Project description:Hypersaline regions are terrestrial analogs of the Earth's primitive ecosystem and extraterrestrial environment. The salt range in Pakistan is considered among a few of the ancient salt deposits in the subcontinent. Karak salt mine is situated at the Northwest end in Pakistan. Despite the fact that halophiles initiated the formation of terrestrial ecosystems, their products and identities remain hidden. Some preliminary studies limited to culture-dependent isolations have been reported. Characterizing the microbiome that spans over centuries of ecosystem development is crucial, given their role in shaping landscape succession and biogeochemical cycles. Here, we used metagenomics techniques to explore the microbial diversity of the Karak salt mine. We used 16S rRNA Illumina amplicon sequencing to characterize the halophilic communities entrapped in Karak mine. The results were interpreted using Illumina Basespace, QIIME, and Cytoscape. Cultures were isolated at 16-25% salinity. Metagenomics data was consistent with our preliminary culturing data, indicating remarkable species to strain-level diversity of unique halophiles. A total of 107,099 (brine) and 122,679 (salt) reads were obtained. 16S rRNA based sequencing revealed a microbiome with bacteria (66% brine and 72% salt) dominated by Bacteroidetes and Proteobacteria with a strikingly high abundance of Archaea (18% brine and 13% salt). Alpha diversity has higher values in salt than in the brine. The study of the halophiles in the Karak salt mine provides clues for species contributing to the maintenance of biogeochemical cycles of the ecosystem. This is the first report of a metagenomic study of any hypersaline region of Pakistan.
Project description:In many habitats, microorganisms are exposed to high or fluctuating salinities and evolved specific acclimation strategies to thrive in those environments. Euryhaline microorganisms can grow in a broad range of salinities, from low ionic strength like freshwater up to twofold seawater salinities. Here, we analyzed the salt acclimation process of the euryhaline model cyanobacterium Synechocystis sp. PCC 6803 in a multi-omics approach by combining transcriptomic, proteomic and metabolomic analyses. The Overall, the comparison of salt-induced proteome and transcriptome changes revealed a good correleation between the proteome and transcriptome that as most the majority of stably up-regulated proteins also showed elevated mRNA transcript levels. However, a dynamic reorganization of the transcriptome occurred during the first hours after salt shock, which probably also involves the action of small regulatory RNAs acting at the post-transcriptional level. In addition to the rapid and stable steady upregulation of compatible solute biochemistry, a dynamic reorganization of the transcriptome occurred during the first hours after salt shock, which probably involves the action of small regulatory RNAs. Moreover, the coordinated induction of several stress proteins known to be involved in iron and oxidative stress responses as well as of mechano-sensitive channels was observed. Based on these data, an extended salt stimulon can be defined comprising many proteins directly or indirectly related to compatible solute metabolism, ion and water movements as well as a defined set of small regulatory RNAs. Moreover, the massive accumulation of the compatible solute glucosylglycerol had large impact on the overall carbon and nitrogen metabolism. Our comprehensive data set provides the basis for future attempts to engineer cyanobacterial salt tolerance and to search for processes regulating this important environmental acclimation process.
Project description:Transcriptional profiling of an El Tor biotype crp mutant The virulent V. cholerae El Tor Ogawa strain C7258 (Peru isolate 1991) and an isogenic deletion mutant (WL7258) lacking DNA sequences encoding the cAMP receptor protein were grown in LB medium to optical density at 600 nm of 1.5. The cultures were chilled in ice, cells quickly collected by centrifugation and total RNA imediately extracted. RNAwas extracted and purified using the Trizol plus RNA purification system (Invitrogen) followed RNEasy miniElute cleanup (Qiagen). RNA samples were conserved at - 80 C and used within a week. Keywords: Genetic modification
Project description:RNA microarrays technology was used to compare hESC-derived cell populations to undifferentiated hESC and to human EL cell populations. Microarrays confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-derived CD144+-EBs, BCs and ECs, respectively, and the similarities between hESC-derived cell populations and human EL equivalent populations.