Isolation Of Bacteria Ecoding Proterhodopsin (PR) From The Fresh Water

Proteorhodopsins (PRs), seven-transmembrane proteins which uses retinal as its chromophore, are light driven proton pumps. These protein were found widely distributed in oceans and some freshwater around world since the first report about ten years ago. PRs generate proton motive force by translocating protons across the cell inner membrane. Calculation shows that PR-bearing bacterium is one of the numerically richest microorganisms on the Earth, accounting for13%of the total in sea surface water, and with averaged2.5×104PR molecules per cell. Recent studies show that proteorhodopsin phototrophy confers a fitness advantage to marine bacteria during periods of resource deprivation at the ocean’s surface.Even though more than10,000different PR variants was identified through genome sequencing, the physiological function study of PR in its host bacteria are relatively lagged, especially bacteria with PR from freshwater has not been reported yet. In order to better understand the function of PR in fresh bacteria, we attempted to isolat the bacteria with PR from fresh water.In order to isolating the bacteria with PR in the fresh water efficiently, we constructed a vector pHot75BE-vc which replaced the300bp conserved sequence of known PR with1.5kb gene fragment of the sodium alanine symporter gene from Vibrio cholerae and tested the feasibility of with known PR. The results show that this vector can be used for isolating PR gene.The constructed vector pHot75BE-vc was used to isolat the bacteria with PR from different water samples. A strain JL-3containing the PR gene from the water samples of Jinniu Lake successly was isolated. The sequence blast of the300bp conserved sequence shows that this gene has86%sequence identity with the PR gene from Exiguobacterium sibiricum255-15. The full length PR gene was amplified using Self-Formed Adaptor PCR method.The JL-3strain was characterized by morphological, physiological and biochemical characteristics and16srDNA sequence analysis. The result showed that JL-3belongs to Exiguobacterium. So we named it Exiguobacterium sp.JL-3.