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

Abstract

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.

Production of extracellular vesicles with light‐induced proton pump activity by proteorhodopsin‐containing marine bacteria

“…Sediminicola sp. YIK13 and N. antaticus AKS622 T strains were isolated from tidal flat sediments on Yeongheung Island at the coast of the West Sea, Republic of Korea (Kwon et al, ) and from a glacial ice core at the coast near King Sejong station on King George Island, Antarctica (Kwon, Yang, Kwon, & Kim, ), respectively. The YIK13 and AKS622 T strains were routinely cultured on Marine agar 2216 (MA; Difco, USA) or ZoBell medium (ZB; 5‐g peptone, 1g yeast extract, 0.01g FePO 4 per liter of 20% distilled water, and 80% aged seawater) and incubated at 30°C and 15°C, respectively, with continuous shaking at 120 rpm.…”

“…Remaining cell cultures were centrifuged, filtered through a 0.2 μm syringe filter, and then vesicle samples were resuspended after ultracentrifugation under the same conditions as described above. The fixed cells and EV numbers were analyzed using a qNano (Izon Science, New Zealand) instrument according to a previously described method (Choi et al, ; Kwon et al, ). Cells and EVs were diluted 1,000‐fold into 0.2‐μm filtered 1 × TBT buffer and were measured using NP1000 (500–2000 nm) and NP150 (75–300 nm) membranes, respectively.…”

“…Proton pump activity was measured according to a previously described method (Kwon et al, ). Cells grown for 72 hr and purified EVs were diluted to a concentration of 2 × 10 9 ml −1 and 2 × 10 11 ml −1 , respectively using the qNano method described above.…”

“…The concentration of all genomic DNA was determined using a NanoDrop 2000 spectrometer (Thermo Scientific, Waltham, USA), and the genomic DNA was preserved at −20°C. PCR amplification of 16S rRNA, pro , blh (15,15′‐ β ‐carotene dioxygenase), and crtI (phytoene dehydrogenase) genes was conducted according to a previously described method (Kwon et al, ). The amplified products were separated by agarose gel electrophoresis.…”

“…Sediminicola sp. YIK13 is one of the several strains containing proteorhodopsin (PR) isolated from marine sediments (Kwon, Kim, Jung, & Kim, ). PR phototrophy is a likely source of significant microbial processes in marine environments, where light‐driven proton pumps convert sunlight to proton gradients that generate energy for processes, such as cell growth and maintenance (Béjà et al, ; Martinez, Bradley, Waldbauer, Summons, & DeLong, ).…”