Project description:Spores of Bacillales and Clostridiales species contain 100s of different mRNAs, and their major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. In new work, RNA was isolated from spores of five Bacillales and one Clostridioides species and relative spore mRNA levels were determined by RNA-seq. Determination of RNA levels in single spores allowed calculation of RNA nt/spore, and assuming mRNA is 3% of spore RNA allowed calculation that only ~6% of spore mRNAs were present at ≥ 1/spore. Bacillus subtilis, Bacillus atrophaeus and Clostridioides difficile spores had 49, 42 and 51 mRNAs at >1/spore, respectively. Numbers of mRNAs at ≥1/spore were ~10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores, respectively, and ~ 4-fold higher in Bacillus megaterium spores. Notably, in all species: i) many of the 60 most abundant spore mRNAs were transcribed by RNA polymerase with forespore-specific s factors; ii) some to many of the most abundant spore mRNAs encoded orthologs of those encoded by abundant B. subtilis spore mRNAs and proteins present in dormant spores ; and iii) some spore mRNAs were likely transcribed in the mother cell compartment of the sporulating cell. Indeed , analysis of the coverage of RNA-seq reads on mRNAs from all six species suggested that abundant spore mRNAs were at least somewhat fragmented. This observation was confirmed by RT-qPCR analysis of three abundant mRNAs each from B. subtilis and C. difficile spores. These data add to a growing body of evidence indicating that the great majority of mRNAs in spores of Firmicutes are degradation and function as a ribonucleotide depot for new RNA synthesis when spores germinate.
Project description:To study the signals and pathways underlying spore germination we examined the global changes in gene expression during this process. We find that the germination process can be divided into two distinct stages. During the first stage, the induced spores respond only to glucose. The transcription program during this stage recapitulates the general transcription response of yeast cells to glucose. Only during the second phase are the cells able to sense and respond to other nutritional components in the environment. Components of the mitotic machinery are involved in spore germination but in a distinct pattern. In contrast to the mitotic cell cycle, growth related events during germination are not coordinated with nuclear events and are separately regulated. Genome-wide expression profiling enables us to follow the progression of spore germination, thus dividing this process into two major stages and to identify germination-specific regulation of components of the mitotic cell cycle machinery. Keywords: Time course
Project description:Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared in a liquid medium. It has also been shown that the more mature a spore is the higher is its heat resistance contributed most likely by the progressive cross-linking of coat proteins. The current study for the first time enlightens the effect of sporulation medium on spore proteins. The 14N spores prepared on solid agar medium and 15N metabolically labelled spores prepared in liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The 14N:15N isotopic ratio of the 1:1 mixture of liquid and solid medium cultured spores exposes that most of the identified inner coat and crust proteins are significantly upregulated while most of the outer coat proteins are significantly downregulated for the spores prepared on solid medium relative to the spores prepared in the liquid medium. Medium specific differences in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that also the coat protein cross-linking may be medium specific. The spores prepared on solid medium show a higher thermal resistance than the spores prepared in liquid medium. Since the core DPA content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and assembly. Supporting the proteomic analyses, the electron microscopy analyses show a significantly thinner outer coat layer of the spores cultures on the solid medium.
Project description:In this project moderate high pressure superdormant spores were isolated after a high pressure treatement at 150 MPa, 37°C for 4 min. Superdormant spores were defined as those who did not germinate after the pressure treatment. The proteins present in the superdormant spores were quantitatively compared to the initial dormant spore population. The aim of this project was to identify proteins which are potentially involved in moderate high pressure superdormancy.
Project description:Using RNA-seq, we cataloged messenger RNAs in highly purified dormant Bacillus subtilis spores prepared either on plates or in liquid. Almost all of the most abundant spore mRNAs are encoded by genes expressed only in the developing spore late in sporulation under control of the forespore-specific RNA polymerase sigma factor, sG. Given the levels of the ~40 most abundant mRNAs in dormant spores, we calculated the great majority of the low abundance mRNAs can be present in only a small fraction of the spore population.
Project description:Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30 % of the B. subtilis genome. Keywords: time course, spore outgrowth
Project description:Heat-treated spores show delayed and slower germination and outgrowth compared to untreated spores presumably due to spore damage repair. This study was performed to identify genes possibly involved in spore damage repair in B. cereus. In this study we compared the transcriptomic profiles of untreated and heat-treated spores during germination and outgrowth in BHI at 30C.
Project description:Fungal spores, abundant in the environment, are a major cause of asthma. But the precise host response that triggers fungal allergic airway inflammation remains unclear. We found that CD11c+ DCs and CD4+ T cells are essential for development of airway inflammation in mice when repeatedly exposed to inhaled spores. To delinate which DC subsets are mediating fungal allergic inflammation we undertook single cell RNAseq of DCs isolated from the lungs of mice exposed to fungal spores. This identified precise subsets altered upon spore exposure and following targeted removal identified distinct DC subsets (Mgl2+ cDC2s) that are essential for fungal allergic airway inflammation.
Project description:Bacterial spores play an important role in disease initiation, transmission and persistence. The outermost spore layer, the exosporium, is important as it is the first point of contact between the spore and the environment and may be involved in spore adherence, protection and germination. Clostridium sordellii is a highly lethal, spore forming pathogen that causes soft-tissue infections, enteritis and toxic-shock syndrome. Despite the importance of C. sordellii spores in disease, spore proteins from this bacterium have not been defined or interrogated functionally. In this study, we identified the C. sordellii outer spore proteome and two of the identified proteins, CSA and CSB, were characterised using a genetic and phenotypic approach. Both proteins were essential for the correct formation and positioning of the C. sordellii spore coat and exosporium. The absence of CSA reduced sporulation levels and increased spore sensitivity to heat, sodium hydroxide and hydrochloric acid. By comparison, CSB was required for normal levels of spore adherence to cervical, but not vaginal, cells, with csb mutant spores having increased adherence properties. The establishment of a mouse infection model of the gastrointestinal tract for C. sordellii allowed the role of CSA and CSB to be interrogated in an infected host. Following the oral administration of spores to mice, the wild-type strain efficiently colonized the gastrointestinal tract, with the peak of bacterial numbers occurring at one day post-infection. Colonization was reduced by two logs at four days post-infection. By comparison, mice infected with the csb mutant did not show a reduction in bacterial numbers. The absence of CSB therefore allows the csb mutant to persist within the gastrointestinal tract. We conclude that C. sordellii outer spore proteins are important for the structural and functional integrity of spores, and for colonization and persistence during infection. Finally, Clostridium difficile, Bacillus cereus and Bacillus anthracis encode proteins with homology to CSA and CSB but these bacteria produce spores that are structurally dissimilar to those of C. sordellii, and the function of the proteins in these hosts is different to that in C. sordellii. These findings suggest that, despite their homology, spore proteins can have variable functions in different bacterial species which highlights the necessity of studying each spore protein in the cognate species from which it originates.