Project description:Bacillus weihenstephanensis is a subspecies of the Bacillus cereus sensu lato group of spore forming bacteria known to cause food spoilage or food poisoning. The key distinguishing phenotype of B. weihenstephanensis is its ability to grow below 7°C or, from a food safety perspective, to grow and potentially produce toxins in a refrigerated environment. In order to gain insight into to the mechanistic basis of its psychrotolerant phenotype, as well as elucidate relevant aspects of its toxigenic profile, the proteome profiles of cells grown at either 6°C or 30°C were compared.
Project description:BackgroundIn Bacillus mycoides, as well as in other members of the B. cereus group, the tubulin-like protein of the division septum FtsZ is encoded by the distal gene of the cluster division and cell wall (dcw). Along the cluster the genes coding for structural proteins of the division apparatus are intermingled with those coding for enzymes of peptidoglycan biosynthesis, raising the possibility that genes with this different function might be coexpressed. Transcription of ftsZ in two model bacteria had been reported to differ: in B. subtilis, the ftsZ gene was found transcribed as a bigenic mRNA in the AZ operon; in E. coli, the transcripts of ftsZ were monogenic, expressed by specific promoters. Here we analyzed the size and the initiation sites of RNAs transcribed from ftsZ and from other cluster genes in two B. mycoides strains, DX and SIN, characterized by colonies of different chirality and density, to explore the correlation of the different morphotypes with transcription of the dcw genes.ResultsIn both strains, during vegetative growth, the ftsZ-specific RNAs were composed mainly of ftsZ, ftsA-ftsZ and ftsQ-ftsA-ftsZ transcripts. A low number of RNA molecules included the sequences of the upstream murG and murB genes, which are involved in peptidoglycan synthesis. No cotranscription was detected between ftsZ and the downstream genes of the SpoIIG cluster. The monogenic ftsZ RNA was found in both strains, with the main initiation site located inside the ftsA coding sequence. To confirm the promoter property of the site, a B. mycoides construct carrying the ftsA region in front of the shortened ftsZ gene was inserted into the AmyE locus of B. subtilis 168. The promoter site in the ftsA region was recognized in the heterologous cellular context and expressed as in B. mycoides.ConclusionsThe DX and SIN strains of B. mycoides display very similar RNA transcription specificity. The ftsZ messenger RNA can be found either as an independent transcript or expressed together with ftsA and ftsQ and, in low amounts, with genes that are specific to peptidoglycan biosynthesis.
Project description:Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.
Project description:BACKGROUND:Bacillus mycoides Flügge, a Gram-positive, non-motile soil bacterium assigned to Bacillus cereus group, grows on agar as chains of cells linked end to end, forming radial filaments curving clock- or counter-clockwise (SIN or DX morphotypes). The molecular mechanism causing asymmetric curving is not known: our working hypothesis considers regulation of filamentous growth as the prerequisite for these morphotypes. RESULTS:SIN and DX strains isolated from the environment were classified as B. mycoides by biochemical and molecular biology tests. Growth on agar of different hardness and nutrient concentration did not abolish colony patterns, nor was conversion between SIN and DX morphotypes ever noticed. A number of morphotype mutants, all originating from one SIN strain, were obtained. Some lost turn direction becoming fluffy, others became round and compact. All mutants lost wild type tight aggregation in liquid culture. Growth on agar was followed by microscopy, exploring the process of colony formation and details of cell divisions. A region of the dcw (division cell wall) cluster, including ftsQ, ftsA, ftsZ and murC, was sequenced in DX and SIN strains as a basis for studying cell division. This confirmed the relatedness of DX and SIN strains to the B. cereus group. CONCLUSIONS:DX and SIN asymmetric morphotypes stem from a close but not identical genomic context. Asymmetry is established early during growth on agar. Wild type bacilli construct mostly uninterrupted filaments with cells dividing at the free ends: they "walk" longer distances compared to mutants, where enhanced frequency of cell separation produces new growing edges resulting in round compact colonies.
Project description:The Bacillus cereus group comprises genetical closely related species with variable toxigenic characteristics. However, detection and differentiation of the B. cereus group species in routine diagnostics can be difficult, expensive and laborious since current species designation is linked to specific phenotypic characteristic or the presence of species-specific genes. Especially the differentiation of Bacillus cereus and Bacillus thuringiensis, the identification of psychrotolerant Bacillus mycoides and Bacillus weihenstephanensis, as well as the identification of emetic B. cereus and Bacillus cytotoxicus, which are both producing highly potent toxins, is of high importance in food microbiology. Thus, we investigated the use of a machine learning approach, based on artificial neural network (ANN) assisted Fourier transform infrared (FTIR) spectroscopy, for discrimination of B. cereus group members. The deep learning tool box of Matlab was employed to construct a one-level ANN, allowing the discrimination of the aforementioned B. cereus group members. This model resulted in 100% correct identification for the training set and 99.5% correct identification overall. The established ANN was applied to investigate the composition of B. cereus group members in soil, as a natural habitat of B. cereus, and in food samples originating from foodborne outbreaks. These analyses revealed a high complexity of B. cereus group populations, not only in soil samples but also in the samples from the foodborne outbreaks, highlighting the importance of taking multiple isolates from samples implicated in food poisonings. Notable, in contrast to the soil samples, no bacteria belonging to the psychrotolerant B. cereus group members were detected in the food samples linked to foodborne outbreaks, while the overall abundancy of B. thuringiensis did not significantly differ between the sample categories. None of the isolates was classified as B. cytotoxicus, fostering the hypothesis that the latter species is linked to very specific ecological niches. Overall, our work shows that machine learning assisted (FTIR) spectroscopy is suitable for identification of B. cereus group members in routine diagnostics and outbreak investigations. In addition, it is a promising tool to explore the natural habitats of B. cereus group, such as soil.