Using a DNA fragment containing the principal sigma factor gene hrdB of Streptomyces aureofaciens, we identified two sigma70-like genes in a library of Brevibacterium flavum. Sequence analysis of the complete genes revealed two ORFs coding for gene products of 498 and 331 amino acid residues, which showed the greatest similarity to SigA and SigB sigma factors from Brevibacterium lactofermentum. We designated them similarly sigA and sigB. Transcription of B. flavum sigA and sigB has been investigated by S1-nuclease mapping by using RNA from different growth phases and after exposure to several stress conditions. Both genes are transcribed from a single promoter with transcription start points of 368 bp and 25 bp upstream from the proposed translation initiation codon of the sigA and sigB genes, respectively. Whereas sigA is transcribed almost constitutively during growth and after stress conditions, expression of sigB is significantly induced after several stress conditions, like acid stress, ethanol shock, and cold shock. Expression of both genes is significantly reduced after heat shock. Considering these transcriptional results, and also on the basis of the similarity to other principal sigma factor genes, sigA probably encodes the functional principal sigma factor, and sigB might have a function in stress response.
Acetylxylan esterase from Schizophyllum commune was purified using ion-exchange and hydrophobic chromatography. The enzyme has a molecular mass of 31 kDa, as determined by SDS/PAGE, or 18 kDa, according to gel filtration. Glycosylation of the enzyme was not detected. Acetylxylan esterase is relatively stable under laboratory conditions; it retains full activity at pH 6.2-8.5 upon incubation at 25 degrees C for 7 h, but loses nearly the whole activity upon incubation at 60 degrees C for 30 min. The pH optimum of the enzyme activity is 7.7 and its temperature optimum lies between 30 and 45 degrees C. Ca2+ and Co2+ inhibit markedly the activity of acetylxylan esterase at a concentration of 10 mM, as do Mn2+, Zn2+, Fe2+ and Cu2+ at a concentration of 1 mM.
We have previously cloned a gene encoding a SigB, a principal-like sigma factor in Brevibacterium flavum, which was induced by several stress conditions. To clarify the in vivo function of this sigma factor, the sigB gene was disrupted by a homologous recombination, replacing the internal essential coding region in B. flavum chromosome by a kanamycin resistance marker gene. This mutation dramatically decreased vegetative growth rates of B. flavum. Studies of the effect of the sigB mutation on growth and viability of the cells under conditions of stress showed that the sigB mutant had increased susceptibility to acid, salt, alcohol, heat and cold stress. The plasmid-born wild-type sigB gene complemented the mutation. Based on the results, we propose that SigB has a role in vegetative growth and in response to various environmental stresses.
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