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|標題:||Analysis of the AAA+ chaperone clpB gene and stress-response expression in the halophilic methanogenic archaeon Methanohalophilus portucalensis||作者:||Shih, C.J.
|關鍵字:||molecular chaperones;escherichia-coli;glycine-betaine;substrate;recognition;methanosarcina-mazei;sulfolobus-shibatae;sequence;alignment;coiled coils;sp nov.;proteins||Project:||Microbiology-Sgm||期刊/報告no：:||Microbiology-Sgm, Volume 153, Page(s) 2572-2583.||摘要:||
ClpB is a member of the protein-disaggregating chaperone machinery belonging to the AAA+ superfamily. This paper describes a new clpB gene from the halophilic methanoarchaeon Methanohalophilus portucalensis, which has not been reported previously in Archaea. The partial sequence of clpB was identified from the investigation of the salt-stress response of Meh. portucalensis by differential-display IRT-PCIR (DDRT-PCR). Furthermore, the complete clpB sequence (2610 nt) and its upstream genes encoding the type I chaperonin GroEL/ES were obtained through inverse PCR1 Southern hybridization and sequencing. The G + C ratio of clPB is 49.6 mol%. The predicted CIpB polypeptide contains 869 aa and possesses a long central domain and a predicted distinctly discontinuous coiled-coil motif separating two nucleotide-binding domains (NBD1 and NBD2). NBD1 has a single Walker A and two Walker B motifs and NBD2 has only one of each Walker motif, a characteristic of HSP100 proteins. Two repeated Clp amino-terminal domain motifs (ClpN) were identified in CIpB. The putative amino acid sequence shared 75.6 % identity with the predicted clpB homologue annotated as ATPase AAA-2 of Methanococcoides burtonii DSM 6242. Preliminary phylogenetic analysis clustered Meh. portucalensis CIpB (MpClpB) with the low G + C Gram-positive bacteria. Stress response analysis of clpB by Northern blotting showed up to 1.5-fold increased transcription levels in response to both salt up-shock (from 2.1 to 3.1 M NaCl) and down-shock (from 2.1 to 0.9 M NaCl). Both clpB and groEL/ES transcript levels increased when the temperature was shifted from 37 degrees C to 55 degrees C. Under heat stress cIpB transcription was repressed by the addition of the osmolyte betaine (1 mM). In conclusion, a novel AAA+ chaperone clpB gene from a halophilic methanogen that responded to the fluctuations in temperature, salt concentration and betaine has been identified and analysed for the first time.
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