Molecular cloning and characterization of groESL operon in Streptococcus pneumoniae

GroEL is a major target of the immune defense in infection and seems to be negatively regulated by HrcA in gram-positive organisms. However, HrcA's me chanism has not been elucidated. To elucidate the role of groEL in Streptac occus pneumoniae, the groESL operon was cloned in Escherichia coli. The pro moter region of the pneumococcal groESL operon contained a sigma (A) type p romoter and an inverted repeat (CIRCE). A Northern blot analysis of the gro EL operon demonstrated that the groESL operon is transcribed as a bf cistro nic mRNA, and reached maximum expression 7.5 to 10 min after heat shock. A primer extension analysis showed a potential transcription start point at 1 55 bp upstream of the translation start site, preceding the groES gene. The putative negative regulator of the groEL gene, hrcA, of S. pneumoniae was recovered by PCR-based chromosomal walking from grpE locus. A sequence anal ysis showed a sigma (A) type promoter flanked by 2 CIRCE elements. His-tagg ed HrcA was overexpressed as a soluble form in E. coil and bound to the CIR CE regions in the promoter of both groESL and dnAK operons in vitro. Additi onally, a helix-loop helix motif, a putative DNA binding domain, was found at the C-terminal of HrcA, These results will help to determine the nature of HrcA in the groESL repression.