Project description:Omics approaches employed to study biological molecules have revolutionized environmental microbiology and elevated our understanding of ecological processes in the biosphere. However, conventional omics applications often lead to the loss of information about macromolecular organization of the molecules within the cellular context. This can be a significant disadvantage since many proteins are functional only as macromolecular complexes in particular structural forms. A great example is bacterial contractile injection system (CIS), a syringe-like protein complex whose function is the translocation of molecules into a target cell to affect its state, often inducing lysis. Since the function of CIS is tightly linked to its intracellular localization, proteomics cannot confidently predict the function of novel uncharacterized CISs in natura. To overcome this challenge, we have developed a cryo-electron tomography workflow as a technique complementing metagenomics and proteomics. Additionally, we developed an immuno-electron microscopy protocol to identify and quantify CIS particles in environmental samples. Using this approach, we discovered a novel bacterial CIS in thermophilic multicellular Chloroflexota bacteria populating hot spring mats worldwide. We found that this system is similar phylogenetically and structurally to a recently described cytoplasmic CIS, which was found in multicellular Streptomyces and has been shown to be involved in cell cycle regulation. Interestingly, using our approaches, we have discovered that Chloroflexota cells produce different numbers of CIS particles depending on the mat micro-niches they occupy. In agreement with this, we observed that CIS was also non-constitutively expressed under laboratory conditions. Motivated by this discovery, we searched and analyzed similar CIS in extremophilic bacteria from other lineages. Overall, we have gained an understanding that bacterial cytoplasmic CIS is an overlooked cellular feature of the extremophilic bacteria, which is potentially involved in the cell fate control or intraspecies interaction within microbial community.
Project description:The evolutionarily ancient Aquificales bacterium Sulfurihydrogenibium spp. dominates filamentous microbial mat communities in shallow, fast-flowing, and dysoxic hot-spring drainage systems around the world. In the present study, field observations of these fettuccini-like microbial mats at Mammoth Hot Springs in Yellowstone National Park are integrated with geology, geochemistry, hydrology, microscopy, and multi-omic molecular biology analyses. Strategic sampling of living filamentous mats along with the hot-spring CaCO3 (travertine) in which they are actively being entombed and fossilized has permitted the first direct linkage of Sulfurihydrogenibium spp. physiology and metabolism with the formation of distinct travertine streamer microbial biomarkers. Results indicate that, during chemoautotrophy and CO2 carbon fixation, the 87-98% Sulfurihydrogenibium-dominated mats utilize chaperons to facilitate enzyme stability and function. High-abundance transcripts and proteins for type IV pili and extracellular polymeric substances (EPSs) are consistent with their strong mucus-rich filaments tens of centimeters long that withstand hydrodynamic shear as they become encrusted by more than 5 mm of travertine per day. Their primary energy source is the oxidation of reduced sulfur (e.g., sulfide, sulfur, or thiosulfate) and the simultaneous uptake of extremely low concentrations of dissolved O2 facilitated by bd-type cytochromes. The formation of elevated travertine ridges permits the Sulfurihydrogenibium-dominated mats to create a shallow platform from which to access low levels of dissolved oxygen at the virtual exclusion of other microorganisms. These ridged travertine streamer microbial biomarkers are well preserved and create a robust fossil record of microbial physiological and metabolic activities in modern and ancient hot-spring ecosystems.