Background: Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cellbased reporting of oxygen limitation. Because there are greater than 10 9 cells in a fermentor, one can outfit a vessel with more than 10 9 sensors. Our concept was tested in high density, lab-scale (5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency -one verses four impellers.
Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria. It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.
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