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Abstract
Proteorhodopsin (PR) is a widespread form of photoheterotrophy that enables a variety of heterotrophic microbes to subsist in part on light energy. This light activated, retinal-containing transmembrane proton pump can be used for a variety of functions such as generating ATP, flagellar movement, or nutrient transport. Although PR is widespread both phylogenetically and spatially, we have been dependent on secondary proxies to understand its distribution and role in microbial physiology and carbon cycling. These proxies include quantifying the retinal chromophore, DNA and transcript sequencing, qPCR, and spectroscopy. Quantification of protein-level PR expression in environmental and pure culture physiology experiments is necessary to understand how and to what extent PR-based photoheterotrophy contributes to the metabolic energy budget of microbial communities in marine environments. Here, we first developed a method for quantifying protein-level PR expression in pure culture samples. Using an E. coli PR expression construct and wildtype Vibrio campbellii CAIM 519, we enriched our samples for membrane-localized proteins using a carbonate extraction method. We subsequently targeted hydrophobic, transmembrane peptide regions with a chymotrypsin-based digestion approach. When PR expression is relatively high, digestion with chymotrypsin and trypsin before isotopic peptide labeling resulted in consistent quantification of PR. When PR expression is lower, synthetic peptide standards for quantification after membrane enrichment and dual-protease digestion is more reliable.
We then applied our protein-level PR quantification method to understand protein-level gene expression responses to carbon and nitrogen limitation in V. campbellii CAIM 519. We found that PR expression is higher under carbon-limitation than nitrogen-limitation but that V. campbellii does not exhibit growth or survival advantages in the light. Under C-limitation, cultivability and membrane integrity is maintained despite cell dwarfing, the glyoxylate shunt and anaplerotic C fixation is employed, and a stringent response is mediated by the Pho regulon. Under N-limitation, cultivability and membrane integrity are rapidly lost, the central carbon flux through the Entner-Doudoroff pathway is increased, and ammonium is assimilated via the GS-GOGAT pathway. Overall, while protein-level proteorhodopsin expression in V. campbellii is responsive to nutrient limitation, photoheterotrophy does not appear to play a central role in the survival physiology of this organism under these nutrient stress conditions.
Finally, we investigated the impact of subcellular fractionation on our understanding of protein expression and microbial physiology. We analyzed both the cytosolic and membrane fractions from the carbonate extraction method to see if biochemical protein properties such as isoelectric point or hydrophobicity were good predictors for quantification in the membrane or cytosolic fraction. We found that this method declutters highly abundant cytosolic and membrane proteins in their respective fractions, which enables the detection and quantification of less abundant proteins, including PR.