Progress 01/01/05 to 12/31/08
Outputs
OUTPUTS: David Popham presented research results during two seminars at Virginia Tech, Department of Biochemistry, 8/27/07, and at the University of Georgia, Department of Microbiology, 10/4/07. Benjamin Orsburn presented research results at the Virginia Tech Department of Biological Sciences Research Day, 2/24/07, 2/23/08 AND 2/21/09; at the International Conference on Gram Positive Pathogens, Omaha, NE, 10/06 AND 10/08; and at the Mid-Atlantic Microbial Pathogenesis Meeting, Wintergreen, VA, 2/09. New fundamental knowledge produced includes a better understanding of which spore structural factors contribute to high levels of heat resistance, and a resulting greater probability of causing food poisoning. A novel pathway for the synthesis of dipicolinic acid, a major spore solute requried for heat resistance, in C. perfringens was identified. Methods developed include those for analysis of C. perfringens spore peptidoglycan wall structure and highly sensitive measurement of dipicolinic acid content using mass spectrometry. PARTICIPANTS: Individuals who worked on the project, describe the role the person played in the project and how this person participated in the project. Include: (1) principal investigator(s)/project director(s) (PIs/PDs); and (2) each person who has worked at least one person month per year on the project during the reporting period and received salary, wages, a stipend, or other support from the agency (a person month equals approximately 160 hours of effort). Include any that have been added to the project since initiation of the project or activity. PI: David Popham PI: Stephen Melville Graduate Student: Benjamin Orsburn Graduate Student: Katherine Rodgers Lab Technician: Katie Sucre Lab Technician: Jessica McElligott Two graduate students received training in their progress towards Ph.D. degrees. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Knowledge of factors that contribute to spore heat resistance will allow screening of isolates from environmental samples for the presence of strains with greater probability of causing food poisoning. This will lead to the identification of the sources of C. perfringens food poisoning contaminants, which are currently poorly understood. This can eventually lead to improved guidelines for safe food handling.
Publications
- Orsburn, B., S. B. Melville, and D. L. Popham. (2008). Factors contributing to heat resistance of Clostridium perfringens endospores. Appl. Environ. Microbiol. 74:3328-3335.
- Orsburn, B., K. Sucre, D. L. Popham, and S. B. Melville. (2009). The SpmA/B and DacF proteins of Clostridium perfringens play important roles in spore heat resistance. FEMS Microbiol Lett. 291:188-194.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: David Popham presented research results during two seminars at Virginia Tech, Department of Biochemistry, 8/27/07, and at the University of Georgia, Department of Microbiology, 10/4/07. Benjamin Orsburn presented research results in a poster at the Virginia Tech Department of Biological Sciences Research Day, 2/24/07. New fundamental knowledge produced includes a better understanding of which spore structural factors contribute to high levels of heat resistance, and a resulting greater probability of causing food poisoning. Methods developed include those for analysis of C. perfringens spore peptidoglycan wall structure and highly sensitive measurement of dipicolinic acid content using mass spectrometry.
PARTICIPANTS: David L. Popham, P.I. Stephen B. Melville. P.I. Benjamin Orsburn, Graduate Student. Katherine Rodgers, Graduate Student. Katie Sucre, Laboratory technician. Jessica McElligott, Laboratory technician. Sarah Sieb, Work study undergraduate wage employee
Impacts
Knowledge of factors that contribute to spore heat resistance will allow screening of isolates from environmental samples for the presence of strains with greater probability of causing food poisoning. This may lead to the identification of the sources of C. perfringens food poisoning contaminants, which are currently poorly understood. This can eventually lead to improved guidelines for safe food handling.
Publications
- Pending: Orsburn, B., S.B. Melville, and D.L. Popham (2008). Factors contributing to the heat resistance of Clostridium perfringens endospores. Submitted 11-20-07.
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Progress 01/01/06 to 01/01/07
Outputs
Spores have been prepared from ten different strains of C. perfringens, five CPE+ strains and five CPE- strains. Each of these spore preps has been analyzed for relative heat resistance, dipicolinic acid content, core wet density, and spore peptidoglycan structure. The data has revealed a potential correlation between spore heat resistance and the degree of spore peptidoglycan deacetylation. We have begun efforts to create knockout mutants lacking gene predicted to encode peptidoglycan deacetylases and to express a heterologous deacetylase in order to alter the level of deacetylation. Accumulation of these data for the entire series of strains will reveal correlations between spore physical properties and heat resistance. The dacB gene, encoding a carboxypeptidase important in spore wall modification, and the spmAB genes, which are involved in spore dehydration in Bacillus subtilis, have been insertionally disrupted in a C. perfringens strain, and the effects of these
mutations on spore structure and resistance properties are under study. Both of these strains have reduced heat resistance, dramatically in the spmAB mutant. Study of multiple mutant strains will reveal correlations between spore genetic properties and heat resistance. We have completed several steps in the construction of a C. perfringens genomic library in order to identify an apparently novel dipicolinic acid synthesis enzyme by complementation of a B. subtilis dipicolinate synthetase mutant.
Impacts
An understanding of the physical and genetic factors that determine whether Clostridium perfringens strains are able to produce spores with significant heat resistance, will point the way towards methods for evading this agent of food poisoning. A general understanding of the factors influencing heat resistance among all spore-producing bacterial will help in the development of improved food sterilization methods.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Spores have been prepared from eight different strains of C. perfringens, four CPE+ strains and four CPE- strains. Each of these spore preps has been analyzed for relative heat resistance, dipicolinic acid content, and core wet density. Samples have been collected for analyses of spore metal ion contents in the near future. In addition, spore peptidoglycan has been extracted from each preparation in order to carry out structural analyses. An initial analysis of spore peptidoglycan structure has been carried out for one strain. Most of the resulting peptidoglycan fragments have been identified. The use of a new mass spectrometer in the lab is speeding along the complete characterization of peptidoglycan structure, so that it can be applied to samples from all strains. Accumulation of these data for the entire series of strains will reveal correlations between spore physical properties and heat resistance. The dacB gene, encoding a carboxypeptidase important in spore
wall modification, has been insertionally disrupted in one C. perfringens strain, and the effects of this mutation on spore structure and resistance properties are under study. Plasmid constructs have been obtained for the disruption of several other genes expected to play roles in spore heat resistance. Study of multiple mutant strains will reveal correlations between spore genetic properties and heat resistance.
Impacts
An understanding of the physical and genetic factors that determine whether Clostridium perfringens strains are able to produce spores with significant heat resistance, will point the way toward methods for evading this agent of food poisoning. A general understanding of the factors influencing heat resistance among all spore-producing bacterial will help in the development of improved food sterilization methods.
Publications
- No publications reported this period
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