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MIC 329 The Gram-positive World*

MIC 329 The Gram-positive World*. *(Well, really a talk about my favorite Gram (+) organism, Bacillus subtilis ). The changing definition of Bacillus : Any rod-shaped bacterium then Gram-positive rods then Aerobic Gram (+) rods then along came 16S sequences…. Sporosarcina.

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MIC 329 The Gram-positive World*

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  1. MIC 329The Gram-positive World* *(Well, really a talk about my favorite Gram (+) organism, Bacillus subtilis)

  2. The changing definition of Bacillus: Any rod-shaped bacterium then Gram-positive rods then Aerobic Gram (+) rods then along came 16S sequences…...

  3. Sporosarcina Listeria Enterococcus Sporolactobacillus

  4. The ongoing schism of the genus Bacillus • Geobacillus • Thermobacillus • Virgibacillus • Salibacillus • Paenibacillus • Gracibacillus

  5. Why study Bacillus subtilis? • Best-characterized Gram-positive bacterium • Biochem., metabolism well-studied • Good genetic system (transformation, transduction) • Advanced molecular biology techniques • Entire genome sequenced / annotated • Easy to grow, manipulate in culture • Widely used in industry, agriculture • “Simple” model for cellular differentiation

  6. Bacillus subtilis differentiation cycle *Repair *Protection *Photochemistry

  7. B. subtilis spore anatomy outer coat inner coat cortex membranes core nucleoid oc ic

  8. Endospores are resistant to: • Heat (both wet and dry) • Ultraviolet (UV) radiation • Extreme desiccation (including vacuum) • Lysozyme • Chemicals (organic solvents, oxidizing agents, etc.)

  9. Spore Protective Mechanisms Environmental Factors Spore Resistance Repair of Damage Genetic Factors Sporulation/ Germination Physiology

  10. Abundance of spores in extreme locales

  11. Sonoran Desert Environment: • Solar UV: • ~10 J /m2 sec UV-B (noon) • ~25 J/m2 sec UV-A (noon) • Temperature extremes: • Avg. -7 to +46 ˚C (air) • ~70-80 ˚C (surfaces) • Desiccation: • Avg. 13%-30% RH • Avg. 28 cm rainfall / year

  12. Spores are 1-2 orders of magnitude more UV resistant than vegetative cells (254-nm UV-C)

  13. Solar UV Spectrum vs. Laboratory UV

  14. DNA Protective Factors in Spores • Spore coat proteins • Spore pigment in coat • Dipicolinic acid in core • SASP in core

  15. The spore coat layers protect spores from solar UV wavelengths Riesenman and Nicholson. AEM 66: 620. 2000.

  16. Spore pigment offers significant protection against environmentally-relevant UV wavelengths Wild-type (+) CuSO4 Wild-type (-) CuSO4 DcotA (+) CuSO4 Hullo, et al. J. Bacteriol. 183: 5426. 2001.

  17. Dipicolinic acid (pyridine-2,6-dicarboxylate) • Unique to spore core • Exists as Ca+2-chelate • Abundant (up to 10% of dry weight • Important in heat resistance

  18. DPA is especially important for spore resistance to UV-B radiation Slieman and Nicholson. AEM 67: 1274. 2001.

  19. Spore photochemistry is due to SASP-DNA interaction • SASP are Small, Acid-soluble Spore Proteins • SASP are synthesized at Stage III of sporulation • SASP bind to DNA and shift its conformation from B to A • UV irradiation of SASP-DNA complexes results in formation of SP and not T<>T

  20. Bacillus subtilis differentiation cycle SP Repair UV--> SP produced in DNA SASP production

  21. The UV photochemistry of DNA in vegetative cells and spores is different

  22. Saran Wrap 1/2” Plate glass Poly- styrene A. Filter lid B. 3x dried spore spots C. Microscope slide D. Platform E. Box

  23. Solar UV, not heat or desiccation, determines spore survival

  24. SP is repaired in germinating spores by SP lyase and NER LD90 (254-nm UV-C) vegetative cells

  25. Spores of B. subtilis DNA repair mutants respond differently to lab UV and Solar UV w.t. uvrB42 splB1 Yaming Xue Appl. Environ. Microbiol. 62: 2221-2227. 1996.

  26. Do spores exposed to solar UV accumulate different types of DNA damage(s)?

  27. Probing DNA damages with EndoV and alkali

  28. B. subtilis spore DNA exposed to sunlight accumulates ss breaks, ds breaks and cyclobutane dimers in addition to SP. 0.8% neutral agarose 0.8% alkaline agarose

  29. Summary of DNA Damage in solar UV-irradiated spores. Tony Slieman Appl. Environ. Microbiol. 66:199-205. 2000.

  30. SP is repaired in germinating spores by SP lyase and NER LD90 (254-nm UV-C) vegetative cells

  31. SP lyase-mediated DNA repair in B. subtilis • Encoded by splB gene. • Synthesized at Stage III of sporulation, packaged in the dormant spore. • Active during spore germination. • Direct reversal of SP to thymines in situ. • “Dark repair” process.

  32. Organization and expression of the splAB operon in B. subtilis Patricia Fajardo J. Bact. 175:1735.1993. Curr. Micro.34:133.1997. J.Bact. 176: 3983.1994. MGG 255:587.1997. J.Bact.177: 4402. 1995. J.Bact. 182:555.2000. Mario Pedraza

  33. “Radical SAM” Model for SP repair 1. SplB dimerizes via a [4Fe-4S] center. 2. Specific binding to SP in DNA. 3. SAM split by electron donation from Fe-S center, producing 5’-adenosyl radical. 4. Radical abstracts proton from C-6 of SP, reverses SP back to 2 T’s. Tony Slieman Roberto Rebeil J.Bact. 180:4879. 1998. PNAS 98: 9038. 2001. J.Bact. 182: 6412. 2000.

  34. In the laboratory: Spores are highly UV resistant. SP is the major DNA damage. CPD, ss, ds breaks negligible at biol. relevant UV doses. SP lyase > NER during germination. CONCLUSIONS • In the environment: • Spores are highly UV resistant. • SP is still the major DNA damage. • CPD, ss, ds breaks are significant at biol. relevant UV doses. • SP lyase = NER during germination. • Heat not a significant lethal component of sunlight.

  35. Survival and persistence of bacterial endospores in extreme environments Patricia Fajardo Mario Pedraza Lilian Chooback Roberto Rebeil Heather Glanzberg Paul Riesenman Jocelyn Law Tony Slieman Rachel Mastrapa Yubo Sun Heather Maughan Yaming Xue

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