| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 90 === By screening developmentally morphological mutants generated by the restriction enzyme-mediated integration (REMI) mutagenesis, we found a mutant that formed trails that lift the slug off substratum and with age could collect droplets of fluid along their length on bacterial lawn agar. The mutant cells were delayed to aggregate and had a defect in culmination (slugger mutant). The full-length gene was constructed from the plasmid rescued fragments and predicted to encode a membrane transporter, MtpA, containing fourteen transmembrane domains. The mtpA gene is single copy gene and the temporal expression is a very low level at vegetative stage, increases substantially about 12-16 hr and then decreases. In synergy experiments, they participated efficiently in fruiting bodies formation when developing along with wild-type cells, suggesting that MtpA affects the production or release the intracellular signal pathways. The act15::lacZ-marked mtpA- cells were excluded from the prestalk zone in slug stage and distributed over up and low cup at culmination stage, indicating that the mtpA- cells should have an inherent defect in the prestalk differentiation. The development of mtpA- cells was hypersensitive to the change of the environmental ammonia and propionate concentration but its aggregation-ability was slightly rescued by the azide. Disrupted mtpA gene leads to cytosolic acidification by FACS measurements. It is likely the reason to cause to delay to aggregate and over-sensitive to the environmental change. Knocked out mtpA gene in rapidly developing and sporogenous rdeC mutant HTY217 that lacks functional PKA regulatory and therefore has constitutive PKA catalytic activity, the phenotype of the double mutant HTY217/ mtpA- is partially like to the parental strain but is able to form spores. Our results indicate that MtpA plays as a regulator of the ammonia in the early aggregation via possibly regulating the intracellular pH and is required for the intercellular signal that controls the terminal differentiation of the prespore and prestalk cells via regulating the PKA activity.
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