Diversity and functional analysis of light-driven pumping rhodopsins in marine Flavobacteria.

The aims of this study are the description of diversity for proteorhodopsin (PR)-containing flavobacteria in marine environments, the finding of novel photoreceptive membrane proteins, and the elucidation of the effect of light on the growth of three rhodopsin genes containing flavobacterium. We investigated novel sodium ion rhodopsin (NaR) and halorhodopsin (HR) genes from PR-containing flavobacteria that were previously isolated from diverse aquatic sites, mainly from tidal flat sediment (62.5%). In 16 PR-containing isolates, three new types of genes were found. Among these three isolates, one (Nonlabens sp. YIK11 isolated from sediment) contained both the NaR and chloride ion rhodopsin (ClR) - HR type of gene. The sequences showed that the DTE (proton pump), NDQ (sodium ion pump) and NTQ (chloride ion pump) motifs corresponding to the D85, T89, and D96 positions in bacteriorhodopsin (BR) were well conserved. Phylogenetic analysis indicated that three NaR and one ClR grouped within the same clade, as previously reported. Illumination of cell suspensions showed the change in proton pump activity, supporting that one or more rhodopsins are functional. The qRT-PCR study revealed that three rhodopsin genes, especially NaR, are highly induced when they are incubated in the presence of light or in the absence of sufficient nutrients. The expression levels of the DTE, NDQ, and NTQ motif-containing rhodopsin genes in YIK11 correlate positively with illumination, but negatively with nutrient levels. Based on those results, we concluded that light has a positive impact on the relative expression levels of the three rhodopsin genes in the flavobacterium, Nonlabens sp. YIK11, but with no apparent positive impact on growth. Consequently, light did not stimulate the growth of YIK11 as determined by cell numbers in a nutrient-limited or -enriched medium, although it contains and induces three rhodopsins.