Progress 10/01/02 to 10/14/03
Outputs
This project is being terminated due to the PI leaving the University of Arizona to start a faculty position in the department of Microbiology and Cell Science in CALS at the University of Florida-Gainesville, effective 10/14/03. During the final period of this project we validated Bacillus subtilis spores as surrogates for UV inactivation of Bacillus anthracis spores(Nicholson and Galeano, 2003). We also demonstrated the efficacy of siver/zinc zeolite coated stainless steel for inactivation of vegetative cells, but not spores, of B. subtilis, B. cereus, and B. anthracis (see Galeano, Korff, and Nicholson, 2003).
Impacts
The research will impact the fields of Agrobioterrorism, Homeland Security, and infection control.
Publications
- Nicholson, W.L. and B. Galeano. 2003. UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne. Appl. Environ. Microbiol. 69: 1327-1330.
- Galeano, B., E. Korff, and W.L. Nicholson. 2003. Inactivation of vegetative cells, but not spores, of Bacillus anthracis, B. cereus, and B. subtilis on stainless steel surfaces coated swith an antimicrobial silver/zinc-containing zeolite formulation. Appl. Environ. Microbiol. 69: 4329-4331.
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Progress 01/01/02 to 12/31/02
Outputs
For the 2002 year, we have continued our researches into the environmental resistance of Bacillus subtilis spores, and have begun studies of the molecular evolution of spore resistance properties during long-term culture in the laboratory. We have uncovered unique rifampicin-resistance mutations in the rpoB gene of B. subtilis spores which resemble alleles found in clinical isolates of Mycobacterium tuberculosis and which alter the global pattern of gene expression.
Impacts
Understanding spore longevity in the environment is expected to yield valuable information which can be used to predict and manipulate the environmental half-lives of spore-containing biopesticides.
Publications
- Nicholson, W.L. and H. Maughan. 2002. The spectrum of spontaneous rifampicin resistance mutations in the rpoB gene of Bacillus subtilis 168 spores differs from vegetative cells and resembles that of Mycobacterium tuberculosis. J. Bacteriol. 184: 4936-4940.
- Maughan, H., C.W. Birky, W.L. Nicholson, W.R. Rosenzweig, and R.H. Vreeland. 2002. The paradox of the "ancient" bacterium which contains "modern" protein-coding genes. Mol. Biol. Evol. 19: 1637-1639.
- Nicholson, W.L., P. Fajardo-Cavazos, R. Rebeil, T.A. Slieman, P.J. Riesenman, J.F. Law, and Y. Xue. 2002. Bacterial endospores and their significance in stress resistance. Antonie van Leeuwenhoek 81: 27-32.
- Nicholson, W.L. 2002. Roles of Bacillus spores in the environment. In A. Driks (ed.). Development in Bacteria: spore formation in Bacillus subtilis. Cell. Mol. Life Sci. 59:410-416.
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Progress 01/01/01 to 12/31/01
Outputs
During the 2001 reporting period, we discovered that the spore-specific compound pyridine-2,6-dicarboxylate (dipicolinic acid or DPA) exerts a dramatic protective effect on spore resistance to solar radiation, particularly to UV-B solar wavelengths. We also elucidated the subunit structure and catalytic mechanism of the enzyme spore photoproduct (SP) lyase, a spore-specific DNA repair enzyme and an important determinant of spore UV resistance. We found that SP lyase is a dimeric [4Fe-4S] enzyme and operates by a novel radical mechanism. We initiated studies to identify conserved amino acid residues in SP lyase which are essential for [4Fe-4S] cluster formation and catalysis.
Impacts
Both findings deepen our understanding of the factors which influence bacterial spore longevity in the environment.
Publications
- Slieman, T.A. and W.L. Nicholson. 2001. Role of dipicolinic acid in resistance of Bacillus subtilis spores exposed to artificial and solar UV radiation. Appl. Environ. Microbiol.67:1274-1279.
- Rebeil, R. and W.L. Nicholson. 2001. The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase. Proc. Natl. Acad. Sci. USA 98: 9038-9043.
- Nicholson, W.L., P. Fajardo-Cavazos, R. Rebeil, T. A. Slieman, P.J. Riesenman, J.F. Law, and Y. Xue. 2001. Bacterial endospores and their significance in stress resistance. Antonie van Leeuwenhoek (in press).
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Progress 01/01/00 to 12/31/00
Outputs
During the project period, we advanced our knowledge of bacterial spore resistance to solar UV by: (i) identifying and characterizing the splA gene as encoding a trans-acting negative protein which regulates expression of the SP lyase DNA repair system of Bacillus subtilis spores, (ii) Characterizing binding and catalytic parameters of SP lyase on a synthetic SP-containing DNA substrate, (iii) identifying novel UV photoproducts in the DNA of spores exposed to sunlight, and (iv) characterizing the role of the spore coat proteins in spore resistance to both laboratory-generated and solar UV. We also directed and published a multi-author review on the subject of spore resistance to extreme environments.
Impacts
Our work during the past 12 months has attracted considerable attention from both scientific and commercial interests, and prospects look good for bringing grant and contract money into the College of Agriculture for continuation of these studies.
Publications
- Fajardo-Cavazos, P. and W.L. Nicholson, W.L. 2000. The TRAP-like SplA protein is a trans-acting negative regulator of spore photoproduct (SP) lyase synthesis during Bacillus subtilis sporulation. J. Bacteriol 182: 555-560.
- Slieman, T.A. and W.L. Nicholson. 2000. DNA in dormant Bacillus subtilis spores exposed to artificial UV sources and to solar radiation accumulates single-strand breaks and cyclobutane pyrimidine dimers in addition to spore photoproduct. Appl. Environ. Microbiol.66: 199-205.
- Riesenman, P.J. and W.L. Nicholson. 2000. Role of the spore coat layers in resistance of Bacillus subtilis spores to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation Appl. Environ. Microbiol. 66:620-626.
- Slieman, T.A.*, R. Rebeil*, and W.L. Nicholson. 2000. Spore photoproduct (SP) lyase from Bacillus subtilis specifically binds to and cleaves SP (5-thyminyl-5,6-dihydrothymine) and not cyclobutane dimers in UV-irradiated DNA. J. Bacteriol. 182: 6412-6417. (*denotes equal contribution by both authors).
- Nicholson, W.L., N. Munakata, G. Horneck, H.J. Melosh, and P. Setlow. 2000. Resistance of bacterial endospores to extreme terrestrial and extraterrestrial environments. Microbiol. Mol. Biol. Rev. 64: 548-572.
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Progress 01/01/99 to 12/31/99
Outputs
During the period covered, our work from 1998 was published in the Journal of Microbiological Methods (see below). In 1999, the resistance of spores of the common soil bacterium to solar UV radiation was investigated through the use of artificial air-dried biofilms. Two separate studies were undertaken. In the first study, the UV photochemistry of spore DNA exposed to solar radiation was probed using a combination of alkaline-agarose gel electrophoresis and digestion of DNA with enzymes which recognize specific DNA damages. We found that in addition to the unique "spore photoproduct" spores exposed to sunlight also accumulated single-strand breaks, double-strand breaks and cyclobutane pyrimidine dimers, but not abasic sites. A manuscript describing the work was submitted to Applied and Environmental Microbiology. In the second study, the contribution of the proteinaceous spore coat to solar UV resistance was determined by exposing spores various coat-defective strains
of B. subtilis to solar UV and quantitating spore survival. We found that the inner coat layer appears to be an important determinant of spore survival to solar radiation. A manuscript describing the work was submitted to Applied and Environmental Microbiology.
Impacts
Bacterial spores are important components of various biological control agents (pesticides, fungicides, and nematocides). The research sets important constraints on the survivability and persistence of applied spores in field settings.
Publications
- Nicholson, W.L. and J.F. Law. 1999. Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168. J. Microbiol. Methods 35: 13-21.
- Slieman, T.A. and W.L. Nicholson. 1999. DNA in dormant Bacillus subtilis spores exposed to artificial UV sources and to solar radiation accumulates single-strand breaks and cyclobutane pyrimidine dimers in addition to spore photoproduct. Submitted to Applied and Environmental Microbiology.
- Riesenman, P.J. and W.L. Nicholson. 1999. Essential role of the spore coats in resistance of Bacillus subtilis spores to hydrogen peroxide, UV-B, and solar radiation. Submitted to Applied and Environmental Microbiology.
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Progress 01/01/98 to 12/31/98
Outputs
The past year (1998) saw the publication of a current review of spore environmental UV resistance, which the PI wrote in 1997 (Ref 1). In addition, a the PI published a method developed in his lab for isolation and purification of dormant bacterial spores from 3 distinct Sonoran desert soil types. Endospores of Bacillus spp. were purified from three Sonoran desert soil samples by Chelex extraction and NaBr density gradient centrifugation, and their UV resistances compared with that of B. subtilis laboratory strain 168. Natural spore populations exhibited tight adherence to rapidly-sedimenting soil particles which was not readily overcome by the extraction and purification procedure. It was observed that spores purified from soil exhibited 2-3 fold higher resistance to UV (as measured by the 90% lethal dose, LD90) than did B. subtilis 168 grown on NSM, a standard laboratory sporulation medium, and purified by the same extraction procedure. Cultivation of spore-forming
bacteria isolated from soil on NSM resulted in production of spores with essentially identical UV resistance as strain 168, suggesting that spore UV resistance is influenced by the environment in which spores are produced. The publication resulting from this work (Ref. 2) will appear in 1999 due to printing delay at the Journal.
Impacts
(N/A)
Publications
- 1. Nicholson, W.L. and P. Fajardo-Cavazos. 1997. DNA repair and the UV resistance of bacterial spores: from the laboratory to the environment. Recent Res. Devel. Microbiol. 1: 125-140.
- 2. Nicholson, W.L. and J.F. Law. 1998. Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168. J. Microbiol. Methods (in press).
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Progress 01/01/97 to 12/31/97
Outputs
(i) A technique for isolation and purification of bacterial spores from Sonoran desert soils has been devised. Pasteurized soil is subjected to Chelex-100 extraction, differential centrifugation, and NaBr gradient centrifugation. Spores adhere strongly to rapidly-sedimenting soil particles, and purified spores obtained from soil exhibited 3x-higher UV resistance (as judged by LD-90 values) than reference laboratory strain Bacillus subtilis 168. (ii) The response of expression of the SP lyase DNA repair system to various environmental conditions prevailing during sporulation of B. subtilis is being tested using an splB-lacZ gene fusion. The fusion is expressed at higher levels in liquid than on the corresponding solid medium, and expression appears to respond to oxygen availability. Experiments are in progress to define the regulatory system responsible.
Impacts
(N/A)
Publications
- 1. Nicholson, W.L. and P. Fajardo-Cavazos. 1997. DNA repair and the UV resistance of bacterial spores: from the laboratory to the environment. In, Recent Research Developments in Microbiology, Research Signpost, Trivandrum, India (in press).
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