ISSN:
1365-2958
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
,
Medicine
Notes:
Sequencing of N-terminal and internal peptide fragments of the purified 17kDa Bacillus subtilis peptldyl-prolyl cls-trans isomerase (PPlase) revealed sequence identity to conserved regions of a number of eukaryotic and prokaryotic cyclophilins. Using two oligonucleotide primers corresponding to the N-terminus and a highly conserved internal amino acid sequence, polymerase chain reactions (PCR) with B. subtilis genomic DNA were carried out. The resultant PCR fragment of 335 bp was cloned, sequenced and subsequently used as a probe for screening a λZap II gene library of B. subtilis. Two overlapping positive clones of 5 and 7 kb containing the B. subtilis PPlase gene (ppiB), which is 432 bp in length and encodes a protein of 144 amino acid residues, were identified and two distinct transcriptional initiation sites at the 5′ end of ppiB were mapped. The entire region (35 kb) between spoVA and serA was recently sequenced in B. subtilis, and an open reading frame (ORF) that encodes a putative peptidyl-prolyl cis-trans isomerase at about 210° on the B. subtilis genetic map was located. This putative PPlase is identical to PPiB. We have overexpressed the ppiB gene in Escherichia coli, purified the encoded protein to apparent homology and shown that it exhibits PPlase activity. In addition, the recombinant PPiB shows a significant inhibition of PPlase activity by cyclosporin A (CsA) at a level comparable to that observed for the B. subtilis enzyme. Interestingly the B. subtilis PPlase shows about 40% identity to eukaryotic PPlases and less similarity to those of Gram-negative bacteria (27–32% identity). Like other interruption mutants of yeast and Neurospora, which iack a functionai cyclophilin gene, a B. subtiiis mutant containing ppiBv.cat, a caf-interrupted copy of ppiB in the chromosome, is viable.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1365-2958.1994.tb00384.x
