RESUMO
The Gram-negative soil micro-organism Myxobacter sp. AL-1 possesses at least five extracellular cellulases, the production of which is regulated by the growth cycle. We cloned the complete gene for one of these cellulases, termed cel9, which encoded a 67-kDa modular family 9 endoglycohydrolase, which was produced during the stationary phase of growth and was strongly enhanced by avicel. The predicted product of cel9 matches the structural architecture of family 9 cellulases such as Thermonospora fusca endo/exocellulase E4. Cel9 protein was synthesized in Escherichia coli from a multicopy plasmid and in Bacillus subtilis from the isopropyl thiogalactoside-inducible Pspac promoter and was purified from the culture medium. Thermal stability, optimum pH and temperature dependence of Cel9 were similar when expressed from either source, and were indistinguishable from related cellulases produced by thermophilic bacteria. Downstream from cel9 was found a partial ORF, designated cel48, the deduced product of which was highly similar to bacterial exocellobiohydrolases and processive endoglucanases belonging to family 48 of the glycosyl hydrolases. The cel9 and cel48 genes appear to be arranged as part of an operon.
Assuntos
Bactérias/metabolismo , Celulase/genética , Celulose/metabolismo , Óperon , Sequência de Aminoácidos , Sequência de Bases , Celulase/química , Celulase/metabolismo , Celulose 1,4-beta-Celobiosidase , Clonagem Molecular , DNA Bacteriano , Hidrólise , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
EsigmaG-dependent transcription of the splAB operon in the forespore at stage III of Bacillus subtilis sporulation initiates from two promoters, P1 preceding splA (major) and P3 preceding splB (minor). To explore the possible role of splA in controlling splB-encoded spore photoproduct lyase expression, we measured beta-galactosidase from splB-lacZ fusions integrated at the SPbeta prophage locus which contained point mutations or deletions which either inactivated or physically removed P1 and/or splA. Paradoxically, inactivation of P1 by point mutation or its removal by deletion from upstream resulted in elevated beta-galactosidase expression of the resulting splB-lacZ fusion, as did an in-frame deletion of splA which left P1 and P3 intact;however, expression of all fusions remained sporulation specific and EsigmaG dependent.
Assuntos
Bacillus subtilis/genética , Desoxirribodipirimidina Fotoliase/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Regiões Promotoras Genéticas , Proteínas , Bacillus subtilis/fisiologia , Deleção de Genes , Óperon Lac , Mutação , Esporos Bacterianos/fisiologiaRESUMO
Bacterial spores are highly resistant to killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA results in formation of spore photoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine. Repair of SP occurs during germination of Bacillus subtilis spores by two distinct routes, either by the general nucleotide excision repair (uvr) pathway or by a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are synthesized during sporulation and are packaged in the dormant spore. To test this hypothesis, the expression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation. beta-Galactosidase expression from the spl-lacZ fusion was silent during vegetative growth and was not DNA damage inducible, but it was activated at morphological stage III of sporulation specifically in the forespore compartment, coincident with activation of expression of the stage III marker enzyme glucose dehydrogenase. Expression of the spl-lacZ fusion was shown to be dependent upon the sporulation-specific RNA polymerase containing the sigma-G factor (E sigma G), as spl-lacZ expression was abolished in a mutant harboring a deletion in the sigG gene and restored by expression of the sigG gene in trans. Primer extension analysis of spl mRNA revealed a major extension product initiating upstream from a small open reading frame of unknown function which precedes spl, and it revealed two other shorter minor extension products. All three extension products were present in higher quantities during sporulation and after sigG induction. The three putative transcripts are all preceded by sequences which share homology with the consensus sigma-G factor-type promoter sequence, but in vitro transcription by purified sigma-G RNA polymerase was detected only from the promoter corresponding to the major extension product. The open reading frame-spl operon therefore appears to be an additional member of the sigma-G regulon, which also includes as members the small, acid-soluble spore proteins which are in large part responsible for spore DNA photochemistry. Therefore, sporulating bacteria appear to coordinately regulate genes whose products not only alter spore DNA photochemistry but also repair the major spore-specific photoproduct during germination