The temperature sensitivity of Bacillus subtilis DB1005 is due to insufficient activity, rather than insufficient concentration, of the mutant delta A factor.
The delta 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 delta factor is responsible for the temperature sensitivity of B. subtilis DB1005. An investigation was conducted into how the mutant delta A could cause temperature-sensitive (Ts) cell growth by analysing its structural stability, cellular concentration and transcriptional activity. The mutant delta A was unstable even at the permissive temperature of 37 degrees C (t1/2 59 min), whereas the wild-type counterpart was fairly stable under the same conditions (t1/2 > 600 min). However, neither wild-type delta A nor mutant delta A was stable at 49 degrees C (t1/2 34 min and 23 min, respectively). Analyses of the rates of delta A synthesis revealed that B. subtilis DB1005 was able to compensate for unstable delta A by elevating the level of delta A at 37 degrees C but not at 49 degrees C. Moreover, overexpression of the mutant delta A at 49 degrees 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 delta A concentration in the cell. The greater decline of an already reduced activity of the mutant delta A at 49 degrees C suggests that the temperature sensitivity of B. subtilis DB1005 is instead the result of a very low activity of delta A; probably below a critical level necessary for cell growth.
Chang
Ban-Yang
BY
Agricultural Biotechnology Laboratories, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.
Liao
Chao-Tsai
CT
Agricultural Biotechnology Laboratories, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.
Wen
Yu-Der
YD
Agricultural Biotechnology Laboratories, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.
Wang
Wen-Horng
WH
Agricultural Biotechnology Laboratories, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.
D001412:Bacillus subtilis / Q000502:physiology*; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D006207:Half-Life; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D017433:Protein Structure, Secondary; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D014158:Transcription, Genetic
D018834:Chaperonin 60; D012808:Sigma Factor; C063505:RNA polymerase sigma A; D012321:DNA-Directed RNA Polymerases