Progress 09/01/06 to 08/31/10
Outputs
OUTPUTS: Listeria monocytogenes is a foodborne pathogen with the unusual but critical ability to grow at refrigeration temperatures. This is a significant factor in its propensity to contaminate ready-to-eat meats leading to morbidity and mortality and expensive product recalls. This project has been directed towards improving the control of the growth of this bacterium, including studies of pertinent basic science.In order to grow at refrigeration temperatures the cytoplasmic membrane of a bacterium must maintain appropriate fluidity. This is mainly a property of the fatty acids. In L. monocytogenes the branched-chain fatty acid anteiso C15:0 plays a critical role.We have studied the mechanism of branched-chain fatty acid switching from iso to anteiso at low temperatures. This was achieved by cloning the gene for the key enzyme in the appropriate part of fatty acid biosynthesis, FabH, over expressing it, and purifying the protein. The FabH enzyme was then used for detailed study of its kinetics at different temperatures with various substrates including 2-methylbutyryl CoA, the precursor of anteiso C15:0.We sought to explore ways in which we could decrease the production of fatty acid anteiso C15:0, and thereby reduce (control) the growth of the organism at low temperatures. To this end we studied the impact of various straight and branched-chain carboxylic acids and branched-chain amino acids on the growth and fatty acid composition of L. monocytogenes. Several of the compounds studied are known food preservatives.One of three major alternative programs is recommended for the control of L. monocytogenes in ready-to-eat meat and poultry products. These are: a post-lethality treatment and an antimicrobial agent or process limiting the growth of L. monocytogenes throughout the shelf-life of the product; an antimicrobial agent or process without a post-lethality treatment; or sanitation control in the processing environment. Severe alkaline conditions such as generated by caustic and quaternary ammonium compounds are commonly used in the food industry for decontamination and sanitization purposes. L. monocytogenes is often able to persist and/or grow under these moderate and severe alkaline conditions.We have studied the alkali shock and alkali tolerance mechanisms of L. monocytogenes through the study of mutants, and by transcriptomic and proteomic approaches.In summary, outputs are completion of studies of the mechanism of switching of branched-chain fatty acids from iso to anteiso branching. Then, application of this knowledge via study of precursor feeding to prevent formation of anteiso C15:0 fatty acids and thereby inhibit growth. Several studies have been completed on the alkali shock and tolerance responses of L. monocytogenes. These outputs have been disseminated via publication in the open scientific literature and by attendance and presentation of findings at scientific conferences such as ISOPOL and the annual meetings of the American Society for Microbiology. PARTICIPANTS: Individuals. P.I. Dr Brian J. Wilkinson. Post doctoral fellow Atul K. Singh. PhD students Mudcharee Julotok, Efstathios Giottis. Partner organizations. University of Ulster, A. T. Still University of Health Sciences, University of Illinois. Collaborators and contacts. E. S. Giotis, D. A. McDowell,I. S. Blair, A. K. Singh, A. Muthaiyan, S. Natesan, C. Gatto, C. O. Rock, R. K. Jayaswal, K. Zhu. Training or professional development. Post doctoral fellow A. K. Singh. PhD students M. Julotok, E. S Giotis. TARGET AUDIENCES: The target audiences for this work are professionals in the field of food safety in academic, government and industry laboratories. Presentation at meetings attended by these target audiences and publications in scholarly journals read be these audiences have been the major ways to communicate new knowledge of the control of Listeria monocytogenes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
FabH, a key enzyme in the biosynthesis of branched-chain fatty acid anteiso C15:0, showed a marked preference for 2-methylbutyryl CoA, the precursor of fatty acid anteiso C15:0 at 30 C,that was markedly increased at 10 C. This implies that a temperature dependent substrate selectivity of FabH is involved in branching switching from iso to anteiso, and in an increased content of fatty acid anteiso C15:0.The fatty acid composition of L. monocytogenes could be modulated by the feeding of branched-chain amino acid, C4, C5, and C6 branched-chain carboxylic acid, and C3 and C4 straight-chain carboxylic acid fatty acid precursors. Several of these precursors decreased the amount of anteiso branched-chain fatty acids, decreased calculated membrane fluidity and growth. However, short branched-chain carboxylic acids were significantly more inhibitory than branched-chain amino acids, even though they had similar effects on fatty acid composition. All short chain carboxylic acids tested inhibited growth significantly, but acetate and compounds such as pyruvate and lactate metabolized via acetate had minimal impact on fatty acid composition. Combining a short-chain carboxylic acid with a compound decreasing the amount of anteiso C15:0 may be a particularly potent anti-listerial combination. The anteiso branched-chain fatty acids increased in alkali stress of L. monocytogenes and were shown to be important in tolerance of alkaline pH through mutant and precursor feeding studies. Exposure of L. monocytogenes to pH 9.5 for 1 hour led to an alkali-tolerance response in that after this exposure cells were protected from normally lethal alkali stress of pH 12.0. Changes in expression of genes involved in virulence, the general stress response, cell division and changes in cell wall structure occurred during alkali adaptation. A role for the alternative sigma factor, SigB, was shown in the alkali tolerance response, particularly against pH 12 stress, but not against subsequent ethanol stress.The genome-wide transcription patterns of alkali-shocked and alkali-adapted L. monocytogenes were studied using DNA microarrays provided by the Pathogen Functional Genomics Resource Center of the National Institutes of Health. Large numbers of genes were altered in their expression both up and down in alkali shock and alkali adaptation. The gene expression patterns involved in alkali shock and adaptation differed. The former reflected a more immediate response of the cell. The letter reflected the changes in gene expression involved in long term growth at high pH.Outcomes of this project are 8 papers published in prestigious journals, plus two further manuscripts in preparation. In terms of impact these papers have been cited a total of more than 32 times already, and the first paper was only published in 2007. The resources provided by the grant led to the hiring of PhD student Efstathios Giotis and Post Doctoral Fellow Atul K. Singh. Also, the financial support was critical in providing funds for laboratory consumables and attendance at meetings.
Publications
- Giotis, E. S., A. Muthaiyan, I. S. Blair, B. J. Wilkinson and D. A. McDowell. 2008. Genomic and proteomic analysis of the alkali-tolerance response (AITR) in Listeria monocytogenes 10403S. BMC Microbiol. 8: 102.
- Giotis, E. S., M. Julotok, B. J. Wilkinson, I. S. Blair, and D. A. McDowell. 2008. Role of sigB factor in the alkaline tolerance response of Listeria monocytogenes 10403S and cross-protection against subsequent ethanol and osmotic stress. J. Food Protect. 71: 1481-1485.
- Singh, V. K., E. S. Giotis, N. R. Chamberlain, M. K. Stuart, and B. J. Wilkinson. 2008. Insertional inactivation of branched-drain α-keto acid dehydrogenase (BKD) in Staphylococcus aureus leads to decreased branched-chain membrane fatty acids content and increased susceptibility to certain stresses. Appl. Environ. Microbiol. 74: 5882-5890.
- Singh, A. K., Y. M. Zhang, K. Zhu, C. Subramanian, Z. Li, R. K. Jayaswal, C. Gatto, C. O. Rock, and B. J. Wilkinson. 2009. FabH selectivity for anteiso branched-chain fatty acid precursors in low temperature adaptation in Listeria monocytogenes. FEMS Microbiol. Lett. 301: 188-192.
- Ledala, N., M. Sengupta, A. Muthaiyan, B. J. Wilkinson, and R. K. Jayaswal. 2010. Transcriptomic response of Listeria monocytogenes to iron limitation and fur mutation. Applied Environ. Microbiol. 76: 406-416.
- Julotok, M., A. K. Singh, C. Gatto, and B. J. Wilkinson. 2010. Influence of fatty acid precursors, including food preservatives, on the growth and fatty acid composition of Listeria monocytogenes at 37 and 10 C. Applied Environ. Microbiol. 76: 1423-1432.
- Giotis, E. S., A. Muthaiyan, B. J. Wilkinson, and D. A. McDowell. 2010. Transcriptome analysis of alkali shock and alkali adaptation in Listeria monocytogenes 10403S. Foodborne Patho. Dis. 7: 1147-1157.
- Singh, A. K., A. V. Ulanov, Z. Li, R. K. Jayaswal, and B. J. Wilkinson. 2010. Metabolomes of the psychrotolerant bacterium Listeria monocytogenes 10403S grown at 37 and 8 C. Under revision for resubmission.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Outputs: Further experiments have been undertaken on analyzing the kinetics of FabH, modeling the activity of the enzyme in the presence of multiple substrates. FabH is a critical enzyme in the biosynthesis of branched-chain fatty acids, particularly anteiso C15:0, which are critical in the growth of the organism at refrigeration temperatures. The fatty acid composition of a branched-chain fatty acid deficient mutant grown in the presence of different precursors has been determined to validate in vivo inferences from studies of FabH kinetics in vitro. Further studies on the influence of various fatty acid precursors, including food preservatives, on the fatty acid composition and growth of Listeria monocytogenes have been carried out. Manuscripts describing these studies are in preparation for publication. An extensive metabolomics study of the small molecular weight compounds present in L. monocytogenes grown at 37 and 8 degrees C have been carried out. I attended the USDA CSREES Principal Investigator's meeting in Washington, DC in November and reported on our FabH and fatty acid studies. Studies have been initiated on the growth of L. monocytogenes on turkey slices with a view to studies of gene expression in this food environment. PARTICIPANTS: Dr Brian J. Wilkinson, Professor of Microbiology, Dr Atul Singh, Postdoctoral Fellow, Mudcharee Julotok, PhD student, all at Illinois State University, Normal, IL. Collaborator: Dr Charles Rock, Saint Jude Research Hospital, Memphis, TN. TARGET AUDIENCES: Target audiences are food scientists interested in food safety in academia, government, and industry. Two publications have appeared this year and a presentation has been made to CSREES principal investigators. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The affinity (Km) of FabH for 2 methylybutyryl CoA increased at low temperatures thus ensuring the high content of fatty acid anteiso C15:0, which is important for growth of the organism at low temperatures. Other CoA substrates effectively competed with 2 methylbutyryl CoA at 30 degrees C, but were much less effective at 10 degrees C. Consistent with the kinetic properties of FabH, only a limited number of fatty acid precursors, related to 2 methylybutyrate, were able to modulate the fatty acid composition and inhibit the growth of Listeria at low temperatures. Combinations of precursors may be an effective means of controlling the growth of Listeria at low temperatures. Major changes were noted in the metabolome of L. monocytogenes grown at 8 compared to 37 degrees C. This helps us understand the mechanisms involved in the psychrotolerance of this foodborne pathogen. Two papers were published on the alkali tolerance response of L. monocytogenes. Alkali stress induced extensive changes in gene and protein expression. The alternative sigma factor, SigB, played a role in the alkali stress response. Alkali stress induced cross protection against osmotic and ethanol challenges. This has implications for food safety and preservation given the role of these conditions in achieving them.
Publications
- Giotis, E. S., Muthaiyan, A., Blair, I.S., Wilkinson, B. J., and McDowell, D.A. (2008). Genomic and proteomic analysis of the alkali-tolerance response (AlTR) in Listeria monocytogenes 10403S. BMC Microbiology 8: 102.
- Giotis, E. S., Julotok, M., Wilkinson, B. J., Blair, I. S., and McDowell, D. A. (2008). Role of sigma factor B in the alkaline tolerance response of Listeria monocytogenes 10403S and cross-protection against subsequent ethanol and osmotic stress. J. Food Protection 71: 1481-1485.
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Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: Experiments have been undertaken to clone, overexpress, and purify the enzyme FabH, which is a key enzyme in fatty acid biosynthesis. Biosynthesis of fatty acid anteiso C15:0, which imparts an essential fluidity to the cytoplasmic membrane, is a critical aspect of the growth of the foodborne pathogen Listeria monocytogenes at refrigeration temperatures. The kinetics and substrate specificities of the enzyme at 10 and 30 degrees C have been analysed. The ability of various fatty acid precursors to modulate the fatty acid composition of L. monocytogenes have been determined with a view to reducing growth at low temperatures. Experiments that had been carried out by a PhD student from the University of Ulster on a 6 month visit have been analysed and prepared for publication. The mechanism of the alkali tolerance response of L. monocytogenes was studied. I attended ISOPOL XVI, the 16th International Symposium on Problems of Listeriosis in Savannah, GA in March 2007.
PARTICIPANTS: Participants: Dr Brian J. Wilkinson, Professor of Microbiology, Dr Atul Singh, Postdoctoral Fellow, Mudcharee Julotok, PhD student, all at Illinois State University, Normal, IL . Collaborator: Dr Charles Rock, Saint Jude Children Research Hospital, Memphis, TN. Training and Professional Development: Efstathios Giotis a PhB student at the University of Ulster spent 6 months in the laboratory studying the mechanism of alkali tolerance in Listeria monocytogenes.
TARGET AUDIENCES: Target audiences are food scientists in academia, geovernment and industry. A publication and an abstract resulted from work in this forst year.
Impacts
The study of the kinetics of FabH activity showed that at low temperatures the affinity of the enzyme for 2-methylbutyrate, the precursor of fatty acid anteiso C15:0, increases two fold, and the turnover number of the enzyme increases. This provides a plausible mechanistic explanation of the switching of fatty acid branching to anteiso branching that occurs at low temperatures. The fatty acid composition of L. monocytogenes was found to be subject to modulation by inclusion of fatty acid precursors in growth medium. In general, precursors of odd- and even- numbered iso fatty acids, and straight chain fatty acids increased the proportion of iso C15 and C17, and iso C14 and C16, and C14 and C16 fatty acids in the lipids of the organism respectively. Correlations were noted in the effects on fatty acid composition and growth inhibitory properties of some of the precursors, particularly at low temperatures. It was shown that anteiso branched chain fatty acids are important
in the tolerance of alkaline stress by L. monocytogenes. Proteomic and transcriptional profiling studies revealed changes in the expression of various genes and proteins in L. monocytogenes exposed to alkali stress. The alternative sigma factor SigB that plays a role in controlling transcription was shown to have a role in alkali tolerance.
Publications
- Giotis, E. S., McDowell, D. A., Blair, I. S., and Wilkinson, B. J. (2007). Role of branched-chain fatty acids in pH stress tolerance in Listeria monocytogenes. Applied and Environmental Microbiology, 73: 997-1001.
- Zhu, K., Bayles, D.O., Bigelow, L.J., and Wilkinson, B. J. (2007). Identification of potential cold-related genes in Listeria monocytogenes. Abstracts of ISOPOL XVI The 16th International Symposium on Problems of Listeriosis, Savannah, GA March 20-23, 2007.
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