Using lasers to probe the transient light absorption by proteorhodopsin in marine bacterioplankton.

We constructed an experimental apparatus that used lasers to provide the probe beams for measuring the transient absorption kinetics of bacterioplankton that contain proteorhodopsin, a microbial protein that binds retinal and is analogous to animal rhodopsin. With this approach we were able to observe photocycles characteristic of functioning retinylidene ion pumps. Using light from lasers instead of broadband sources as transmittance probe beams can be advantageous when examining optically dense, highly scattering samples such as concentrated microbial cultures. Such a laser-based approach may prove useful in shipboard studies for identifying proteorhodopsin in whole cell suspensions concentrated from seawater.

The SAR92 clade: an abundant coastal clade of culturable marine bacteria possessing proteorhodopsin.

Proteorhodopsin (PR) is a protein that is abundant in marine bacterioplankton. PR is hypothesized to be a light-dependent proton pump, thus creating a proton gradient that can be used for energy production without electron transport. Currently, the only culture that has been reported to possesses PR is the highly abundant alphaproteobacterium "Candidatus Pelagibacter ubique" (SAR11 clade), but surprisingly, its growth in batch culture was not enhanced by light. Here, we present the first cultured gammaproteobacterium that possesses a PR gene. Genome sequencing and analysis of HTCC2207 showed that the PR gene is present as a lone transcriptional unit directly followed by an operon containing genes that are presumably involved in the synthesis of retinal, the chromophore of PR. Half-time decay times of different PR intermediates in native HTCC2207 cells ranged between 2 and 15 ms, and the absorbance maximum of PR was determined to be 528 nm. Proteorhodopsin was identified in three additional strains, using a specific PCR assay on other cultured members of the SAR92 clade. Phylogenetic analyses of the PR genes determined that they form a deeply rooting cluster not closely related to any PR genes recovered so far. Fluorescence in situ hybridization and RNA blots showed that the SAR92 clade reaches up to 10% of the total bacterial population in surface waters close to the Oregon coast and decreases over depth and distance from the shore. Although the growth of HTCC2207 is limited by the amount of available carbon that is present in the medium applied, these cultures do not grow at higher rates nor do they have higher growth yields when incubated under light.