Metatranscriptomic and physiological analyses of proteorhodopsin-containing marine flavobacteria

Abstract

Proteorhodopsin (PR) is a seven-helix integral membrane protein that uses retinal as a chromophore. PRs transport protons from the cytoplasmic (CP) to the extracellular (EC) side of the cell membrane utilizing the energy from light. Since PR was first discovered in marine Gammaproteobacteria, similar types of rhodopsins have been found in all three domains of life (archaea, bacteria, and eukaryotes). Recent studies have suggested that some flavobacteria showed a light-dependent increase in cell yield and growth rate of cultures grown in low carbon media. Although their function as proton pumps with energy-yielding potential has been suggested in some strains, the photophysiological role of proteorhodopsins remains largely unexplored. This thesis describes the functional characterization of PR-containing flavobacteria previously identified from a (Gomez- Consarnau et al. 2007; Yoshizawa et al. 2012). We describe here experiments performed to help understand how PR-containing marine flavobacteria respond to varied DOC concentrations during light-dependent growth, using growth curve observations, inhibitor experiments and transcriptomic analyses. The light-dependent growth effects demonstrated a dependence on carbon concentration, decreasing at increasing carbon concentration in all PR-harboring strains examined in this study. Interestingly however, the inverse results were observed at high carbon concentration (48.5 mM C) which resulted in higher cell yields when grown in the dark than in the light. Growth experiments using 2-(4-methylphenoxy)triethylamine (MPTA) as an inhibitor of f-carotene synthesis were performed for the representative isolates, Dokdonia sp. MED134 and Gilvibacter sp. SZ-19, at low and high concentrations of DOC. These experiments showed that inhibition of retinal biosynthesis abolished the lightstimulated growth response at low DOC concentrations. Transcriptomic experiments were designed to determine the effect of DOC concentration on gene expression of PR-containing MED134 under light and darkness. The results show the both PR and retinal biosynthetic enzymes exhibit significant upregulation in the low carbon condition when they exposed to the light. Among protein-coding transcripts of high carbon concentration, beta-oxidation-associated proteins were expressed at significantly higher levels in the dark. This work furthers our understanding of the details of light-enhanced growth rates and cell yields in diverse marine flavobacterial isolates, and demonstrate proteorhodopsinassociated light-dependent growth effects at various carbon concentrations in several different flavobacterial proteorhodopsin photosystems.