Analysis of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores.
ABSTRACT: Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of B. cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth was assessed at pH 5.5 without and with 0.75, 1.5 and 3.0mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from delays in germination and outgrowth to complete blockage of germination at 3.0mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multi-drug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed. Per concentration of undissociated sorbic acid (0, 0.75, and 1.5mM) four exposure times (10, 30, 60, and 120 minutes) were each compared with dormant spores (i.e., t0). The experiments were performed in duplicate and the duplicate samples were hybridized with a dye-swap
Project description:Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of B. cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth was assessed at pH 5.5 without and with 0.75, 1.5 and 3.0mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from delays in germination and outgrowth to complete blockage of germination at 3.0mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multi-drug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed. Overall design: Per concentration of undissociated sorbic acid (0, 0.75, and 1.5mM) four exposure times (10, 30, 60, and 120 minutes) were each compared with dormant spores (i.e., t0). The experiments were performed in duplicate and the duplicate samples were hybridized with a dye-swap
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 Overall design: Time series of outgrowing B subtilis spores (cy-3) with common reference (RNA vegetative cells, TSB OD 0.4, Cy-5). Quality of the spotted oligoarray allows single dye dataanalysis.
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. Overall design: Matrix is compatible with the Agilent-017343 array design. Elermeiers with BHI were inoculated with untreated or heat-treated (1 min at 95C) B. cereus spores (final volume 20ml, final spore concentration 10^8 sp/ml) and incubated with aeration (200rpm) at 30C untill harvesting. Elermeiers were harvested for untreated spores 10, 20, 30 and 50 min and for heat-treated 50, 90, 120 and 150 minutes after adding BHI. Samples from experimental duplicates were hybridized on the arrays with at least 3 technical replicates for each experimantal replicate.
Project description:Specificity of interaction between a microRNA (miRNA) and its targets crucially depends on the seed region located in its 5’-end. It is often implicitly considered that two miRNAs sharing the same biological activity should display similarity beyond the strict six nucleotide region that forms the seed, in order to form specific complexes with the same mRNA targets. We have found that expression of hsa-miR-147b and hsa-miR-210, though triggered by different stimuli (i.e. lipopolysaccharides and hypoxia, respectively), induce very similar cellular effects in term of proliferation, migration and apoptosis. Hsa-miR-147b only shares a “minimal” 6-nucleotides seed sequence with hsa-miR-210, but is identical with hsa-miR-147a over 20 nucleotides, except for one base located in the seed region. Phenotypic changes induced after heterologous expression of miR-147a strikingly differ from those induced by miR-147b or miR-210. In particular, miR-147a behaves as a potent inhibitor of cell proliferation and migration. These data fit well with the gene expression profiles observed for miR-147b and miR-210, which are very similar, and the gene expression profile of miR-147a, which is distinct from the two others. Bioinformatics analysis of all human miRNA sequences indicates multiple cases of miRNAs from distinct families exhibiting the same kind of similarity that would need to be further characterized in terms of putative functional redundancy. Besides, it implies that functional impact of some miRNAs can be masked by robust expression of miRNAs belonging to distinct families. To compare the set of transcripts targeted by hsa-miR-147a, hsa-miR-147b and hsa-miR-210, we overexpressed these miRNAs in human lung adenocarcinoma A549 cells by transfecting them with synthetic pre-miRNAs or a synthetic “negative” pre-miRNA as a control (miR-Neg). RNA samples were harvested at 48 hours post-transfection and 3 independent experiments were carried out. 48 hours post-transfection, 3 independent experiments were performed in dye-swap: hsa-miR-147a versus miR-Neg; hsa-miR-147b versus miR-Neg; hsa-miR-210 versus miR-Neg.
Project description:A comparative transcriptome approach was used to assess genes involved in metabolism and pathogenesis that are specifically activated during anaerobic growth of the spore-forming food-borne human pathogen Bacillus cereus ATCC 14579. Growth under anaerobic conditions in Brain Heart Infusion broth revealed a reduced growth rate and a lower yield as compared to that under aerobic conditions. Comparative transcriptome analysis of cells harvested at early- and mid-exponential growth phase, transition phase and stationary phase, subsequently showed hundreds of genes to be induced under anaerobic condition. These included novel genes identified for anaerobic growth of B. cereus, encoding metabolic pathways, such as the arginine deiminase pathway (ArcABDC), a formate dehydrogenase (FdhF) and a pyruvate fomate lyase (Pfl), and alternative respiratory proteins, such as arsenate reductases. Furthermore, the nitrosative stress response was induced in the anaerobic transition phase of growth, conceivably due to the production of nitric oxide as a by-product of nitrite and nitrate respiration. Notably, both hemolytic enzyme and enterotoxin encoding genes were activated in different oxygen limiting conditions, i.e. hemolytic enzyme encoding genes were induced during anaerobic growth, whereas enterotoxin encoding genes were induced in the transition and stationary phase of aerobic cultures reaching a high cell density. These data point to metabolic rearrangements, stress adaptation and activation of the virulent status of B. cereus under anaerobic conditions, such as encountered in the human GI-tract. B. cereus ATCC 14579 was grown in BHI in 50 ml. Aerobic in a Erlenmeyer flask, shaking at 200 rpm. Anaerobic in a closed flask, flushed with Nitrogen-gas for 30 min, also shaking at 200 rpm. Transcriptome analyses Phase compared to mid-exponential phase Anaerobic (OD600) 0.2 compared to 0.4 Early-exponential 1.0 compared to 0.4 Transition 1.1 compared to 0.4 Stationary Aerobic (OD600) 0.2 compared to 0.8 Early-exponential 4.0 compared to 0.8 Transition 8.0 compared to 0.8 Stationary Aerobic to anaerobic (OD600) Anaerobic 0.6 to aerobic 0.6
Project description:Spermatophyte pollen tubes and root hairs have been used as single-cell-type model systems toward understanding the molecular processes underlying polar growth of plant cells. Horsetail (Equisetum arvense L.) is a perennial herb species in Equisetopsida, which creates separately growing spring and summer stems in its life cycle. The mature chlorophyllous spores produced from spring stems can germinate without dormancy. Here we report the cellular features and protein expression patterns in five stages of horsetail spore germination (mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Using 2-DE combined with mass spectrometry, 80 proteins were found to be differentially expressed upon spore germination. Among them, proteins involved in photosynthesis, protein turnover, and energy supply were over-represented. Thirteen proteins appeared as isoforms on the gels, indicating the potential importance of post-translational modification. In addition, the dynamic changes of ascorbate peroxidase, peroxiredoxin, and dehydroascorbate reductase implied that reactive oxygen species homeostasis is critical in regulating cell division and tip-growth. The time course of germination and diverse expression patterns of proteins in photosynthesis, energy supply, lipid and amino acid metabolism indicated that heterotrophic and autotrophic metabolism were necessary in light-dependent germination of the spores. Twenty-six proteins were involved in protein synthesis, folding, and degradation, indicating that protein turnover is vital to spore germination and rhizoid tip-growth. Furthermore, the altered abundance of small G protein Ran1, 14-3-3 protein, actin, and Caffeoyl-CoA O-methyltransferase revealed that signaling transduction, vesicle trafficking, cytoskeleton dynamics, and cell wall modulation were critical to cell division and polar growth. These findings provide up-to-date evidences for understanding fern spore asymmetric division and rhizoid polar growth.
Project description:Clostridium difficile is the leading cause of hospital acquired diarrhoea in industrialised countries. Under conditions that are not favourable for growth, the pathogen produces metabolically dormant endospores via asymmetric cell division. These are extremely resistant to both chemical and physical insult and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants. Spores are the primary infective agent and must germinate to allow for vegetative cell growth and toxin production. While spore germination in Bacillus is well understood, little is known about C. difficile germination and outgrowth. Here we use genome-wide transcriptional analysis to elucidate the temporal gene expression patterns in C. difficile 630 endospore germination. We have optimized methods for large scale production and purification of spores. The germination characteristics of purified spores have been characterized and RNA extraction protocols have been optimized. Gene expression was highly dynamic during germination and outgrowth, and was found to involve a large number of genes. Using this genome-wide, microarray approach we have identified 511 genes that are significantly up- or down-regulated during C. difficile germination (p less than or = 0.01). A number of functional groups of genes appeared to be co-regulated. These included transport, protein synthesis and secretion, motility and chemotaxis as well as cell wall biogenesis. These data give insight into how C. difficile re-establishes its metabolism, re-builds the basic structures of the vegetative cell and resumes growth. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-145]
Project description:Bacterial endospores, the transmissible forms of pathogenic bacilli and clostridia, are heterogeneous multilayered structures composed of proteins. These proteins protect the spores against variety of stresses, thus helping spore survival, and assist in germination, by interacting with the environment to form vegetative cells. Owing to the complexity, insolubility, and dynamic nature of spore proteins, it has been difficult to obtain their comprehensive protein profiles. The intact spores of Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile and their vegetative counterparts were disrupted by bead-beating in 6M urea under reductive conditions. The heterogeneous mixture was then double-digested with LysC and trypsin. Next, the peptide mixture was pre-fractionated with Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) followed by reverse phase LC-FT-MS analysis of the fractions. ‘One-pot’ method is a simple, robust method that yields identification of >1000 proteins with high confidence, across all spore layers from Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile. This method can be employed for proteome-wide analysis of non-spore-forming as well as spore-forming pathogens. Analysis of spore protein profile will help to understand the sporulation and germination processes and to distinguish immunogenic protein markers.
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 Overall design: Wild type SK1 cells (DS1) were grown to saturation in YPD (2% yeast extract, 4% bactopeptone, 4% glucose) at 30 C for 24 hours, washed in sterile water and plated on sporulation plates (0.25% yeast extract, 1.5% Potassium acetate, 0.05% glucose supplemented with all amino acids). 3 days old asci were harvested and suspended in YPD at 30 C to initiate spore germination. RNA for microarray hybridization was extracted at high temporal resolution (15-minutes intervals for 7.5 hours, total of 31 samples)