The temperature sensitivity of Bacillus subtilis DB1005 is due to insufficient activity, rather than insufficient concentration, of the mutant σA factor
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 (t1/2 600 min). However, neither wild-type σA nor mutant σA was stable at 49°C (t1/2 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.
ChakK. F.,
de LencastreH.,
LiuH.-M.,
PiggotP. J.1982; Facile in vivo transfer of mutations between the Bacillus subtilis chromosome and a plasmid harbouring homologous DNA.. J Gen Microbiol 128:2813–2816
ChangB.-Y.,
DoiR. H.1993b; Effects of amino acid substitutions in the promoter –10 binding region of the σA factor on growth of Bacillus subtilis.. J Bacteriol 175:2470–2474
ChangB.-Y.,
ChenK.-Y.,
WenY.-D.,
LiaoC.-T.1994; The response of a Bacillus subtilis temperature-sensitive sigA mutant to heat stress.. J Bacteriol 176:3102–3110
DanielsD.,
ZuberP.,
LosickR.1990; Two amino acids in an RNA polymerase σ factor involved in the recognition of adjacent base pairs in the –10 region of the cognate promoter.. Proc Natl Acad Sci USA 87:8075–8079
FukudaR.,
DoiR. H.1977; Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation.. J Bacteriol 129:422–432
GrossmanA. D.,
BurgessR. R.,
WalterW. A.,
GrossC. A.1983; Mutations in the Ion gene of E. coli K12 phenotypically suppress a mutation in the sigma subunit of RNA polymerase.. Cell 32:151–159
HailingS. M.,
Sanchez-AnzaldoF. J.,
FukudaR.,
DoiR. H.,
MearesC. F.1977; Zinc is associated with the β subunit of DNA-dependent RNA polymerase of Bacillus subtilis.. Biochemistry 16:2880–2884
HeX.-S.,
ShuT.-S.,
FukudaR.,
DoiR. H.1991; Construction and use of a Bacillus subtilis mutant deficient in multiple protease genes for the expression of eucaryotic genes.. Ann NY Acad Sci 646:60–77
hicksK. A.,
GrossmanA. D.1995; Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth.. Annu Rev Biochem 177:3736–3742
IglesiasA.,
TrautnerT. A.1983; Plasmid transformation in Bacillus subtilis: symmetry of gene conversion in transformation of a hybrid plasmid containing chromosomal DNA.. Mol Gen Genet 189:73–76
KahnD.,
DittaG.1991; Modular structure of FixJ: homology of the transcriptional activator domain with the –35 binding domain of sigma factor.. Mol Microbiol 5:987–997
KiodeY.,
NakamuraA.,
UozumiT.,
BeppuT.1986; Cloning and sequencing of the major intracellular serine protease gene of Bacillus subtilis.. J Bacteriol 167:110–116
LiebkeH.,
GrossC. A.,
WalterW. A.,
BurgessR. R.1980; A new mutation, rpoD800, affecting the sigma subunit of E. coli RNA polymerase is allelic to two other sigma mutants.. Mol Gen Genet 177:277–282
QiF.-X.,
HeX.-S.,
DoiR. H.1991; Localization of a new promoter, P5, in the sigA operon of Bacillus subtilis and its regulation in some spore mutant strains.. J Bacteriol 173:7050–7054
SiegeleD.A.,
HuJ.-C.,
WalterW. A,
GrossC. A.1989; Altered promoter recognition by mutant forms of the σ70 subunit of Escherichia coli RNA polymerase.. J Mol Biol 206:591–603
TaborS.1990; Expression using the T7 RNA polymerase/ promoter system. In Current Protocols in Molecular Biology, pp.. 16.2.1–16.2.11 Edited by F. A. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Greene Publishing & Wiley Interscience..
TattiK. M.,
JonesC. H.,
MoranC. P.1991; Genetic evidence for interaction of σE with the spoIIID promoter in Bacillus subtilis.. J Bacteriol 173:7828–7833
TaylorW. E.,
StrausD. B.,
GrossmanA. D.,
BurtonZ. F.,
GrossC. A.,
BurgessR. R.1984; Transcription from a heat-inducible promoter causes heat shock regulation of the sigma subunit of E. coli RNA polymerase.. Cell 38:371–381
WangL.-F.,
DoiR. H.1986; Nucleotide sequence and organization of Bacillus subtilis RNA polymerase major sigma (σ43) operon.. Nucleic Acids Res 14:4293–4307
YuanG.,
WongS.-L.1995; Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK.. J Bacteriol 177:6462–6468
The temperature sensitivity of Bacillus subtilis DB1005 is due to insufficient activity, rather than insufficient concentration, of the mutant σA factor