Genetic study on the interaction between ribose-binding protein and the membrane permease

Abstract

A number of binding-proteins located in the periplasmic space are required for active transport of nutrients and chemotactic signal transmission into the cell. Among them, ribose-binding protein bound with ribose interacts with Trg and ABC transporter, permease embedded the inner membrane. To elucidate the original mechanism of chemotaxis and transport that are mediated by ribose-loaded RBP at periplasmic space a new mutants screening experiment is conducted, unlike conventional screening, utilizing trz, chimeric receptor. Trz is transmembrane, composed of N-domain of Trg, hemoreceptor and C-domain of EnvZ, osmosensor. Transmission of signal through Trz into cell is the process which ribose-loaded RBP interact with Trg domain of Trz, to express ompC-lacZ fusion gene of chromosome through EnvZ domain of Trz and ompR. As lacZ is connected to ompC, if signal is to be transmitted normally, red colony can be formed at McConkey plate, and if RBP is to be defected due to mutation, white colony can be formed. This phenotype is utilized for the isolation of taxis mutant on the McConkey plate added allose. For the isolation of transport mutation, the following characteristics are adopted: when a defect is caused by RBP mutation at the interaction between RBP and permease, cells fails to uptake the ribose that came out leaked into periplasm, it lead to the binding of these ribose with mutated RBP, and red colony can be formed by sending signal into cytoplasm of cell on the McConkey plate without ribose. The isolated taxis mutants showed somewhat weak defects, or many of taxis and transport had defects. The taxis mutations were located in the area of either binding-cleft or hinge. All transport mutations failed to form ring and also failed with uptake of radioactive ribose. Concerning mutational changes, many of the same mutation were obtained at 3 sites and all of them were located on the surface. Therefore, it is suggested that these residue occupied considerably importa...