| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 98 === Environmental microorganisms have to deal with the fluctuations of osmolarity often. To maintain homeostasis in cytoplasm, the osmoregulation by accumulating compatible solutes is a widespread strategy in Archaea, Bacteria, and Eukarya. Among all of the compatible solutes, the most adopted ones are proline and glycine betaine. Accumulation of compatible solutes for osmoadaptation is also found in methanogenic archaea species Methanohalophilus portucalensis strain FDF1, which can de novo synthesize osmolyte glycine betaine and Nε-acetyl-beta-lysine and also transport glycine betaine through ABC transporter bta system. In this study, a secondary transporter gene MpputP from M. portucalensis FDF1 was detected by Southern Blot with partial Na+/ proline symporter probe. The 1589 bp partial MpputP gene was obtained which included most sequence (99%) except the stop terminal area as compared to the similar sequence found in Methanohalophilus mahii Mmah_1119 (1599 bp). The MpputP can be translated into 529 amino acids, and the transmembrane numer is predicted to be 13 by hydropathy plot, which is the same as Escherichia coli Na+/ proline symporter PutP. The residues Arg40, Ser57, Asp187, Ser340, and Thr341 in E. coli PutP which are proved to be or nearby the sodium binding site are conserved in MpPutP. To understand if MpputP is involved in the osmoregulation like the PutP (OpuE) in Bacillus subtilis, the transcriptional levels of MpputP is measured under osmotic stress. The relative transcriptional levels of MpputP measured by Northern Blot showed that the expression of MpputP was retained at a stabile level and reduced during higher osmotic stress (3.3 M), whether with or withour proline. To understand if the MpputP is also involved in the general metabolism in the growth of M. portucalensis FDF1, its expression is measured from lag, early log, mid log,late log and stationary phases. The results showed that M. porucalensis FDF1 maintained a steady transcriptional levels of MpputP during log phases and reduced by three- fold at the stationary phase. MpputP gene might play a role in cellular metabolism, not related to the salt shock response.
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