Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing, China.
FEMS Microbiol Lett. 2013 Jul;344(1):10-7. doi: 10.1111/1574-6968.12144. Epub 2013 May 1.
Proteorhodopsins (PRs), light-driven proton pumps, constitute the largest family of the microbial rhodopsins. PRs are widely distributed in the oceanic environment and freshwater, but no bacteria with PRs have been isolated from freshwater so far. To facilitate isolation of the bacteria with PR genes, we constructed a vector system that can be used to clone potential PR genes and render color changes when overexpressed in Escherichia coli. Using this method, we successfully isolated a strain with PR gene from freshwater and identified it as Exiguobacterium sp. JL-3. The full length PR gene was then cloned using the SEFA PCR method. Protein sequence alignment showed that JL-3_PR shares high sequence identity (84-89%) with the PRs from Exiguobacterium strains, but low sequence identity (< 38%) with other PRs. Surprisingly, we could not detect any proton-pumping activity in the native JL-3 cells and protoplasts, but the recombinant JL-3_PR do pump protons when overexpressed in E. coli. Sequence analysis further revealed that the PRs from Exiguobacterium had an unusual lysine as the proton donor instead of the typical acidic residue. These data suggest that JL-3_PR is a sensory PR rather than a proton pump.
细菌视紫红质（PRs）是光驱动质子泵，构成微生物视紫红质中最大的家族。PRs 广泛分布于海洋和淡水中，但迄今为止尚未从淡水中分离出具有 PRs 的细菌。为了便于分离具有 PR 基因的细菌，我们构建了一个载体系统，可用于克隆潜在的 PR 基因，并在大肠杆菌中过表达时发生颜色变化。使用这种方法，我们成功地从淡水中分离出一株具有 PR 基因的菌株，并将其鉴定为极端嗜盐古菌 JL-3。然后使用 SEFA PCR 方法克隆全长 PR 基因。蛋白序列比对表明，JL-3_PR 与极端嗜盐古菌菌株的 PRs 具有很高的序列同一性（84-89%），但与其他 PRs 的序列同一性较低（<38%）。令人惊讶的是，我们在天然 JL-3 细胞和原生质体中均未检测到任何质子泵活性，但重组 JL-3_PR 在大肠杆菌中过表达时确实可泵出质子。序列分析进一步表明，极端嗜盐古菌的 PRs 具有不寻常的赖氨酸作为质子供体，而不是典型的酸性残基。这些数据表明，JL-3_PR 是一种感应 PR，而不是质子泵。
