Combined scanning transmission X-ray and electron microscopy for the characterization of bacterial endospores

Endospores (also referred to as bacterial spores) are bacterial structures formed by several bacterial species of the phylum Firmicutes. Spores form as a response to environmental stress. These structures exhibit remarkable resistance to harsh environmental conditions such as exposure to heat, desiccation, and chemical oxidants. The spores include several layers of protein and peptidoglycan that surround a core harboring DNA as well as high concentrations of calcium and dipicolinic acid (DPA). A combination of scanning transmission X-ray microscopy, scanning transmission electron microscopy, and energy dispersive spectroscopy was used for the direct quantitative characterization of bacterial spores. The concentration and localization of DPA, Ca2+, and other elements were determined and compared for the core and cortex of spores from two distinct genera: Bacillus subtilis and Desulfotomaculum reducens. This micro-spectroscopic approach is uniquely suited for the direct study of individual bacterial spores, while classical molecular and biochemical methods access only bulk characteristics

Megahertz single-particle imaging at the European XFEL

The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate.We acknowledge European XFEL in Schenefeld, Germany, for provision of X-ray free-electron laser beamtime at Scientific Instrument SPB/SFX and would like to thank the instrument group and facility staff for their assistance. We acknowledge the use of the XBI biological sample preparation laboratory, enabled by the XBI User Consortium. The results of the work were obtained using Maxwell computational resources operated at Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany, and computational resources of MCC NRC “Kurchatov Institute.” This research used resources of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. We acknowledge the support of funding from: the Swedish Foundation for International Cooperation in Research and Higher Education (STINT); Helmholtz Associations Initiative and Networking Fund and the Russian Science Foundation grant HRSF-0002/18-41-0600; the Russian Science Foundation grant 18-14-00321; European Research Council, “Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS),” ERC-2013-SyG 609920; Fellowship from the Joachim Herz Stiftung; Singapore National Research Foundation Grant number NRF-CRP16-2015-05; Ministry of Education, Science, Research and Sport of the Slovak Republic and by grant APVV-18-0104; the project CZ.02.1.01/0.0/0.0/16_019/0000789 (ADONIS) from European Regional Development Fund, Chalmers Area of Advance; Material Science and the Ministry of Education, Youth and Sports as part of targeted support from the National Programme of Sustainability II; US National Science Foundation (NSF) Science and Technology Center BioXFEL Award 1231306; Helmholtz Initiative and Networking Fund through the Young Investigators Group Program and Deutsche Forschungsgemeinschaft, project B03/SFB755; VR starting grant (2018-03387); FORMAS future research leader (2018-00421); KVA Biosciences 2018 (BS2018-0053); NSF 1231306; German Ministry for Education and Research, BMBF (grant No. 05K2016—Visavix); the Heinrich Pette Institute, Leibniz Institute for Experimental Virology is supported by the Free and Hanseatic City of Hamburg and the Federal Ministry of Health; NSF STC BioXFEL grant 1231306; The National Research Foundation (NRF) of Korea (Grant No. 2017K1A3A7A09016380); the Röntgen-Ångström Cluster; the Swedish Research Council; the Swedish Foundation for Strategic Research. We thank Arwen Pearson for critical reading of the manuscript. Open access funding provided by Uppsala University

Expression, purification and X-ray crystallographic analysis of thioredoxin from Streptomyces coelicolor

The structures of thioredoxins from several species, including man and E. coli, are known. The bacterium S. coelicolor contains three thioredoxins with unknown physiological functions. TrxA from S. coelicolor has been overexpressed, purified and crystallized and its crystal structure determined at 1.5 Å resolution

Spores and Spore Formers

Bacterial spore formers have been the focus of intense study for almost half a century centered primarily on Bacillus subtilis. This research has given us a detailed picture of the genetic, physiological and biochemical mechanisms that allow bacteria to survive harsh environmental conditions by forming highly robust spores. Although, many basic aspects of this process are now understood in great detail, bacterial sporulation still continues to be a highly attractive model for studying various cell processes at a molecular level. There are several reasons for such scientific interest. First, some of the complex steps in sporulation are not fully understood and/or only are only described by 'controversial' models. Second, intensive research on unicellular development of a single microorganism, B. subtilis, left us largely unaware of the multitude of diverse sporulation mechanisms in many other Gram-positive endospore and exospore formers. This diversity would likely increase if we were to include sporulation processes in the Gram-negative spore formers. In addition, spore formers have great potential in applied research. Spore forming bacteria are becoming increasingly important in the areas of probiotics, vaccine technology and biotechnology. This Research Topic in Frontiers in Microbiology details the most recent advances in basic science of spore research and cover also emerging areas of scientific importance involving the use of spores

Chromate tolerance and removal of bacterial strains isolated from uncontaminated and chromium-polluted environments

Investigation of bacterial chromate tolerance has mostly focused on strains originating from polluted sites. In the present study, we isolated 33 chromate tolerant strains from diverse environments harbouring varying concentrations of chromium (Cr). All of these strains were able to grow on minimal media with at least 2 mM hexavalent chromium (Cr(VI)) and their classification revealed that they belonged to 12 different species and 8 genera, with a majority (n = 20) being affiliated to the Bacillus cereus group. Selected B. cereus group strains were further characterised for their chromate tolerance level and the ability to remove toxic Cr(VI) from solution. A similar level of chromate tolerance was observed in isolates originating from environments harbouring high or low Cr. Reference B. cereus strains exhibited the same Cr(VI) tolerance which indicates that a high chromate tolerance could be an intrinsic group characteristic. Cr(VI) removal varied from 22.9% (strain PCr2a) to 98.5% (strain NCr4). Strains NCr1a and PCr12 exhibited the ability to grow to the greatest extent in Cr(VI) containing media (maximum growth of 65.3% and 64.9% relative to that in the absence of Cr(VI), respectively) accompanied with high chromate removal activity (73.7% and 74.4%, respectively), making them prime candidates for the investigation of chromate tolerance mechanisms in Gram-positive bacteria and Cr(VI) bioremediation applications

Forces and Kinetics of the <i>Bacillus subtilis</i> Spore Coat Proteins CotY and CotX Binding to CotE Inspected by Single Molecule Force Spectroscopy

Spores are uniquely stable cell types that are produced when bacteria encounter nutrient limitations. Spores are encased in a complex multilayered coat, which provides protection against environmental insults. The spore coat of <i>Bacillus subtilis</i> is composed of around 70 individual proteins that are organized into four distinct layers. Here we explored how morphogenetic protein CotE guides formation of the outermost layer of the coat, the crust, around the forespore by focusing on three proteins: CotE, CotY, and CotX. Single molecule force spectroscopy (SMFS) was used to investigate the interactions among CotE, CotY, and CotX at the single-molecule level. Direct interactions among these three proteins were observed. Additionally, the dissociation kinetics was also studied by measuring the unbinding forces of the complexes at different loading rates. A series of kinetic data of these complexes were acquired. It was found that the interaction of CotE and CotY was stronger than that of CotE and CotX

The Structure and Interactions of SpoIISA and SpoIISB, a Toxin-Antitoxin System in Bacillus subtilis*

Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase σ factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, σF. Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 Å spacing, revealing a CSpoIISA2·SpoIISB2 heterotetramer. CSpoIISA has a single domain α/β structure resembling a GAF domain with an extended α-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA·SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed