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.