The DNA region of the Vibrio harveyi chromosome containing the heat-shock genes groES and groEL was cloned, and the genes were sequenced. These genes are arranged in the chromosome in the order groES-groEL. Northern hybridization experiments with RNA from V. harveyi and a DNA probe carrying both groES and groEL genes showed a single, heat-inducible transcript of approximately 2200 nt, indicating that these genes form an operon. Primer extension analysis revealed a strong, heat-inducible transcription start site 59 nt upstream of groES, preceded by a sequence typical for the Escherichia coli heat-shock promoters recognized by the s 32 factor, and a weak transcription start site 25 nt upstream the groES gene, preceded by a sequence typical for s 70 promoters. Transcription from the latter promoter occurred only at low temperatures. The V. harveyi groE operon cloned in a plasmid in E. coli cells was transcribed in a s 32 -dependent manner; the transcript size and the s 32 -dependent transcription start site were as in V. harveyi cells. Comparison of V. harveyi groE transcription regulation with the other well-characterized groE operons of the c subdivision of proteobacteria (those of E. coli and Pseudomonas aeruginosa) indicates a high conservation of the transcriptional regulatory elements among these bacteria, with two promoters, s 32 and s 70 , involved in the regulation. The ability of the cloned groESL genes to complement E. coli groE mutants was tested: V. harveyi groES restored a thermoresistant phenotype to groES bacteria and enabled l phage to grow in the mutant cells. V. harveyi groEL did not abolish thermosensitivity of groEL bacteria but it complemented the groEL mutant with respect to growth of l phage. The results suggest that the GroEL chaperone may be more species-specific than the GroES co-chaperone.
INTRODUCTIONAll organisms analysed so far respond to a sudden increase in temperature by transiently enhanced synthesis of heatshock proteins (Hsps) (Neidhardt et al., 1984). Most prominent among the Hsps are molecular chaperones and ATP-dependent proteases, which help to restore homeostasis by either refolding or degradation of the non-native proteins (Bukau & Horwich, 1998). The primary structure of most Hsps is highly conserved during evolution, suggesting that they serve similar and very important functions in all organisms, from bacteria to man (Lindquist & Craig, 1988).The chaperonin GroEL (Cpn60) and co-chaperonin GroES (Cpn10) constitute the GroE chaperone machine, which takes part in the process of folding and assembly of proteins (reviewed by Hartl & Hayer-Hartl, 2002;Houry, 2001) and is found in bacteria, mitochondria and chloroplasts (Ellis & van der Vies, 1991). The GroE chaperone system assists the folding of polypeptides in compact conformations, recognizing intermediates exposing hydrophobic surfaces (Hartl & Hayer-Hartl, 2002). Although purified GroEL is able to bind to a wide range of proteins, in vivo there is evidence that it has high affinity for a defined set of substrates. While ...