Marina Raguse

Resistance of Bacillus subtilis Spore DNA to Lethal Ionizing Radiation Damage Relies Primarily on Spore Core Components and DNA Repair, with Minor Effects of Oxygen Radical Detoxification

ABSTRACT The roles of various core components, including α/β/γ-type small acid-soluble spore proteins (SASP), dipicolinic acid (DPA), core water content, and DNA repair by apurinic/apyrimidinic (AP) endonucleases or nonhomologous end joining (NHEJ), in Bacillus subtilis spore resistance to different types of ionizing radiation including X rays, protons, and high-energy charged iron ions have been studied. Spores deficient in DNA repair by NHEJ or AP endonucleases, the oxidative stress response, or protection by major α/β-type SASP, DPA, and decreased core water content were significantly more sensitive to ionizing radiation than wild-type spores, with highest sensitivity to high-energy-charged iron ions. DNA repair via NHEJ and AP endonucleases appears to be the most important mechanism for spore resistance to ionizing radiation, whereas oxygen radical detoxification via the MrgA-mediated oxidative stress response or KatX catalase activity plays only a very minor role. Synergistic radioprotective effects of α/β-type but not γ-type SASP were also identified, indicating that α/β-type SASPs binding to spore DNA is important in preventing DNA damage due to reactive oxygen species generated by ionizing radiation.

Antibacterial Activity of Blue Light against Nosocomial Wound Pathogens Growing Planktonically and as Mature Biofilms

ABSTRACT The blue wavelengths within the visible light spectrum are intrinisically antimicrobial and can photodynamically inactivate the cells of a wide spectrum of bacteria (Gram positive and negative) and fungi. Furthermore, blue light is equally effective against both drug-sensitive and -resistant members of target species and is less detrimental to mammalian cells than is UV radiation. Blue light is currently used for treating acnes vulgaris and Helicobacter pylori infections; the utility for decontamination and treatment of wound infections is in its infancy. Furthermore, limited studies have been performed on bacterial biofilms, the key growth mode of bacteria involved in clinical infections. Here we report the findings of a multicenter in vitro study performed to assess the antimicrobial activity of 400-nm blue light against bacteria in both planktonic and biofilm growth modes. Blue light was tested against a panel of 34 bacterial isolates (clinical and type strains) comprising Acinetobacter baumannii, Enterobacter cloacae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, and Elizabethkingia meningoseptica. All planktonic-phase bacteria were susceptible to blue light treatment, with the majority (71%) demonstrating a ≥5-log10 decrease in viability after 15 to 30 min of exposure (54 J/cm2 to 108 J/cm2). Bacterial biofilms were also highly susceptible to blue light, with significant reduction in seeding observed for all isolates at all levels of exposure. These results warrant further investigation of blue light as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications. IMPORTANCE Blue light shows great promise as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications (e.g., wound closure during surgery). This warrants further investigation.

Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws.

A special focus area of planetary protection is the monitoring, control, and reduction of microbial contaminations that are detected on spacecraft components and hardware during and after assembly. In this study, wild-type spores of Bacillus pumilus SAFR-032 (a persistent spacecraft assembly facility isolate) and the laboratory model organism B. subtilis 168 were used to study the effects of low-pressure plasma, with hydrogen alone and in combination with oxygen and evaporated hydrogen peroxide as a process gas, on spore survival, which was determined by a colony formation assay. Spores of B. pumilus SAFR-032 and B. subtilis 168 were deposited with an aseptic technique onto the surface of stainless steel screws to simulate a spore-contaminated spacecraft hardware component, and were subsequently exposed to different plasmas and hydrogen peroxide conditions in a very high frequency capacitively coupled plasma reactor (VHF-CCP) to reduce the spore burden. Spores of the spacecraft isolate B. pumilus SAFR-032 were significantly more resistant to plasma treatment than spores of B. subtilis 168. The use of low-pressure plasma with an additional treatment of evaporated hydrogen peroxide also led to an enhanced spore inactivation that surpassed either single treatment when applied alone, which indicates the potential application of this method as a fast and suitable way to reduce spore-contaminated spacecraft hardware components for planetary protection purposes.

Understanding of the importance of the spore coat structure and pigmentation in the Bacillus subtilis spore resistance to low-pressure plasma sterilization

Low-pressure plasmas have been evaluated for their potential in biomedical and defense purposes. The sterilizing effect of plasma can be attributed to several active agents, including (V)UV radiation, charged particles, radical species, neutral and excited atoms and molecules, and the electric field. Spores of Bacillus subtilis were used as a bioindicator and a genetic model system to study the sporicidal effects of low-pressure plasma decontamination. Wild-type spores, spores lacking the major protective coat layers (inner, outer, and crust), pigmentation-deficient spores or spore impaired in encasement (a late step in coat assembly) were systematically tested for their resistance to low-pressure argon, hydrogen, and oxygen plasmas with and without admixtures. We demonstrate that low-pressure plasma discharges of argon and oxygen discharges cause significant physical damage to spore surface structures as visualized by atomic force microscopy. Spore resistance to low-pressure plasma was primarily dependent on the presence of the inner, and outer spore coat layers as well as spore encasement, with minor or less importance of the crust and spore pigmentation, whereas spore inactivation itself was strongly influenced by the gas composition and operational settings.

Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies.

ABSTRACT Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 × 107 spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques.

Identification of a conserved 5'-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair.

Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs.

VUV absorption spectroscopy of bacterial spores and DNA components

Low-pressure plasmas can be used to inactivate bacterial spores and sterilize goods for medical and pharmaceutical applications. A crucial factor are damages induced by UV and VUV radiation emitted by the plasma. To analyze inactivation processes and protection strategies of spores, absorption spectra of two B. subtilis strains are measured. The results indicate, that the inner and outer coat of the spore significantly contribute to the absorption of UV-C and also of the VUV, protecting the spore against radiation based damages. As the sample preparation can significantly influence the absorption spectra due to salt residues, the cleaning procedure and sample deposition is tested for its reproducibility by measuring DNA oligomers and pUC18 plasmid DNA. The measurements are compared and discussed with results from the literature, showing a strong decrease of the salt content enabling the detection of absorption structures in the samples.

Simulated Space Radiation: Impact of Four Different Types of High-Dose Ionizing Radiation on the Lichen Xanthoria elegans

This study addresses the viability of the lichen Xanthoria elegans after high-dose ionizing irradiation in the frame of the STARLIFE campaign. The first set of experiments was intended to resemble several types of galactic cosmic radiation (GCR) as present beyond the magnetic shield of Earth. In the second set of experiments, γ radiation up to 113 kGy was applied to test the limit of lichen resistance to ionizing radiation. Entire thalli of Xanthoria elegans were irradiated in the anhydrobiotic state. After STARLIFE 1, the metabolic activity of both symbionts was quantified by live/dead staining with confocal laser scanning microscopy. The photosynthetic activity was measured after the respective irradiation to assess the ability of the symbiotic green algae to restore photosynthesis after irradiation. The STARLIFE campaign complements the results of the LIFE experiments at the EXPOSE-E facility on the International Space Station by testing the model organism Xanthoria elegans on its resistance to hazardous radiation that might be accumulated during long-term space exposure. In addition, the photosynthetic activity of metabolically active lichen was investigated after X-ray irradiation up to 100 Gy (3.3 Gy/min). Since previous astrobiological experiments were mostly performed with anhydrobiotic lichen, these experiments will broaden our knowledge on the correlation of physiological state and astrobiological stressors. Key Words: Astrobiology-Extremotolerance-Gamma rays-Ionizing radiation-Lichens-Viability. Astrobiology 17, 136-144.

Bacillus subtilis DisA helps to circumvent replicative stress during spore revival

The mechanisms that allow to circumvent replicative stress, and to resume DNA synthesis are poorly understood in Bacillus subtilis. To study the role of the diadenylate cyclase DisA and branch migration translocase (BMT) RadA/Sms in restarting a stalled replication fork, we nicked and broke the circular chromosome of an inert mature haploid spore, damaged the bases, and measured survival of reviving spores. During undisturbed ripening, nicks and breaks should be repaired by pathways that do not invoke long-range end resection or genetic exchange by homologous recombination, after which DNA replication might be initiated. We found that DNA damage reduced the viability of spores that lacked DisA, BMT (RadA/Sms, RuvAB or RecG), the Holliday junction resolvase RecU, or the translesion synthesis DNA polymerases (PolY1 or PolY2). DisA and RadA/Sms, in concert with RuvAB, RecG, RecU, PolY1 or PolY2, are needed to bypass replication-blocking lesions. DisA, which binds to stalled or reversed forks, did not apparently affect initiation of PriA-dependent DNA replication in vitro. We propose that DisA is necessary to coordinate responses to replicative stress; it could help to circumvent damaged template bases that otherwise impede fork progression.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.