An official website of the United States government.
We will initiate a series of experiments to determine the global pattern of gene expression during germination of B. subtilis spores both before and after UV irradiation, and further elucidate the role of methionine, ATP, and SAM synthetase in the generation of SAM for SP lyase during spore germination. <P>
To accomplish this, we will utilize a combination of in vivo, in vitro, and genomic experiments: <OL type=A> <LI> In vivo experiments. Questions to be addressed are: 1. Is SAM synthetase produced and packaged in spores? 2. What is the ATP source for SAM production? 3. What is the methionine source for SAM production? <LI> In vitro experiments. Specific tasks include: 1. Directly measure levels of SAM and SAM synthetase germinating spore extracts. 2. Indirectly measure SAM and SAM synthetase using in vitro SP repair assay. <LI> Genomic experiments. Specific tasks include: 1. Identify and characterize homologues of spore DNA protective and repair factors in the genomes of other sporeforming bacteria. 2. Characterize the global germination regulon by microarray analysis. 3. Identify the regulon of genes induced in response to spore DNA damage by microarray analysis of germinating UV-irradiated spores.
NON-TECHNICAL SUMMARY: Sporeforming bacteria are significant in agriculture as animal and human pathogens, and as agents of both biocontrol and bioterror. A major factor determining spore longevity in the environment is resistance to solar UV, which is known to involve DNA repair during germination. The project is to further understand spore UV resistance as a function of germination physiology using a combination of genetic, biochemical and genomic approaches. <P>
APPROACH: A1.We will assay expression of the metK-lacZ gene during sporulation, germination, and outgrowth; transfer the metK-lacZ fusion to our lab's strains, sporulate the strains in NSM and measure beta-galactosidase activity expressed by the fusion. We will transfer the metK1 mutation into our standard uvrB42 lab strain and assess its effects on SP lyase-mediated spore UV resistance and kinetics of SP repair. A2.ATP production can be blocked in vivo during spore germination by NaF or KF, which inhibit enolase. Therefore, fluoride would be predicted inhibit SP lyase function during germination by lowering ATP, hence SAM levels. A3.Mutant spores lacking Gpr are impaired in SASP degradation, hence germination. We will transfer the Delta-gpr mutation into our standard uvrB42 lab strain to test (i) UV resistance of spores and (ii) kinetics of SP lyase-mediated SP repair. B1. We will directly measure levels of SAM synthetase in sporulating cells and in dormant and germinating spores. B2.Using cell-free extracts, we will assay for SAM synthetase activity. We will reconstruct SP repair completely in vitro from purified components and (6His)SP lyase. C1.We are accumulating a rich lode of sequence data from which to mine information about DNA protective and repair functions. Examples include SASP and SP lyase. We plan to continue accumulating and comparing sequence data on SASP and SP lyase as they become available, and also to initiate compiling data on other factors such as: cot genes encoding spore coat proteins; and dpaAB and spoVA involved in DPA synthesis and localization. C2.Currently available is the entire microarray of 4,200 genes in the B. subtilis genome spotted in duplicate on glass slides for $375 per slide, and the entire reverse primer set for probing the array for $250 (Eurogentec, Belgium). We have 2 microarray facilities available on-campus, each of which offers technical support. For more detailed protocols, the reader is referred to the following web sites: http://gatc.arl.arizona.edu/info/microarray.html (UA-ARL's Laboratory of Molecular Systematics and Evolution) and http://150.135.37.182/galbraith/ (Dr. David Galbraith's faculty page). Briefly, total RNA will be isolated from B. subtilis, labeled, and used to probe the B. subtilis microarray. RNA populations to be isolated will include: (1) dormant B. subtilis spores, to obtain the RNA population present in dormant spores; and (2) germinated spores. Images of the microarrays will be scanned and quantitated using standard software. Subtraction of the signals obtained from population (1) from those of (2) will yield (3) the regulon of genes whose transcription is specifically induced by triggering of germination. C3. In addition, (4) UV-irradiated spores will be germinated, RNA isolated, labeled, and used to probe the B. subtilis microarray. Image (3) will be subtracted from Image (4) to identify genes whose expression is specifically induced during germination of spores carrying UV-induced DNA damage. These experiments will identify genes encoding new factors important in spore recovery from DNA damage.