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 mechanism has not been elucidated. To elucidate the role of groEL in Streptacoccus pneumoniae, the groESL operon was cloned in Escherichia coli. The promoter region of the pneumococcal groESL operon contained a sigma (A) type promoter and an inverted repeat (CIRCE). A Northern blot analysis of the groEL operon demonstrated that the groESL operon is transcribed as a bf cistronic mRNA, and reached maximum expression 7.5 to 10 min after heat shock. A primer extension analysis showed a potential transcription start point at 155 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 analysis showed a sigma (A) type promoter flanked by 2 CIRCE elements. His-tagged HrcA was overexpressed as a soluble form in E. coli and bound to the CIRCE regions in the promoter of both groESL and dnAK operons in vitro. Additionally, 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.