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Arsenic (As) is a toxic heavy metal widely found in the environment that severely undermines the integrity of water resources. Bioremediation of toxic compounds is an appellative sustainable technology with a balanced cost-effective setup. To pave the way for the potential use of Deinococcus indicus, an arsenic resistant bacterium, as a platform for arsenic bioremediation, an extensive characterization of its resistance to cellular insults is paramount. A comparative analysis of D. indicus cells grown in two rich nutrient media conditions (M53 and TGY) revealed distinct resistance patterns when cells are subjected to stress via UV-C and methyl viologen (MV). Cells grown in M53 demonstrated higher resistance to both UV-C and MV. Moreover, cells grow to higher density upon exposure to 25 mM As(V) in M53 in comparison with TGY. This analysis is pivotal for the culture of microbial species in batch culture bioreactors for bioremediation purposes. We also demonstrate for the first time the presence of polyphosphate granules in D. indicus which are also found in a few Deinococcus species. To extend our analysis, we also characterized DiArsC2 (arsenate reductase) involved in arsenic detoxification and structurally determined different states, revealing the structural evidence for a catalytic cysteine triple redox system. These results contribute for our understanding into the D. indicus resistance mechanism against stress conditions. [ABSTRACT FROM AUTHOR]

Summary: Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)‐encoded type one secretion system (T1SS) and the SPI1‐encoded type three secretion system (T3SS‐1). The SPI4‐encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure–function relationships in SiiA, a non‐canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA‐PD). The crystal structure of SiiA‐PD displays homology to that of MotB and other peptidoglycan (PG)‐binding domains. SiiA‐PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA‐MotB homology is paralleled by a predicted SiiB‐MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion. [ABSTRACT FROM AUTHOR]

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.