טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentPushkarev Alina
SubjectThe Search for New Ways to Harvest Light using Functional
Metagenomics
DepartmentDepartment of Biology
Supervisor Professor Oded Beja
Full Thesis textFull thesis text - English Version


Abstract

Ion translocating retinal binding rhodopsins are widely distributed among marine and freshwater microbes. The translocation is light-driven, contributing to the production of biochemical energy in diverse microbes. Until today, most microbial rhodopsins had been detected using bioinformatics based on homology to other rhodopsins. In the past decade, there has been increased interest in microbial rhodopsins in the field of optogenetics since microbial rhodopsins were found to be the most useful in vertebrate neuronal systems. In this work, three different assays for detecting microbial rhodopsins were developed. In the first case assay we combine polymerase chain reaction (PCR) and chimeric vector expression to scan environmental niches for rhodopsin variety in a rapid protocol to discover different wavelengths of absorption. In the second assay, by using an array of narrow pH electrodes and LED illumination, we were able to screen a metagenomic fosmid library to detect diverse marine proteorhodopsins and an actinorhodopsin based solely on proton pumping activity. In the third assay we utilized the appearance of color in the presence or retinal by expression of random rhodopsins from the environment and discovered a new family of rhodopsins. We report a previously unrecognized, diverse family, named Heliorhodopsin (HeR), which are distantly related to other rhodopsins and assume the opposite membrane topology compared to type-1 and type-2 rhodopsins, with the N-terminus facing the cell cytoplasm. HeRs have slow reaction to light when expressed in Escherichia coli, suggestive of light sensory activity. HeRs are abundant and distributed globally, being detected in soil, freshwater, marine and hypersaline environments, and in psychrophilic, mesophilic and even hyperthermophilic microbes including archaea, bacteria, eukarya and their viruses. Our assays therefore provide a rather simple phenotypic means to enrich our understanding of microbial rhodopsins with little to no prior knowledge of the genomic content of the environmental entities screened.