Cloning and characterization of ppiB, a Bacillus subtilis gene which encodes a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase.
Sequencing of N-terminal and internal peptide fragments of the purified 17 kDa Bacillus subtilis peptidyl-prolyl cis-trans isomerase (PPIase) 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 lambda Zap II gene library of B. subtilis. Two overlapping positive clones of 5 and 7 kb containing the B. subtilis PPIase 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 degrees on the B. subtilis genetic map was located. This putative PPIase 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 PPIase activity. In addition, the recombinant PPiB shows a significant inhibition of PPIase activity by cyclosporin A (CsA) at a level comparable to that observed for the B. subtilis enzyme. Interestingly the B. subtilis PPIase shows about 40% identity to eukaryotic PPIases and less similarity to those of Gram-negative bacteria (27-32% identity). Like other interruption mutants of yeast and Neurospora, which lack a functional cyclophilin gene, a B. subtilis mutant containing ppiB::cat, a cat-interrupted copy of ppiB in the chromosome, is viable.
Philipps-Universität Marburg, Fachbereich Chemie/Biochemie, Germany.
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Marahiel
D000591:Amino Acid Isomerases / Q000187:drug effects / Q000235:genetics*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics*; D003001:Cloning, Molecular; D016572:Cyclosporine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005798:Genes, Bacterial / Q000235:genetics*; D007535:Isomerases / Q000145:classification / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019696:Peptidylprolyl Isomerase; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D012333:RNA, Messenger; D016572:Cyclosporine; D007535:Isomerases; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase