Cloning and characterization of the groE heat-shock operon of the marine bacterium Vibrio harveyi.
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 sigma(32) factor, and a weak transcription start site 25 nt upstream the groES gene, preceded by a sequence typical for sigma(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 sigma(32)-dependent manner; the transcript size and the sigma(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 gamma 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, sigma(32) and sigma(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 lambda 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 lambda phage. The results suggest that the GroEL chaperone may be more species-specific than the GroES co-chaperone.
Kuchanny-Ardigò
Dorota
D
Department of Biochemistry, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
Lipińska
Barbara
B
Department of Biochemistry, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
D001483:Base Sequence; D018835:Chaperonin 10 / Q000235:genetics*; D018834:Chaperonin 60 / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009876:Operon; D011550:Pseudomonas aeruginosa / Q000235:genetics; D013045:Species Specificity; D014158:Transcription, Genetic; D014733:Vibrio / Q000235:genetics*
D018835:Chaperonin 10; D018834:Chaperonin 60; D004269:DNA, Bacterial