The ClpB ATPase of Streptomyces albus G belongs to the HspR heat shock regulon

Abstract

SummaryThe clpB gene of Streptomyces albus was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotides. Transcriptional analysis showed that the clpB gene was heat induced. Primer extension identified a transcription start site preceded by typical vegetative ¹10 and ¹35 hexamer sequences. The Streptomyces HspR repressor is known to bind to three inverted repeat motifs (IR1, IR2, IR3) upstream from the S. coelicolor dnaK operon. We identified an inverted repeat motif identical to IR3 upstream from the S. albus clpB gene. DNA-binding experiments showed that HspR regulates clpB transcription by interacting directly with this motif. Streptomyces albus is the first Gram-positive organism for which the co-regulation of DnaK and ClpB has been described. Such co-regulation suggests that there is a physiological relationship between these two proteins in this bacterium. Genes similar to hspR were also identified in Mycobacterium leprae, M. tuberculosis and in bacteria unrelated to the actinomycetales order, such as Helicobacter pylori and Aquifex aeolicus. HspR binding sites were found in these bacteria upstream from various heat shock genes, suggesting that these genes are regulated by HspR. The HspR binding site, here called HAIR (H spR associated inverted repeat), has the consensus sequence CTTGAGT N7 ACTCAAG.

The lon gene, encoding an ATP-dependent protease, is a novel member of the HAIR/HspR stress-response regulon in actinomycetes

“…In addition to the HAIR motifs associated with dnaK and clpB, a HAIR sequence was found upstream of a gene closely related to the lon gene, encoding the ubiquitous ATPdependent protease. The nucleotide sequence upstream A. Sobczyk and others from lon contains the following sequence : ATTGAGT-N ( -ACTCAAC, which is similar to the HAIR consensus motif CTTGAGT-N ( -ACTCAAG of the HspR-binding site (Grandvalet et al, 1999). This led us to clone the lon gene with oligonucleotides derived from the S. coelicolor sequence.…”

“…RNA was prepared as described previously (Servant & Mazodier, 1996). The transcription start site upstream of lon was located by primer extension using oligonucleotide AB10 (5h-GTCGATGCGCGGGACGAGGA-G-3h) as previously described (Grandvalet et al, 1999).…”

“…The control of groE gene expression involves an invertedrepeat element (called CIRCE) that is highly conserved among eubacteria, and the HrcA repressor (Grandvalet et al, 1998). The dnaK operon and clpB belong to the HspR\HAIR regulon (Bucca et al, 1997 ;Grandvalet et al, 1999). The HspR repressor-HAIR operator system is used in some bacteria (Spohn & Scarlato, 1999) but is not widespread.…”

Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays a aA list of the 100 ORFs most highly induced by heat shock is provided as supplementary data with the online version of this paper (http://mic.sgmjournals.org).

“…We searched the genome for sequences that resembled the HspR binding site, HAIR CTTGAGT-N ( -ACTCAAG (Grandvalet et al, 1999) and compared the locations of potential sites to the gene expression analysis of both heat-shocked M. tuberculosis and M. tuberculosis ∆hspR. In addition to the HAIR sequences already identified upstream of the Hsp70 operon and clpB (Stewart et al, 2001), a HAIR-like domain was present 71 bp upstream of the start codon of Rv0251c (Fig.…”

Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF287346.

“…Total RNA was extracted from B. thuringiensis strain 407 Cry − , and the inhA transcription start site was determined by primer extension as previously described (Grandvalet et al, 1999) using synthetic oligonucleotide GC1 : 5h-TCAGCATATGCAGATTGAGT-GCCAGCTCC-3h. DNA sequencing was performed by the dideoxy-chain-termination method with double-stranded pHT304ΩinhAh-lacZ plasmid as template and the primer GC1.…”