The dnaK operon of Bacillus subtilis is heptacistronic

Abstract

In 1992, we described the cloning and sequencing of the dnaK locus of Bacillus subtilis which, together with transcriptional studies, implied a tetracistronic structure of the operon consisting of the genes hrcA, grpE, dnaK, and dnaJ. We have repeated the Northern blot analysis, this time using riboprobes instead of oligonucleotides, and have detected a heat-inducible 8-kb transcript, suggesting the existence of additional heat shock genes downstream of dnaJ. Cloning and sequencing of that region revealed the existence of three novel heat shock genes named orf35, orf28, and orf50, extending the tetra-into a heptacistronic operon. This is now the largest dnaK operon to be described to date. The three new genes are transcribed as a part of the entire dnaK operon (8.0-kb heptacistronic heat-inducible transcript) and as part of a suboperon starting at an internal vegetative promoter immediately upstream of dnaJ (4.3-kb tetracistronic non-heat-inducible transcript). In addition, the Northern blot analysis detected several processing products of these two primary transcripts. To demonstrate the existence of the internal promoter, a DNA fragment containing this putative promoter structure was inserted upstream of a promoterless bgaB gene, resulting in the synthesis of ␤-galactosidase. Challenging this transcriptional fusion with various stress factors did not result in the activation of this promoter. To assign a biological function to the three novel genes, they have each been inactivated by the insertion of a cat cassette. All of the mutants were viable, and furthermore, these genes are (i) not essential for growth at high temperatures, (ii) not involved in the regulation of the heat shock response, and (iii) sporulation proficient. Blocking transcription of the suboperon from the upstream heat-inducible promoter did not impair growth and viability at high temperatures.The heat shock response occurs when cells growing at a low temperature are shifted to a higher temperature and results in the induction of a subset of proteins called heat shock proteins. This response to temperature upshift is universal among prokaryotes and eukaryotes. The heat shock family of proteins has recently become central to the study of correct folding of nascent polypeptides, assembly of protein complexes, and translocation of proteins into organelles (13). Therefore, some heat shock proteins are widely recognized as molecular chaperones that play crucial roles even under physiological conditions (10, 11).Escherichia coli has some 31 heat shock genes whose expression is regulated positively at the transcriptional level by the 32 polypeptide, the rpoH gene product. 32 is extremely unstable in vivo, with a half-life of Ϸ1 min at normal temperatures (for recent reviews, see references 3, 27, and 49).In contrast to E. coli, there are at least three classes of heat shock genes in Bacillus subtilis (for recent reviews, see references 16 and 40). Class I heat shock genes constitute the CIRCE regulon, and these genes are negatively regulated by a r...

Post-transcriptional regulation of the Bacillus subtilis pst operon encoding a phosphate-specific ABC transporter

“…Transcript size was determined by comparison with DIG-labelled RNA size markers (Roche Diagnostics). The arrows labelled 16S rRNA and 23S rRNA indicate the locations of these rRNA species, which are known to trap smaller RNA species (Homuth et al, 1997).…”

The Bacillus subtilis ABC transporter EcsAB influences intramembrane proteolysis through RasP

“…b-Galactosidase activities and a-amylase activities of culture supernatants were measured as described previously Pummi et al, 2002). Northern blot analysis was performed according to a published procedure (Homuth et al, 1997) with antisense RNAs against yuaG, pbpE and yhaU (Zellmeier et al, 2006). Western blots were performed as described previously (Homuth et al, 1996) using a semi-dry blotting procedure (Bio-Rad, Trans Blot SD).…”

Isolation and Analysis of Mutant Alleles of the Bacillus subtilis HrcA Repressor with Reduced Dependency on GroE Function

“…Transcriptional Analysis and Determination of ␤-Galactosidase Activities-Preparation of total RNA of B. subtilis and Northern blot analysis were performed as described before (9). To easily monitor HrcA activity, the promoter region of the dnaK operon, including the CIRCE operator, was transcriptionally fused to the bgaB reporter gene coding for a heat-stable ␤-galactosidase (21).…”

“…This protein binds to an operator designated CIRCE 1 (Controlling Inverted Repeat of Chaperone Expression) (6), which precedes the heptacistronic dnaK and the bicistronic groE operon (7)(8)(9). Upon a heat shock, HrcA dissociates from its operators leading to a transient induction of the two operons followed by rebinding after about 10 min (7,8,10).…”