Analysis of the AAA+ chaperone clpB gene and stress-response expression in the halophilic methanogenic archaeon Methanohalophilus portucalensis.
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 degrees C to 55 degrees 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.
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
Chao-Jen
CJ
Shih
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
Mei-Chin
MC
Lai
D000222:Adaptation, Physiological / Q000235:genetics*; D000251:Adenosine Triphosphatases / Q000235:genetics; D020816:Amino Acid Motifs; D019843:Archaeal Proteins / Q000096:biosynthesis / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D018835:Chaperonin 10 / Q000235:genetics; D018834:Chaperonin 60 / Q000235:genetics; D018833:Chaperonins / Q000096:biosynthesis / Q000235:genetics*; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D019848:Gene Expression Regulation, Archaeal; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D006358:Hot Temperature; D017019:Methanosarcinaceae / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000378:metabolism
D019843:Archaeal Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D019641:DNA, Archaeal; D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D012965:Sodium Chloride; D000251:Adenosine Triphosphatases; D018833:Chaperonins