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

Abstract

The σA factor of Bacillus subtilis DB1005 contains two amino acid substitutions (1198A and 1202A) in the promoter –10 binding region. It has been confirmed that this σ factor is responsible for the temperature sensitivity of B. subtilis DB1005. An investigation was conducted into how the mutantσA could cause temperature-sensitive (Ts) cell growth by analysing its structural stability, cellular concentration and transcriptional activity. The mutant σA was unstable even at the permissive temperature of 37°C (t1/2 59 min), whereas the wild-type counterpart was fairly stable under the same conditions (t 1/2 600 min). However, neither wild-type σA nor mutant σA was stable at 49°C (t 1/2 34 min and 23 min, respectively). Analyses of the rates of σA synthesis revealed that B. subtilis DB1005 was able to compensate for unstable σ by elevating the level of σA at 37°C but not at 49°C. Moreover, overexpression of the mutant σA at 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 σA concentration in the cell. The greater decline of an already reduced activity of the mutant σA at 49°C suggests that the temperature sensitivity of B. subtilis DB1005 is instead the result of a very low activity of σ A probably below a critical level necessary for cell growth.