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.
Shih
Chao-Jen
CJ
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
Lai
Mei-Chin
MC
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
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