Discovery-oriented teaching: The use of chimeric proteorhodopsins for the development of a lab curriculum in marine microbiology and for the discovery of natural red-shifted proteorhodopsins

Student laboratory courses in microbial ecology are conducted as condensed courses, where theory and wet lab work are combined in a very intensive short time period. During the last decades, the study of marine ecology became molecular-based, most of the research requiring sequencing that is often not available at the course facilities, and takes more time than the course allows. Therefore, students often find themselves obtaining and analyzing results weeks after the course ended. In this work, we describe a protocol combining molecular and functional methods for analyzing microbial rhodopsins, with visible results in only 4-5 days. We created a chimeric plasmid suitable for direct expression of environmentally retrieved proteorhodopsins (PRs) using PCR, and cloned it into Escherichia coli cells for visualization and functional analysis. Using this assay, we discovered several exceptional cases of PRs whose phenotype is different than predicted based on sequence only. We expect this assay to improve the marine Microbiology laboratories experience fors tudents, gaining fast feedback and reward for their work, and to promote the use of functional assays for discovery of marine diversity that was masked by sequence-based methods. Originality-Significance Statement We developed an improved chimeric proteorhodopsin (PR) plasmid suitable for direct expression of environmentally retrieved PRs for visualization and functional analysis. This vector was originally envisioned for better design of lab curriculum in marine microbiology. Using this vector we were also able to detect new natural red-shifted PR variants.


Originality-Significance Statement
We developed an improved chimeric proteorhodopsin (PR) plasmid suitable for direct expression of environmentally retrieved PRs for visualization and functional analysis. This vector was originally envisioned for better design of lab curriculum in marine microbiology. Using this vector we were also able to functional analysis. Using this assay, we discovered several exceptional cases of PRs whose phenotype is different than predicted based on sequence only. We expect this assay to improve the marine 3 microbiology laboratories experience for students, gaining fast feedback and reward for their work, and to promote the use of functional Introduction 4 5 Microbial retinal-based ion pumps were first discovered in the hypersaline dwelling archaea Halobacterium salinarum (Oesterhelt and Stoeckenius, 1971). Since then, rhodopsins were found in various microorganisms, spanning the three domains of the tree of life (Béjà et al., 2013;Pinhassi et al., 2016) and even detected in viruses (Yutin and Koonin, The first bacterial rhodopsin was discovered in the abundant uncultured proteobacterial SAR86 group, and was therefore named proteo-rhodopsin (PR) (Béjà et al., 2000). PRs are light driven proton pumps, that absorb light in the blue or green regions of the visible light spectrum (i.e. spectral tuning), The search for novel rhodopsins is mostly based on sequence homology screens, by utilizing metagenomic data collected from various environments, 6 5 or by PCR performed on environmental DNA samples using degenerate primers designed for conserved regions in rhodopsin proteins. Only two functional screens were employed to search for new rhodopsins, (i) based on 5 colony colour by plating fosmid libraries on retinal containing plates (Martínez et al., 2007), and (ii) based on pH changes of fosmid clones in response to 7 0 illumination (Pushkarev and Béjà, 2016).
In order to combine sequence homology and function based methods, we devised a protocol based on a previously designed chimeric PR construct ( Supplementary Fig. S1). This chimeric construct was used to express individual partial clones recovered from the environment via PCR 7 5 amplification, cloning and sequencing (Choi et al., 2013). Here, we improved the chimeric PR construct to enable the screening of diverse partial PR sequences directly from the environment, enabling rapid visualization of the PR activity. In this manner, we developed a simple way to demonstrate the concept of niche adaptation and spectral tuning to undergraduate and 8 0 graduate students. Student labs in marine microbial ecology or marine microbiology are usually operated as condensed courses ranging between 10 to 30 days. Hence, there is a need for short experiments that can demonstrate some proof of concepts within the timeframe of the course.
In this work we present a protocol ( Fig. 1)  We used a chimeric green PR protein (GPR) vector with designed restriction sites (Choi et al., 2013) and replaced the middle part of the PR with a "stuffer" DNA sequence, to avoid the high background (reddish colonies) observed with the original chimeric GPR vector ( Supplementary Fig. S1). This introduced a stop codon after the downstream restriction site and hence an 9 5 inactive PR.
The DNA samples from all depths (0 to 100 meter with 20 meter intervals), tested positive for PRs, resulted in two PCR amplicons of ~400 bp and ~330 bp ( Supplementary Fig. S2). Two 96-well plates of clones were picked for each depth to detect coloured colonies for further study (Fig. 2).
Out of a total of 1,152 colonies, 45 had visible colour and were chosen for further characterization. As expected, the depth from which the sample was collected from, showed the expected spectral tuning of the PRs; with yellow absorbing PR (YPR; purple colonies) and GPR (red colonies) dominating surface waters, while BPRs (orange colonies) were mostly found in deeper performed in order to understand their spectral tuning mechanism.
In order to verify that the clones are functional proton pumps, a lightdriven proton pumping assay was performed. Proton pumping activity was observed in all chimeric PRs with varying intensities (Supplementary File S2).
This shows that although the clones obtained are chimeric, they are able to 1 3 0 transport protons across the membrane and therefore could be used for teaching and discussing various aspects connected to rhodopsins; the use of uncouplers, change of membrane potential, proton pumping under various wavelengths, the conversion of light energy to potential or chemical energy,

etc.
1 3 5 In this study we employed two strategies for obtaining absorption spectra; whole cell measurement, and purified protein measurement. A comparison of the two methods is presented in supplementary material

Concluding remarks
Expression of the environmental fragments depends on primer matching, correct length of PCR product, frame and compatibility to the chimeric 1 5 0 construct, and therefore it would be interesting to compare the chimeric ligation results to standard TA cloning of the PCR fragments. This would allow the estimation of the rhodopsins "left in the dark" in such an experiment, and comparing the sequences to the ones that underwent expression in the chimeric vector. This could also be useful for altering the primers to better at room temperature for the detection of cell colour. subtraction of negative control signal, and purified protein spectra.

Sequencing and phylogenetic analysis
All clones were extracted using standard alkaline lysis miniprep protocol and sequenced using standard M13R primer (GCGGATAACAATTTCACACAGG,

Acknowledgments
The authors would like to thank the captain and crew of the R/V Sam

Conflict of Interest
The authors declare no conflict of interest.