Cloning, sequencing, and transcriptional analysis of the dnaK heat shock operon of Listeria monocytogenes

The complete dnaK operon of Listeria monocytogenes was isolated by chromoso me walking using the previously cloned dnaK gene as a probe. Molecular anal ysis of the locus identified 6 genes in the order hrcA, grpE, dnaK, dnaJ, o rf35, and orf29. Primer extension analysis revealed 3 transcription start s ites-S1, S2, and S3-upstream of the hrcA, grpE, and dnaJ, respectively. The transcription from S1 was heat inducible. Analysis of the sequences reveal ed the consensus promoter sequences of gram-positive bacteria, P1 and P2 up stream of the hrcA and dnaJ, respectively. The hrcA gene and a regulatory s equence, designated CIRCE (controlling inverted repeat of chaperone express ion), play a role in the regulation of expression of the dnaK focus in resp onse to heat shock in several grampositive bacteria. Their presence upstrea m of the dnaK locus in L monocytogenes suggested a similar regulatory mecha nism for the transcription initiated at the promoter, P1. Northern blot ana lysis led to the detection of 4 mRNA species of 4.9 kb, 3.6 kb, 3.6 kb, and 1.2 kb; the first 2 species were heat inducible. The current results indic ate that 4 distinct transcripts directed by 3 promoters are involved in the expression of the dnaK operon of L. monocytogenes.