Molecular analysis of the Rhodobacter capsulatus chaperonin (groESL) operon: purification and characterization of Cpn60.
The heat-shock protein Cpn60 (chaperonin, GroEL homologue) from the phototrophic bacterium Rhodobacter capsulatus B10 was purified to homogeneity and biochemically characterized. Native Cpn60 from R. capsulatus was shown to be a tetradecamer of 840 kDa similar to that of homologous chaperones characterized so far. Cpn60 possesses ATPase activity and promotes refolding of chaotropically denatured citrate synthase. The groESL operon of R. capsulatus was cloned using a degenerate oligonucleotide and sequenced. Two open reading frames (285 and 1,635 bp) were found; they encode Cpn10 and Cpn60, with corresponding deduced molecular masses of 10.6 and 57.6 kDa. The deduced amino acid sequences coincided perfectly with those of the amino terminus and of three tryptic peptides of purified Cpn60 from R. capsulatus. Strong evidence that R. capsulatus encodes only one copy of the groESL operon was obtained. Primer-extension analysis revealed that the groESL operon is transcribed by a -35/-10-type promoter, and that transcription was initiated from the same positions before and after heat-shock under both chemotrophic and phototrophic conditions. The major initiation site is immediately followed by the inverted repeat structure CIRCE, which has been found upstream of many bacterial heat-shock operons. A second minor transcript starts just after the CIRCE element. Although heat-shock induction of a groEL-lacZ fusion failed because of thermal inactivation of the fusion protein, Western blot analysis revealed a two- to threefold induction of cellular Cpn60 levels 45-75 min after shifting from 28 degrees C to 39 degrees C. Deletion mapping of the groESL promoter identified upstream of the promoter a 19-bp element that enhances groESL transcription eightfold and contains the AT-rich sequence dAAATTTTT, which is found at similar positions in heat-shock operons of other gram-negative bacteria.
Hübner
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P
Department of Microbiology, Biozentrum, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Dame
G
G
Sandmeier
U
U
Vandekerckhove
J
J
Beyer
P
P
Tadros
M H
MH
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics*; D018833:Chaperonins / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D016354:Rhodobacter capsulatus / Q000235:genetics*
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; C097970:GroESL protein, Bacteria; D006360:Heat-Shock Proteins; D000251:Adenosine Triphosphatases; D018833:Chaperonins