Baraúna Rafael A, das Graças Diego A, Nunes Catarina I P, Schneider Maria P C, Silva Artur, Carepo Marta S P
Laboratório de Genômica e Bioinformática, Centro de Genômica e Biologia de Sistemas, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, 66075-110, Brazil.
REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Campus de Caparica, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
BMC Res Notes. 2016 Sep 20;9(1):447. doi: 10.1186/s13104-016-2250-9.
FapR protein from the psychrotrophic species Exiguobacterium antarcticum B7 was expressed and purified, and subsequently evaluated for its capacity to bind to the promoter regions of the fabH1-fabF and fapR-plsX-fabD-fabG operons, using electrophoretic mobility shift assay. The genes that compose these operons encode for enzymes involved in the de novo synthesis of fatty acids molecules. In Bacillus subtilis, FapR regulates the expression of these operons, and consequently has influence in the synthesis of long or short-chain fatty acids. To analyze the bacterial cold adaptation, this is an important metabolic pathway because psychrotrophic microrganisms tend to synthesize short and branched-chain unsaturated fatty acids at cold to maintain cell membrane fluidity.
In this work, it was observed that recombinant protein was able to bind to the promoter of the fully amplified fabH1-fabF and fapR-plsX-fabD-fabG operons. However, FapR was unable to bind to the promoter of fapR-plsX-fabD-fabG operon when synthesized only up to the protein-binding palindrome 5'-TTAGTACCAGATACTAA-3', thus showing the importance of the entire promoter sequence for the correct protein-DNA interaction.
Through this observation, we demonstrate that the FapR protein possibly regulates the same operons as described for other species, which emphasizes its importance to cold adaptation process of E. antarcticum B7, a psychrotrophic bacterium isolated at Antarctica.
表达并纯化了嗜冷菌南极微小杆菌B7的FapR蛋白，随后使用电泳迁移率变动分析评估其与fabH1 - fabF和fapR - plsX - fabD - fabG操纵子启动子区域的结合能力。构成这些操纵子的基因编码参与脂肪酸分子从头合成的酶。在枯草芽孢杆菌中，FapR调节这些操纵子的表达，因此对长链或短链脂肪酸的合成有影响。为了分析细菌的冷适应性，这是一条重要的代谢途径，因为嗜冷微生物在低温下倾向于合成短链和支链不饱和脂肪酸以维持细胞膜流动性。
在这项研究中，观察到重组蛋白能够与完全扩增的fabH1 - fabF和fapR - plsX - fabD - fabG操纵子的启动子结合。然而，当仅合成至蛋白质结合回文序列5'-TTAGTACCAGATACTAA-3'时，FapR无法与fapR - plsX - fabD - fabG操纵子的启动子结合，从而表明整个启动子序列对于正确的蛋白质 - DNA相互作用的重要性。
通过这一观察结果，我们证明FapR蛋白可能调节与其他物种中描述的相同的操纵子，这强调了其对南极微小杆菌B7冷适应过程的重要性，南极微小杆菌B7是一种在南极洲分离的嗜冷细菌。
