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

Abstract

The 8 factor of Bacillus subtilis DB1005 contahs two amino acid substitutions (II98A and IZOZA) in the promoter -10 binding region. It has been confirmed that this c factor is responsible for the temperature sensitivity of B. subtilis DB1005. An investigation was conducted into how the mutant aA could cause temperature-sensitive (Ts) cell growth by analysing its structural stability, cellular concentration and transcriptional activity. The mutant aA was unstable even a t the permissive temperature of 37 "C (t1,* 59 min), whereas the wildtype counterpart was fairly stable under the same conditions (tl12 > 600 min). However, neither wild-type 8 nor mutant 8 was stable a t 49 "C (tl12 34 min and 23 min, respectively). Analyses of the rates of 8 synthesis revealed that B. subtilis DB1005 was able to compensate for unstable 8 by elevating the level of aA a t 37 "C but not at 49 "C. Moreover, overexpression of the mutant 8 a t 49 "C could not suppress the Ts phenotype of B. subtilis DB1005. This indicates that the temperature sensitivity of B. subtilis DB1005 is not due to insufficient 8 concentration in the cell. The greater decline of an already reduced activity of the mutant aA a t 49 "C suggests that the temperature sensitivity of B.subtilis DB1005 is instead the result of a very low activity of 8; probably below a critical level necessary for cell growth.

Structural and functional properties of a Bacillus subtilis temperature‐sensitive σA factor

“…Both A factors were active as measured by in vitro transcription assays. 30 The protein standards used for size-exclusion chromatography were purchased from Boehringer Mannheim. 1-Anilinonaphthalene-8-sulfonate (ANS) was from Sigma Corporation.…”

“…Although there are many possible reasons for the low Ts A activity in B. subtilis DB1005 at elevated temperature, 30 we first chose to investigate the core-binding activity of the Ts A . This is because that binding of the factor to core RNA polymerase occurs before the initiation of transcription.…”

“…However, the temperature sensitivity of this sigA mutant is due to insufficient activity, rather than insufficient concentration, of the Ts A , because overexpression of this mutant factor is unable to suppress the Ts phenotype of B. subtilis DB1005. 30 The insufficient A activity may be ascribed to the structural instability, [31][32][33] the low core-binding activity, 7,8,10 or the change in promoter specificity of the Ts A factor. 12,14,22,23 It could also be attributed to the blocking of transcription initiation by the Ts A , 34 or to the defect of the Ts A in open complex formation.…”

The Importance of Region 2.1 in Sustaining the Functional Structure of the Bacillus subtilis AFactor

“…Preparation and Labeling of the G3b Promoter DNAThe 135-bp G3b promoter DNA was synthesized by PCR using a pCoiZA-derived plasmid containing the G3b promoter DNA as template (36). The two primers used for the synthesis were BC1041-PsiI (5'-GCTGGTCTGCAGAACG-TAGACAACAACC-3') and BClO48-XfeaI (5'-GCGTCGTCT-AGAATTTGTAGACTCTGTATC-3').…”

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

“…Preparation and labeling of G3b promoter DNA for gel retardation assay. The 135-bp linear G3b promoter DNA was synthesized by PCR using the pCoiZAderived plasmid as template (4). The two primers are BC1041-PstI (5Ј-GCTGG TCTGCAGAACGTAGACAACAACC-3Ј) and BC1048-XbaI (5Ј-GCGTCGTC TAGAATTTGTAGACTCTGTATC-3Ј).…”