CLONING AND NUCLEOTIDE-SEQUENCE COMPARISON OF THE GROE OPERON OF PSEUDOMONAS-AERUGINOSA AND BURKHOLDERIA-CEPACIA

By alignment of GroEL amino acid sequences from four distantly related bacteria two highly conserved domains were identified. Two oligonucle otides complementary to the conserved domains were designed based on t he preferred Pseudomonas aeruginosa codon usage. The primers were used in the PCR to amplify a 900-base fragment of the P. aeruginosa groEL gene. The fragment was sequenced and the partial GroEL sequence was ex panded by vectorette PCR upstream and downstream to cover the complete P. aeruginosa groE operon. The same technique was used to sequence th e Burkholderia cepacia (formely Pseudomonas cepacia) groE operon and t he region immediately upstream of groES. The B. cepacia groE operon is preceded by typical -10 and -35 heat shock expression signals. A tota l of 2041 and 2139 bp was sequenced from P. aeruginosa and B. cepacia respectively. Each revealed two open reading frames encoding two prote ins with a predicted molecular mass of 10 and 57 kDa, corresponding to GroES and GroEL respectively. The GroEL proteins show an interspecies amino acid homology of 71%, and 73% with E. coli GroEL. Both GroEL pr oteins are 52% homologous to the corresponding human mitochondrial Gro EL protein. The sequence data confirm the existence of highly conserve d structures, which could be functionally important for the concerted action of GroEL and GroES in the folding and assembly of other protein s, and possibly in the initiation of autoimmune diseases.