The Use of a Chimeric Rhodopsin Vector for the Detection of New Proteorhodopsins Based on Color

Abstract

Student microbial ecology laboratory courses are often conducted as condensed courses in which theory and wet lab work are combined in a very intensive short time period. In last decades, the study of marine microbial ecology is increasingly reliant on molecular-based methods, and as a result many of the research projects conducted in such courses require sequencing that is often not available on site and may take more time than a typical course allows. In this work, we describe a protocol combining molecular and functional methods for analyzing proteorhodopsins (PRs), with visible results in only 4–5 days that do not rely on sequencing. PRs were discovered in oceanic surface waters two decades ago, and have since been observed in different marine environments and diverse taxa, including the abundant alphaproteobacterial SAR11 group. PR subgroups are currently known to absorb green and blue light, and their distribution was previously explained by prevailing light conditions – green pigments at the surface and blue pigments in deeper waters, as blue light travels deeper in the water column. To detect PR in environmental samples, we created a chimeric plasmid suitable for direct expression of PRs using PCR amplification and functional analysis in Escherichia coli cells. Using this assay, we discovered several exceptional cases of PRs whose phenotypes differed from those predicted based on sequence only, including a previously undescribed yellow-light absorbing PRs. We applied this assay in two 10-days marine microbiology courses and found it to greatly enhance students’ laboratory experience, enabling them to gain rapid visual feedback and colorful reward for their work. Furthermore we expect this assay to promote the use of functional assays for the discovery of new rhodopsin variants.

Heterologous expression and cell membrane localization of dinoflagellate opsins (rhodopsin proteins) in mammalian cells

“…Microbial rhodopsin heterologous expression has mostly been conducted using Escherichia coli cell system (Friedrich et al 2002;Inoue et al 2013;Pushkarev and Béjà 2016;Pushkarev et al 2018). E. coli is a good choice for expressing bacterial PRs.…”

Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design

“…Position 93 is known to be diversified in the PR family (the well-known position 105 in PRs). Green-light-absorbing PRs (GPRs) have leucine as a BR, whereas glutamine is conserved in blue-light-absorbing PRs 5 , 26 . This color-tuning effect by the difference between leucine and glutamine is known as the “L/Q-switch” 42 .…”

“…These are successful examples of knowledge-driven experimental approach. Recently, a new method using a chimeric rhodopsin vector and functional assay was reported to screen the λ max and proton transport activities of several microbial rhodopsins that are present in specific environments 26 . This method identified partial sequences of red-shifted yellow (560–570 nm)-absorbing proteorhodopsin (PR), the most abundant outward H + -pumping bacterial rhodopsin subfamily, from the marine environment.…”

Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design

“…Position 93 is known to be diversified in the PR family (the well-known position 105 in PRs). Green-light-absorbing PRs (GPRs) have leucine as a BR, whereas glutamine is conserved in blue-light-absorbing PRs 4,18 . This colour-tuning effect by the difference between leucine and glutamine is known as the “L/Q-switch” 33 .…”