Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK

Abstract

An inverted repeat sequence known as CIRCE (controlling inverted repeat of chaperone expression) in the Bacillus subtilis groE operon has been suggested to function as an operator. To identify the regulatory gene directly or indirectly involved in CIRCE-mediated heat-inducible groE expression, B. subtilis WBG2, carrying an integrated groE-bgaB transcription fusion in the amyE locus, was mutagenized. Dark blue colonies formed at 37؇C represent mutants which constitutively produce BgaB (a thermostable ␤-galactosidase) at high levels. Seven mutants (WBG101 to WBG107) were selected for further characterization. They all overproduced BgaB, GroEL, and DnaK simultaneously at 37؇C. These mutants could be restored to normal by introducing a plasmid carrying a functional copy of orf39, the first gene in the B. subtilis dnaK operon. Genomic sequencing of these mutants demonstrated that they all carried a single mutation in orf39. These mutations can be divided into three groups: (i) Gly-307 to Asp, (ii) Ser-122 to Phe, and (iii) Gly-63 to Glu. By using a binary vector system in E. coli, production of ORF39 was found to negatively regulate the expression of groE-bgaB in a CIRCEspecific manner. Under the heat shock condition, the negative regulation mediated by ORF39 was abolished. Mobility shift of the CIRCE-containing probe was also observed with the crude extract prepared from the E. coli strain that overproduced ORF39. Therefore, ORF39 is the negative regulatory factor which regulates both groE and dnaK expression in B. subtilis. It is likely to function as a CIRCE-specific repressor.Expression of Bacillus subtilis heat shock genes encoding molecular chaperones GroES, GroEL, and DnaK is regulated by a novel mechanism which differs from the well-characterized regulatory mechanism operating in Escherichia coli. In both the B. subtilis groE and dnaK operons, two sequence elements are found in the regulatory region. They are a typical A -type promoter (16,26,33) and an inverted repeat (IR) sequence designated CIRCE (controlling IR of chaperone expression) (39). CIRCE is located downstream from the transcription start site in these operons. CIRCE-like IR sequences can also be found in groE or dnaK operons from many bacteria (28). Alignment of these sequences illustrates that a consensus sequence can be generated (20,33).For groE, identical transcription start sites are observed under both heat shock and non-heat shock conditions. Mutating either the first T nucleotide in the Ϫ10 region or the third G nucleotide in the Ϫ35 region of the groE promoter results in a significant decrease in promoter activity. The activities of these mutated promoters can be restored in an allele-specific manner by specific sigA mutants at both 37 and 48ЊC (37). Transcription studies of groE with the purified A -containing RNA polymerase in vitro and a temperature-sensitive sigA mutant in vivo also support the involvement of the A -containing RNA polymerase in transcribing groE (3). Since A is the major sigma factor produced constitutively, some ot...

The temperature sensitivity of Bacillus subtilis DB1005 is due to insufficient activity, rather than insufficient concentration, of the mutant σA factor

“…The correlation between the markedly lower oA activity and the temperature sensitivity of B. subtilis DB1005 at 49 "C suggests that the level of activity of oA is the limiting factor for DB1005 growth at high temperature. Furthermore, the lack of induction of groE message in vitro in the presence of wild-type oA above 37 "C was probably due to the absence of repressors (Yuan & Wong, 1995) in the system, which thus allowed the groE promoter to express constitutively.…”

A Novel Class of Heat and Secretion Stress-Responsive Genes Is Controlled by the Autoregulated CssRS Two-Component System ofBacillus subtilis

“…The transcription of class I genes, which code for classical chaperones, is A dependent and regulated by the HrcA repressor (14,29,31). The class II genes respond not only to heat but also to other stresses such as exposure to ethanol or salt, starvation for glucose or phosphate, or growth under anaerobic conditions.…”

The hsp Operons Are Repressed by the hrc37 of the hsp70 Operon in Staphylococcus aureus

“…Two working groups reported that the HrcA (ORF39) of B. subtilis regulated the expression of the groE and dnaK genes by associating with the CIRCE element (24)(25)(26). We further analyzed the role of hrc37 in heat-shock response by isolating the hrc37 disrupt mutant.…”