Development of Robust Methods for NMR-Based Microbial Metabolomics Using the Temperature-Dependent Coral Pathogen Vibrio coralliilyticus

 

Arezue F. B. Boroujerdi,1,2 Maria Vizcaino,3,2 Jonathan Jefferies,4 Tracey B. Schock,1,2 Pamela J. Morris,5,2 and Daniel W. Bearden1,2

 

1National Institute of Standards and Technology, Hollings Marine Laboratory, Charleston, SC 29412

2Hollings Marine Laboratory, Charleston, SC 29412

3Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC 29412

4North Carolina Agricultural & Technical State University, Greensboro, NC 27411

5Department of Biology, College of Charleston, Charleston, SC 29412

 

Metabolomics, or the observation of an organism’s metabolome, is an informative way to observe a ‘snapshot’ of a particular metabolic state which is indicative of an organisms’ physiological response to stressors. The bacterium Vibrio coralliilyticus, which has been found in association with corals globally (e.g., Mediterranean, Caribbean, Red Sea, Indian Ocean) and linked to coral disease, exhibits a temperature-dependent pathogenicity similar to many other Vibrio species. The temperature-dependence of V. coralliilyticus in regard to its metabolome is of particular interest and has been investigated by our lab,1 and the results consistently show a distinct metabolic fingerprint based on temperature. In previous work, as well as the work presented here, nuclear magnetic resonance (NMR) spectra were obtained of methanol-water extracts of intracellular metabolites (the endo-metabolome) from multiple samples of the bacteria cultured into late stationary phase at both 27 °C (when this organism exhibits virulence) and 24 °C (an avirulent form). The spectra were subjected to principal components analysis (PCA), and significant temperature-based separations in PC1, PC2, and PC3 dimensions were observed. Betaine, succinate, and glutamate were also consistently identified in loading plots as metabolites that cause the greatest temperature-based separations in the PC scores plots. In order to optimize the analysis of stressor response in this system, we have undertaken efforts to reduce the amount of time required to obtain suitable samples and to address data quality issues. We have shortened the cell culture period from 3.5 days to 2 days. We have examined the source variability within each culture time and also compared to one another. The temperature dependence is present in each experiment (or batch) indicating a fundamental difference between the virulent and avirulent V. coralliilyticus; however, controlling the consistency from batch to batch has been found to be challenging. We report on methods development for developing a robust microbiological platform for metabolomics investigations.

1. Boroujerdi, A.F.B.; Vizcaino, M.I.; Meyers, A.; Pollock, E.C.; Huynh, S.L.; Schock, T.B.; Morris, P.J.; Bearden, D.W. “NMR-Based Microbial Metabolomics and the Temperature-Dependent Coral Pathogen Vibrio coralliilyticusEnvironmental Science & Technology, 2009, 43:7658-7664.