Analysis of the AAA+ chaperone clpB gene and stress-response expression in the halophilic methanogenic archaeon Methanohalophilus portucalensis

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 RT-PCR (DDRT-PCR). Furthermore, the complete clpB sequence (2610?nt) and its upstream genes encoding the type I chaperonin GroEL/ES were obtained through inverse PCR, Southern hybridization and sequencing. The G+C ratio of clpB is 49.6?mol%. The predicted ClpB 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 ClpB. 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 ClpB (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?°C to 55?°C. Under heat stress clpB 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.