Date of Award
Doctor of Philosophy
Shawn R. Campagna
David C. Baker, Craig E. Barnes, Alison Buchan
Quorum sensing (QS) is a phenomenon that allows bacteria to communicate with each other. Small molecules known as autoinducers are synthesized and released by bacteria, and once enough members of the community are around to ensure survival, i.e. quorum, a phenotype, e.g. bioluminescence, is expressed. There are two types of QS molecules, intra- and inter-species.
S-4,5-Dihydroxy-2,3-pentanedione (DPD) is a byproduct of the activated methyl cycle which recycles methionine. This has led to the discussion as to whether DPD is a metabolic byproduct or is the interspecies signal as proposed previously. The detection and quantitation of DPD however, has not been incredibly efficient. We have designed and synthesized a derivatizing agent to detect DPD using SRM based mass spectrometry. Using this technology combined with a previously synthesized isotope version of DPD, we were able to derivatize and quantitate DPD.
Acylhomoserine lactones (AHLs) are a type of intra-species QS molecules in which the acyl chain can be differentiated among species. Previously there has been no easy way to quantitate these molecules despite their ease of detection through mass spectrometry. Herein we have designed an internal standard that incorporates two deuteriums into the conserved portion of the lactone and designed a convergent synthesis that uses D2-Fmoc-Methionine as a common intermediate to make a range of AHLs standards.
Using these technologies for isotope-dilution mass-spectrometry (MS) we were able to quantitate both DPD and AHLs from cultures of Escherichia coli, Vibrio harveyi, and Vibrio fischeri. From these measurements we learned the E. coli does not use DPD as a cell density regulator while V. harveyi does. Using SRM based MS we discovered a new AHL produced by V. fischeri and showed a temporal pattern in synthesis that could be used to determine the phase of growth in this organism.
Lastly, the metabolic effects of DPD and AHLs on lab and mixed microbial cultures from the Atlantic Ocean were studied. Each autoinducer showed opposing effects on the metabolism of these cultures showing that QS has the ability to alter the physiology of these organisms and potentially effecting dissolved organic matter in the ocean.
May, Amanda, "Quorum Sensing and Metabolism in Marine Environments. " PhD diss., University of Tennessee, 2013.
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