated pathogenic bacterial spores

Abstract Objectives Gram-positive sporulated bacilli can cause many different diseases and isolation from environmental samples is difficult. Therefore, the quick detection and diagnosis of these microorganisms have critical importance because of their potentially harmful situation. However, many accepted diagnostic methods exist, and future technology points to immunoassay systems. Immunological methods to detect biological microorganisms require antigen-specific high-affinity antibodies as key materials. Methods In this study, Bacillus anthracis (34F2 sterne) bacterium, which causes anthrax disease, was chosen as a model organism to develop antibodies against bacterial spores. The produced spores were inactivated with gamma irradiation, and the development of monoclonal antibodies against inactivated spores was performed using hybridoma technology. Also, the polyclonal antibody was successfully obtained by immunizing the rabbit. Indirect and sandwich ELISA tests were performed to determine the antigenic properties of inactivated spores and the specific affinity of the developed antibodies. Results The spores, inactivated with 15 kGy, have the best-preserved surface epitopic regions and were selected as immunogen. Developed monoclonal and polyclonal antibodies were shown that there was no cross-reaction with other Bacillus species. Also, it was demonstrated that these antibodies could detect inactivated spores at a concentration of 105 spores/mL in a sandwich ELISA assay. Conclusions These qualified antibodies obtained will be essential in developing antibody-based diagnostic systems for spore detection from various environmental samples. This study suggests that the inactivated spores are a decent immunogen for generation antibodies and may be a candidate component for live vaccine formulation.

Insight into effects of terbium on cell growth, sporulation and spore properties of Bacillus subtilis.

Recovery of rare earth elements (REEs) from wastewater with Bacillus subtilis(B. subtilis)during culture is promising due to its environmental benefits. However, the effects of REEs in the culture media on B. subtilis are poorly understood. This study aims to investigate the effects of the terbium (Tb(III)), a typical rare earth element, on the cell growth, sporulation, and spore properties of B. subtilis. Tb(III) can suppress bacterial growth while enhancing spore tolerance to wet heat. Spore germination and content of dipicolinic acid (DPA) were promoted at low concentrations of Tb(III) while inhibited at a high level, but an inverse effect on initial sporulation appeared. Scanning electron microscope and energy dispersive spectrometer detection indicated that Tb(III) complexed cells or spores and certain media components simultaneously. The germination results of the spores after elution revealed that Tb(III) attached to the spore surface was a key effector of spore germination. In conclusion, Tb(III) directly or indirectly regulated both the nutrient status of the media and certain metabolic events, which in turn affected most of the properties of B. subtilis. Compared to the coat-deficient strain, the wild-type strain grew faster and was more tolerant to Tb(III), DPA, and wet heat, which in turn implied that it was more suitable for the recovery of REEs during cultivation. These findings provide fundamental insights for the recovery of rare earths during the culture process using microorganisms.

Identification of Clinical Isolates of <i>Bacillus cereus</i> Group and Their Characterization by Mass Spectrometry and Electron Microscopy

Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were investigated in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the isolates SRCC 19/16 has an enlarged exosporium, and isolate SRCC 1208 shows large electron-dense inclusions of unclear nature during sporulation. We can assume that it contains a biologically active component with a cytotoxic effect and possibly plays a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

Spore-forming properties and enhanced oxygen tolerance of butyrate-producing Anaerostipes spp.

ObjectivesButyrate producing bacteria are promising candidates for next-generation probiotics. However, they are extremely sensitive to oxygen, which is a significant obstacle to their inclusion in food matrices in a viable form. The present study characterized the spore-forming properties and stress tolerance of human gut butyrate-producing Anaerostipes spp. MethodsSpore formation properties in six species of Anaerostipes spp. were studied by in vitro and in silico tests. ResultsSpores were observed from the cells of three species using microscopic analyses, while the remaining three did not form spores under the tested conditions. Spore-forming properties were confirmed by an ethanol treatment. The spores of Anaerostipes caccae were tolerant to oxygen and survived for 15 weeks under atmospheric conditions. Spores tolerated heat stress at 70 °C, but not at 80 °C. An in silico analysis of the conservation of potential sporulation signature genes revealed that the majority of human gut butyrate-producing bacteria were classified as potential spore formers. Comparative genomics revealed that three spore-forming Anaerostipes spp. specifically possessed the spore formation-related genes of bkdR, sodA, and splB, which may be key genes for different sporulation properties in Anaerostipes spp. ConclusionsThe present study demonstrated the enhanced stress tolerance of butyrate producing Anaerostipes spp. for future probiotic application. Presence of specific gene(s) are possibly keys for sporulation in Anaerostipes spp.

Physico-chemical characterization of single bacteria and spores using optical tweezers

Spore-forming pathogenic bacteria are adapted for adhering to surfaces, and their endospores can tolerate strong chemicals making decontamination difficult. Understanding the physico-chemical properties of bacteria and spores is therefore essential in developing antiadhesive surfaces and disinfection techniques. However, measuring physico-chemical properties in bulk does not show the heterogeneity between cells. Characterizing bacteria on a single-cell level can thereby provide mechanistic clues usually hidden in bulk measurements. This paper shows how optical tweezers can be applied to characterize single bacteria and spores, and how physico-chemical properties related to adhesion, fluid dynamics, biochemistry, and metabolic activity can be assessed.

Analysis of Compounding and Broadband Extinction Properties of Novel Bioaerosols

Artificially prepared microbial spores have excellent electromagnetic attenuation properties due to their special composition and structure. At present, studies on the optical properties of microbial spores have mainly focused on those with a single band or a single germplasm, which has limitations and cannot reveal the optical properties comprehensively. In this paper, 3 kinds of laboratory-prepared microbial spores were selected for compounding, and the spectral reflectivities of single-germplasm biospores and compound biospores were measured in the wavebands of 0.25–2.4 and 3–15 μm. The complex refractive indices (CRIs) were calculated in combination with the Kramers–Kronig (K-K) algorithm. Relying on the smoke box broadband test system, the transmittance of single-germplasm bioaerosols and compound bioaerosols from the ultraviolet (UV) band to the far-infrared (FIR) band was measured, and the mass extinction coefficients were calculated. The results indicate that the trend of the complex refractive indices of the compound spores is consistent with that of the single-germplasm spores with a larger particle size. For the single-germplasm bioaerosols, the lowest transmittance values were 2.21, 5.70 and 6.27% in the visible (VIS), near-infrared (NIR) and middle-infrared (FIR) bands, and the mass extinction coefficients reached 1.15, 0.87 and 0.84 m2/g, respectively. When AO and BB spores were compounded at 4:1, the extinction performance of the bioaerosols somewhat improved in all wavebands. These results can help to comprehensively analyze the optical properties of bioaerosols and provide ideas for the development of new extinction materials.

Identification of Clinical Isolates of the Bacillus cereus Group and Their Characterization by Mass Spectrometry and Electron Microscopy

Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were used in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the SRCC 19/16 isolates has an enlarged exosporium, and the SRCC 1208 isolate has large electron-dense inclusions of an unclear nature during sporulation. We can assume that these contain a biologically active component with a cytotoxic effect and possibly play a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results we obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

Dielectric analysis of Sclerotinia sclerotiorum airborne inoculum by the measurement of dielectrophoretic trapping voltages using a microfluidic platform

Sclerotinia stem rot, caused by the fungal pathogen Sclerotinia sclerotiorum is a devastating crop disease. Various forecasting systems have been developed to provide information about the risk of outbreaks and thus avoid the heavy financial burden caused by over-spraying fungicides. In this study, we experimentally determine the dielectric properties of Sclerotinia sclerotiorum airborne spores, one of the main agents of infection in stem rot. The dielectric properties of spores are important parameters for the development of forecasting systems based on dielectrophoretic filters as it provides information about the dielectrophoretic response of spores without the need for iterative testing. A microfluidic platform was employed to estimate the dielectric parameters based on a dielectrophoretic method and in media of different conductivities. In addition, using the multi-shell model, spores were modeled using a realistic ellipsoidal double-shell model. To validate the methodology and analysis, the dielectric properties of human embryonic kidney 293 were also determined and compared to values reported in the literature. The obtained values for the electrical permittivity and conductivity of the spore interior and membrane were found to be insensitive to the conductivity of the external medium. On the other hand, the dielectric parameters of the outer most layer showed a small variation with the conductivity of the external medium. This study represents the first report on the dielectric properties of Sclerotinia sclerotiorum airborne inoculum, and it aims to contribute to the development of forecasting systems based on dielectrophoretic filters.

Pulsed Electric Field Treatments with Nonlethal Field Strength Alter the Properties of Bacterial Spores

Pulsed electric field (PEF) treatment, an alternative to thermal processing in the food industry, is insufficient to inactivate bacterial spores. Although spores that have been treated in this manner remain alive, specific understanding of their physiological properties is limited. The purpose of this study is to describe the morphology, viability, and germination behavior of Bacillus atrophaeus spores treated with PEF. Our findings indicate that nonlethal PEF treatment results in spore deformation, dipicolinic acid (DPA) leakage, and a shorter and more uniform germination lag time (\(\def\upalpha{\unicode[Times]{x3B1}}\)\(\def\upbeta{\unicode[Times]{x3B2}}\)\(\def\upgamma{\unicode[Times]{x3B3}}\)\(\def\updelta{\unicode[Times]{x3B4}}\)\(\def\upvarepsilon{\unicode[Times]{x3B5}}\)\(\def\upzeta{\unicode[Times]{x3B6}}\)\(\def\upeta{\unicode[Times]{x3B7}}\)\(\def\uptheta{\unicode[Times]{x3B8}}\)\(\def\upiota{\unicode[Times]{x3B9}}\)\(\def\upkappa{\unicode[Times]{x3BA}}\)\(\def\uplambda{\unicode[Times]{x3BB}}\)\(\def\upmu{\unicode[Times]{x3BC}}\)\(\def\upnu{\unicode[Times]{x3BD}}\)\(\def\upxi{\unicode[Times]{x3BE}}\)\(\def\upomicron{\unicode[Times]{x3BF}}\)\(\def\uppi{\unicode[Times]{x3C0}}\)\(\def\uprho{\unicode[Times]{x3C1}}\)\(\def\upsigma{\unicode[Times]{x3C3}}\)\(\def\uptau{\unicode[Times]{x3C4}}\)\(\def\upupsilon{\unicode[Times]{x3C5}}\)\(\def\upphi{\unicode[Times]{x3C6}}\)\(\def\upchi{\unicode[Times]{x3C7}}\)\(\def\uppsy{\unicode[Times]{x3C8}}\)\(\def\upomega{\unicode[Times]{x3C9}}\)\(\def\bialpha{\boldsymbol{\alpha}}\)\(\def\bibeta{\boldsymbol{\beta}}\)\(\def\bigamma{\boldsymbol{\gamma}}\)\(\def\bidelta{\boldsymbol{\delta}}\)\(\def\bivarepsilon{\boldsymbol{\varepsilon}}\)\(\def\bizeta{\boldsymbol{\zeta}}\)\(\def\bieta{\boldsymbol{\eta}}\)\(\def\bitheta{\boldsymbol{\theta}}\)\(\def\biiota{\\boldsymbol{\iota}}\)\(\def\bikappa{\boldsymbol{\kappa}}\)\(\def\bilambda{\boldsymbol{\lambda}}\)\(\def\\bimu{\boldsymbol{\mu}}\)\(\def\binu{\boldsymbol{\nu}}\)\(\def\bixi{\boldsymbol{\xi}}\)\(\def\biomicron{\boldsymbol{\micron}}\)\(\def\bipi{\boldsymbol{\pi}}\)\(\def\birho{\boldsymbol{\rho}}\)\(\def\bisigma{\boldsymbol{\sigma}}\)\(\def\bitau{\boldsymbol{\\tau}}\)\(\def\biupsilon{\boldsymbol{\upsilon}}\)\(\def\biphi{\boldsymbol{\phi}}\)\(\def\bichi{\boldsymbol{\chi}}\)\(\def\bipsy{\boldsymbol{\psy}}\)\(\def\biomega{\boldsymbol{\omega}}\)\(\def\bupalpha{\bf{\alpha}}\)\(\def\bupbeta{\bf{\beta}}\)\(\def\bupgamma{\bf{\gamma}}\)\(\def\bupdelta{\bf{\delta}}\)\(\def\bupvarepsilon{\bf{\varepsilon}}\)\(\def\bupzeta{\bf{\zeta}}\)\(\def\bupeta{\bf{\eta}}\)\(\def\buptheta{\bf{\theta}}\)\(\def\bupiota{\bf{\iota}}\)\(\def\bupkappa{\bf{\kappa}}\)\(\def\\buplambda{\bf{\lambda}}\)\(\def\bupmu{\bf{\mu}}\)\(\def\bupnu{\bf{\nu}}\)\(\def\bupxi{\bf{\xi}}\)\(\def\bupomicron{\bf{\micron}}\)\(\def\buppi{\bf{\pi}}\)\(\def\buprho{\bf{\rho}}\)\(\def\bupsigma{\bf{\sigma}}\)\(\def\buptau{\bf{\tau}}\)\(\def\bupupsilon{\bf{\upsilon}}\)\(\def\bupphi{\bf{\phi}}\)\(\def\bupchi{\bf{\chi}}\)\(\def\buppsy{\bf{\psy}}\)\(\def\bupomega{\bf{\omega}}\)\(\def\bGamma{\bf{\Gamma}}\)\(\def\bDelta{\bf{\Delta}}\)\(\def\bTheta{\bf{\Theta}}\)\(\def\bLambda{\bf{\Lambda}}\)\(\def\bXi{\bf{\Xi}}\)\(\def\bPi{\bf{\Pi}}\)\(\def\bSigma{\bf{\Sigma}}\)\(\def\bPhi{\bf{\Phi}}\)\(\def\bPsi{\bf{\Psi}}\)\(\def\bOmega{\bf{\Omega}}\)\({T_{{\rm{lag}}}}\)), but that there is no change in release duration (\(\Delta {T_{{\rm{release}}}}\)), germination ratio, or viability. Based on our findings, we conclude that an intact morphologic state and DPA content are not prerequisites for germination and full viability and that, in contrast to nutrient-induced germination in which initially slowly released DPA triggers subsequent germination events, leaked DPA during PEF treatment does not. Spores that have been subjected to this procedure remain dormant and preserve their full germinability. We found that PEF-treated spores respond to germinants more quickly and with less heterogeneity, possibly because the tiny cracks formed on the spore surface facilitate the germinants' access to the germination receptors situated on the spore's inner membrane. The consensus view that nonlethal PEF has less impact on spores that are still capable of forming CFUs under proper conditions is one-sided. This research advances our understanding of how spores behave following nonlethal PEF treatment and gives information on the topics of nosocomial sterilization, food safety, and public health.

Reactive oxygen species generated by infrared laser light in optical tweezers inhibits the germination of bacterial spores.

Bacterial spores are highly resistant to heat, radiation and various disinfection chemicals. The impact of these on the biophysical and physicochemical properties of spores can be studied on the single-cell level using optical tweezers. However, the effect of the trapping laser on spores' germination rate is not fully understood. In this work, we assess the impact of 1064 nm laser light on the germination of Bacillus thuringiensis spores. The results show that the germination rate of spores after laser exposure follows a sigmoid dose-response relationship, with only 15% of spores germinating after 20 J of laser light. Under anaerobic growth conditions, the percentage of germinating spores at 20 J increased to 65%. The results thereby indicate that molecular oxygen is a major contributor to the germination-inhibiting effect observed. Thus, our study highlights the risk for optical trapping of spores and ways to mitigate it.

A Sporulation-Specific sRNA Bvs196 Contributing to the Developing Spore in Bacillus velezensis.

Many putative sRNAs have been characterized using bioinformatic analysis and high-throughput sequencing in Gram-positive Bacillus strains, but there are only a few functional studies on the sRNAs involved in the spore formation developmental process. In particular, there is no sRNA confirmed experimentally to regulate the late stages of sporulation. Bvs196 is an sRNA with a length of 294 nucleotides that is abundantly expressed in the stationary phase of several media and independently transcribed in Bacillus velezensis strain PEBA20, as validated by RNA-seq and Northern blot,. It is also confirmed, by qRT-PCR, that Bvs196 is transcribed abundantly throughout the intermediate and late stages of sporulation. Using the gfpmut3a gene transcriptional reporter demonstrates that Bvs196 is expressed specifically in the forespore during sporulation and controlled by σF and σG (mainly by σG). This was observed by fluorescence microscopy and multi-function microplate reader. Further evolutionary conservation analysis found that Bvs196 is widely present in Bacillus with a strongly conserved and stable secondary structure. Resistance phenotypic assays of spores formed from the Bvs196 deletion mutant, the overexpressed Bvs196 mutant, and the wild-type strain revealed that the absence of Bvs196 led to reduced heat and UV resistance and enhanced formaldehyde resistance. We determined, by MST analysis, that Bvs196 can directly interact with spo0A and sspN-tlp mRNAs in vitro, and that short incomplete complementary paired bases affect the binding affinity of Bvs196 to target mRNAs. Our results suggest that Bvs196 is a novel sporulation-specific sRNA of B. velezensis, 294 nt in length, independently transcribed under the control of σF and σG in the forespore during sporulation, and that it affects spore resistance, and is able to directly interact with spo0A and sspN-tlp mRNAs. The remarkable conservation and impressive expression level of Bvs196 imply that it acts as an important conservative regulator, presumably by interacting with many other unknown targets in the forespore, and therefore contributing to spore properties. This work provides new clues for further understanding of the spore formation regulatory network.

Structure of deposits formed by drying of droplets contaminated with Bacillus spores determines their resistance to rinsing and cleaning

The formation of deposits by evaporation of droplets contaminated by Bacillus spores was investigated, focusing on the role of spore and material properties. Droplets containing hydrophilic to hydrophobic spores were deposited on materials (stainless steels, polypropylene, glass). The presence of spores within the droplets, as well as material hydrophobicity plays a major role in the kinetics of the droplet shape during drying while the pattern of the dried deposit was affected by both material and spore properties. The resistance to detachment of the different deposits was then investigated. After a single rinsing procedure, a very high resistance to detachment of adherent spores was observed (up to 90% of residual spores). The hydrophilic Bs PY79 spores were the least resistant to removal, while the differences between materials were not pronounced. The ease of cleaning of the dried deposits was much greater. The least resistant spores were still the Bs PY79 ones, while the detachment was 3–100 times more effective on glass than on other materials. These results highlight the predominant role of hydrophilic/hydrophobic properties of particles and materials on the structure of deposits and their further resistance to rinsing and cleaning procedures.

Mechanisms and Applications of Bacterial Sporulation and Germination in the Intestine.

Recent studies have suggested a major role for endospore forming bacteria within the gut microbiota, not only as pathogens but also as commensal and beneficial members contributing to gut homeostasis. In this review the sporulation processes, spore properties, and germination processes will be explained within the scope of the human gut. Within the gut, spore-forming bacteria are known to interact with the host’s immune system, both in vegetative cell and spore form. Together with the resistant nature of the spore, these characteristics offer potential for spores’ use as delivery vehicles for therapeutics. In the last part of the review, the therapeutic potential of spores as probiotics, vaccine vehicles, and drug delivery systems will be discussed.

Nε-Lysine Acetylation of the Histone-Like Protein HBsu Regulates the Process of Sporulation and Affects the Resistance Properties of Bacillus subtilis Spores.

Bacillus subtilis produces dormant, highly resistant endospores in response to extreme environmental stresses or starvation. These spores are capable of persisting in harsh environments for many years, even decades, without essential nutrients. Part of the reason that these spores can survive such extreme conditions is because their chromosomal DNA is well protected from environmental insults. The α/β-type small acid-soluble proteins (SASPs) coat the spore chromosome, which leads to condensation and protection from such insults. The histone-like protein HBsu has been implicated in the packaging of the spore chromosome and is believed to be important in modulating SASP-mediated alterations to the DNA, including supercoiling and stiffness. Previously, we demonstrated that HBsu is acetylated at seven lysine residues, and one physiological function of acetylation is to regulate chromosomal compaction. Here, we investigate if the process of sporulation or the resistance properties of mature spores are influenced by the acetylation state of HBsu. Using our collection of point mutations that mimic the acetylated and unacetylated forms of HBsu, we first determined if acetylation affects the process of sporulation, by determining the overall sporulation frequencies. We found that specific mutations led to decreases in sporulation frequency, suggesting that acetylation of HBsu at some sites, but not all, is required to regulate the process of sporulation. Next, we determined if the spores produced from the mutant strains were more susceptible to heat, ultraviolet (UV) radiation and formaldehyde exposure. We again found that altering acetylation at specific sites led to less resistance to these stresses, suggesting that proper HBsu acetylation is important for chromosomal packaging and protection in the mature spore. Interestingly, the specific acetylation patterns were different for the sporulation process and resistance properties of spores, which is consistent with the notion that a histone-like code exists in bacteria. We propose that specific acetylation patterns of HBsu are required to ensure proper chromosomal arrangement, packaging, and protection during the process of sporulation.

Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I–III. We propose two distinct sporulation strategies used by C. botulinum Groups I–III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

Visualization and characterization of spore morphogenesis in Paenibacillus polymyxa ATCC39564.

Paenibacillus polymyxa is a spore-forming Gram-positive bacterial species. Both its sporulation process and the spore properties are poorly understood. Here, we investigated sporulation in P. polymyxa ATCC39564. When cultured at 37℃ for 24 h in sporulation medium, more than 80% of the total cells in the culture were spores. Time-lapse imaging revealed that cellular morphological changes during sporulation of P. polymyxa were highly similar to those of B. subtilis. We demonstrated that genetic deletion of spo0A, sigE, sigF, sigG, or sigK, which are highly conserved transcriptional regulators in spore forming bacteria, abolished spore formation. In P. polymyxa, spo0A was required for cell growth in sporulation medium, as well as for the initiation of sporulation. The sigE and sigF mutants formed abnormal multiple asymmetric septa during the early stage of sporulation. The sigG and sigK mutants formed forespores in the sporangium, but they did not become mature. Moreover, fluorescence reporter analysis confirmed compartment-specific gene expression of spoIID and spoVFA in the mother cell and spoIIQ and sspF in the forespore. Transmission electron microscopy imaging revealed that P. polymyxa produces multilayered endospores but lacking a balloon-shaped exosporium. Our results indicate that spore morphogenesis is conserved between P. polymyxa and B. subtilis. However, P. polymyxa genomes lack many homologues encoding spore-coat proteins that are found in B. subtills, suggesting that there are differences in the spore coat composition and surface structure between P. polymyxa and B. subtilis.

A new role for SR1 from Bacillus subtilis: regulation of sporulation by inhibition of kinA translation.

SR1 is a dual-function sRNA from Bacillus subtilis. It inhibits translation initiation of ahrC mRNA encoding the transcription activator of the arginine catabolic operons. Base-pairing is promoted by the RNA chaperone CsrA, which induces a slight structural change in the ahrC mRNA to facilitate SR1 binding. Additionally, SR1 encodes the small protein SR1P that interacts with glyceraldehyde-3P dehydrogenase A to promote binding to RNase J1 and enhancing J1 activity. Here, we describe a new target of SR1, kinA mRNA encoding the major histidine kinase of the sporulation phosphorelay. SR1 and kinA mRNA share 7 complementary regions. Base-pairing between SR1 and kinA mRNA decreases kinA translation without affecting kinA mRNA stability and represses transcription of the KinA/Spo0A downstream targets spoIIE, spoIIGA and cotA. The initial interaction between SR1 and kinA mRNA occurs 10 nt downstream of the kinA start codon and is decisive for inhibition. The sr1 encoded peptide SR1P is dispensable for kinA regulation. Deletion of sr1 accelerates sporulation resulting in low quality spores with reduced stress resistance and altered coat protein composition which can be compensated by sr1 overexpression. Neither CsrA nor Hfq influence sporulation or spore properties.