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

Agricultural Biotechnology Laboratories, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China

Author for correspondence: Ban-Yang Chang. Tel: +886 4 287 4754. Fax: +SS6 4 286 1905. e-mail: bychang@:mail.nchu.edu.tw

ABSTRACT

The ^A factor of Bacillus subtilis DB1005 contains two amino acid substitutions (I198A and I202A) in the promoter -10 binding region. It has been confirmed that this factor is responsible for the temperature sensitivity of B. subtilis DB1005. An investigation was conducted into how the mutant^A could cause temperature-sensitive (Ts) cell growth by analysing its structural stability, cellular concentration and transcriptional activity. The mutant ^A was unstable even at the permissive temperature of 37°C (t_1/2 59 min), whereas the wild-type counterpart was fairly stable under the same conditions (t_1/2 > 600 min). However, neither wild-type ^A nor mutant ^A was stable at 49°C (t_1/2 34 min and 23 min, respectively). Analyses of the rates of ^A synthesis revealed that B. subtilis DB1005 was able to compensate for unstable by elevating the level of ^A at 37°C but not at 49°C. Moreover, overexpression of the mutant ^A 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 ^A concentration in the cell. The greater decline of an already reduced activity of the mutant ^A at 49°C suggests that the temperature sensitivity of B. subtilis DB1005 is instead the result of a very low activity of ^A probably below a critical level necessary for cell growth.

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