Identification and characterization of a stress‐responsive promoter in the macromolecular synthesis operon of Bacillus subtilis

Abstract

SummaryBacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant. Induction of s A has been observed exclusively in this mutant harbouring extra copies of the plasmid-borne Ts sigA gene transcriptionally controlled by the P1P2 promoters of the B. subtilis macromolecular synthesis (MMS; rpoD or sigA) operon. Investigation of the mechanisms leading to the induction has allowed us to identify a s B -type promoter, P7, in the MMS operon for the ®rst time. Therefore, at least seven promoters in total are responsible for the regulation of the B. subtilis MMS operon, including the four known s A -and s H -type promoters, as well as two incompletely de®ned promoters. The P7 promoter was activated in B. subtilis after the imposition of heat, ethanol and salt stresses, indicating that the MMS operon of B. subtilis is subjected to the control of general stress. The signi®cant heat induction of P7 in B. subtilis DB1005 harbouring a plasmid-borne Ts sigA gene can be explained by a model of competition between s A and s B for core binding; very probably, the s B factor binds more ef®ciently to core RNA polymerase under heat shock. This mechanism may provide a means for the expression of the B. subtilis MMS operon when s A becomes defective in core binding.

Transcription Regulation ofezrAand Its Effect on Cell Division ofBacillus subtilis

“…To locate exactly the position of the promoter(s) about 100 bp upstream of ezrA, primer extension analysis of total RNA extracted from B. subtilis DB430 harboring pEZ6 was performed. The protocol used for primer extension was similar to that reported previously (28). The primer used is 22 bases long, starting from the base immediately upstream of the initiation codon ( Table 1).…”

“…The symbol, ⌬, e, छ, or E indicates the expression of lacZ in the corresponding strain of B. subtilis. (6,8,28,39), and reconstituted with core RNA polymerase (20). Subsequently, each RNA polymerase holoenzyme or a mixture of two different RNA polymerase holoenzymes was examined for activity on transcribing the pKM3 plasmid containing the DNA sequence encompassing both P1 and P2 (Table 2).…”

Properties of Bacillus subtilis σ A Factors with Region 1.1 and the Conserved Arg-103 at the N Terminus of Region 1.2 Deleted

“…The two primers are BC1041-PstI (5Ј-GCTGG TCTGCAGAACGTAGACAACAACC-3Ј) and BC1048-XbaI (5Ј-GCGTCGTC TAGAATTTGTAGACTCTGTATC-3Ј). To label the G3b promoter, we suspended 2.5 g of the DNA fragment recovered by electroelution in 100 l of The arrowheads indicate the mRNA transcripts (451 and 291 bases for the G3b promoter; 505 and 348 bases for the P1P2 promoter) terminated at the T1T2 tandem terminator (19). The value below each lane is the relative transcription activity of the assayed RNA polymerase, with that of the WT A -RNA polymerase referred to as 100%.…”

“…2C). Since the nucleotide sequences of the G3b and P1P2 promoters are different, especially at the Ϫ35 region (7,19), it was assumed that the transcription defect caused by deletion of Arg-103 in SND104 is promoter independent. Moreover, our results clearly manifested that the conserved Arg-103 is an essential N-terminal boundary amino acid residue for the truncated A ; the loss of this arginine, rather than its preceding amino acid sequence, is detrimental to A function.…”

“…The molar ratio of core to A was 1:10. Afterward, 20 l (0.3 g) of pCT20 or pCT24 plasmid harboring the P1P2 promoter of the B. subtilis sigA operon or the G3b promoter of the B. subtilis 29 phage (7,19), respectively, was added to the reconstituted holoenzyme. The mixture was then incubated on ice for 10 min before being transferred to a temperature environment of 37°C.…”