Sequence and transcriptional analysis of groES and groEL genes from the thermophilic bacterium Clostridium...

The groESL operon from Clostridium thermocellum (Ct) has been isolated and sequenced, revealing two ORFs of 285 and 1626 nt, separated by 48 nt. The first ORF encoded a 94-aa 10.6-kDa GroES homologue; the second encoded a 541-aa polypeptide of 57.6 kDa, that exhibited 61% and 77% sequence identity with GroEL from Escherichia coli (Ec) and Clostridium acetobutylicum (Ca), respectively. A putative tsp, preceded by -10 and -35 consensus promoters, was identified upstream of groES. This was followed by an inverted repeat observed previously in bacterial heat shock genes. A 15-nt palindrome characteristic of a Rho-independent transcription terminator, was located downstream of groEL. The first nt of the groES translational start codon was preceded (7 nt) by a putative RBS (AGGAGG); a second RBS sequence was located 8 nt upstream of the groEL start. Production of GroE homologues by Ct was constitutive, but was enhanced significantly during a temperature upshift from 60 degrees C to 70 degrees C. The Ct GroEL, expressed in Ec as a fusion protein with GST, was purified, free of contaminating Ec GroEL.