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Workshop on CBRN Defence – 22-24 October 2013 – Brussels
“Destruction of Bacterial Spores by Solar UV Radiation”
Dr. Ralf Möller (representing the DEBACS project team)
German Aerospace Center (DLR e.V.)
Institute of Aerospace Medicine
Radiation Biology Department, Cologne, Germany
Collaborative Linkage Grant (CLG) number: CBP.EAP.CLG.983747
status: completed (2009-2011)
Brussels, Belgium, 23 October 2013
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Project: « Sporicidal effects of UV »
• Microbial inactivation via UV radiation: impact for CBRN defence program
• Bacillus subtilis spores – biological dosimeter for testing UV radiation sources
• Spore resistance to germicidal and environmental UV
Overview
Overview
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Project: « Sporicidal effects of UV »
- determination of spore resistance to germicidal and environmental UV radiation
- characterization of the mechanisms allowing spores to survive/resist UV radiation
- recording of fluence-effect correlation and inactivation rates (decimal reduction value)
- characterization of UV-induced DNA photoproducts
- studying the (potential) mutagenic effects of UV radiation
Objectives
Objectives
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Sky
Soil Hay Desert Rocks
Deep surface
Pathogens Insects
Where can we find spores of Bacillus spp.?
B. stratosphericus (above 24 km)
B. subtilis („hay“-Bacillus)
B. infernus (2700 m below surface)
B. sonorensis (Sonoran Desert,
Arizona, USA)
B. simplex (500 spores/g rock) B. thermoterrestis
(egypt. soil, 55°C)
B. cereus (food-poising)
B. anthracis (the bioterrorist)
B. thuringiensis (the exterminator)
Food
Description: background
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UV radiation areas and reactivity
UV radiation Reactivity
- different λ area (10-400 nm)
- environmental (290-400 nm)
[natural insolation]
- artificial (254 nm) [industrial / military / clinical application]
> selective absorption by
molecules (DNA, RNA,
proteins)
> interaction and
biochemical changes
> major DNA damage
(dimers, SSB, DSB)
Description: background
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Portrait of a Bacillus subtilis spore
1) DNA profile
• A-DNA conformation
• one single genome copy
• toroidal-shaped chromosome
• SASPs binding
2) Spore interior
• rRNA, ribosomes
• low water content
• Ca2+-DPA complex
• minerals, 3-PGA
3) Spore exterior
• spore coat and pigments
• crust and exosporium
Nicholson et al., Microbiol. Mol. Biol. Rev. (2000)
Description: background
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Direct or indirect radiation damage
Horneck et al., Microbiol. Mol. Biol. Rev. (2010)
(i) repair
(ii) detoxification
Description: effects of radiation
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Tools, approaches and methods to
study UV radiation effects Galactic Cosmic Rays (GCR):
high energy protons and heavy ions 87 % Protons 12 % -Particles 1 % heavy ions
UV radiation
(254 nm)
Biological model system:
- Spores of Bacillus subtilis (wild-type, mutant (DPA, SASP formation, increased core water content, pigmentation, coat assembly) and DNA repair deficient-strains (HR, NHEJ, AP, SP lyase, TLS))
UV-C lamps and sunlight simulators
Experimental approach:
Assays:
Description: objectives
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Working flow from the spore exposure to environmental UV
radiation until survival, mutation screening and UV-radiation
damage analyses.
Description: workflow of project
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WP1: Response to UV-C radiation (254 nm)
Galactic Cosmic Rays (GCR):
high energy protons and heavy ions 87 % Protons 12 % -Particles 1 % heavy ions
Setlow, JAM (2006), Moeller et al., J. Bacteriol. (2007,08,09,11) Rivas-Castillo et al., Curr. Microbiol (2011), Nicholson et al., MMBR (2000)
Protection: SASP, core water content, DPA Repair: SP lyase, NHEJ, HR, AP, TLS
Outcome and results: radiation resistance
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WP1: Spore resistance to environmental
relevant UV radiation
290-400 nm 320-400 nm
Protection: SASP, DPA, coat layers, pigm., core water Repair: SP lyase, NHEJ, HR, AP
Moeller et al. in J. Bacteriol. (2007,09,11)
Outcome and results: radiation resistance
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WP2: UV generated DNA lesions
Spore DNA fragmentation after exposure to terrestrial UV
Outcome and results: DNA damage
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WP2: DNA photoproducts (UV-C)
DNA-protein interaction (SASP: small, acid-soluble spore proteins)
wild-type: 1 molecule SASP
every 5 bp spore DNA
SASP mutant:
1 molecule SASP
every 20-25 bp spore DNA
in cooperation with T. Douki and J. Cadet
Setlow, JAM (2006), Moeller et al., Int. Microbiol. (2007), Lee et al., PNAS (2008)
same induction rate of total DNA photoproducts
Outcome and results: DNA damage
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WP2: DNA photoproducts (UV-C)
different spectra of DNA lesions (SP, CTP, 6-4) Setlow, JAM (2006)
Moeller et al., Int. Microbiol. (2007)
Outcome and results: DNA damage
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WP3: Mutagenicity of UV radiation
Moeller et al., J. Bacteriol. (2007,08,09,11)
254-nm UV-C 290-400 UV-(A+B) 320-400 UV-A
Error prone vs. free DNA repair: AP Protection: SASP
NalR hot spots: in GyrA: S63F/L, S84L, E88Q
Outcome and results: mutation induction
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WP3: Mutagenicity of UV radiation
LB medium / LB with 50 µg/ml Rif
• increase UV
• increase RifR
• antibioticR & UVR
= costs of spore inactivation?
Outcome and results: mutation induction
UV-C UV-(A+B)
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Summary and outlook
Project-related outcome
Insights in the UV resistance/inactivation of bacterial spores (CBRN defence)
Determination of types and nature of DNA lesions / combined treatment
Occurrence of antibiotic resistant strains (UVR) / general health concern
Further research focus: suggestions
Support of studies on combined treatments (heat, chemicals and radiation)
Funding (start-up projects, teaching, education, workshop, exchanges for young researchers (PhD students, PostDocs)
Project evaluation: direct scientific and educational successes
• Publications in microbiology journals (3 published, 1 in progress)
• Research exchange => Guest scientist (incl. further work on DNA repair) regional & international cooperation (involvement & networking)
• Stimulation of new aspects: antibiotic resistant strains & resistance
Project evaluation, impact, ideas for future SPS activities in CBRN defence