Barbieri Giulia, Albertini Alessandra M, Ferrari Eugenio, Sonenshein Abraham L, Belitsky Boris R
Dipartimento di Biologia e Biotecnologie Lazzaro Spallanzani, Università di Pavia, Pavia, Italy.
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.
J Bacteriol. 2016 Jan 4;198(6):907-20. doi: 10.1128/JB.00894-15.
AprE and NprE are two major extracellular proteases in Bacillus subtilis whose expression is directly regulated by several pleiotropic transcriptional factors, including AbrB, DegU, ScoC, and SinR. In cells growing in a rich, complex medium, the aprE and nprE genes are strongly expressed only during the post-exponential growth phase; mutations in genes encoding the known regulators affect the level of post-exponential-phase gene expression but do not permit high-level expression during the exponential growth phase. Using DNA-binding assays and expression and mutational analyses, we have shown that the genes for both exoproteases are also under strong, direct, negative control by the global transcriptional regulator CodY. However, because CodY also represses scoC, little or no derepression of aprE and nprE was seen in a codY null mutant due to overexpression of scoC. Thus, CodY is also an indirect positive regulator of these genes by limiting the synthesis of a second repressor. In addition, in cells growing under conditions that activate CodY, a scoC null mutation had little effect on aprE or nprE expression; full effects of scoC or codY null mutations could be seen only in the absence of the other regulator. However, even the codY scoC double mutant did not show high levels of aprE and nprE gene expression during exponential growth phase in a rich, complex medium. Only a third mutation, in abrB, allowed such expression. Thus, three repressors can contribute to reducing exoprotease gene expression during growth in the presence of excess nutrients.
The major Bacillus subtilis exoproteases, AprE and NprE, are important metabolic enzymes whose genes are subject to complex regulation by multiple transcription factors. We show here that expression of the aprE and nprE genes is also controlled, both directly and indirectly, by CodY, a global transcriptional regulator that responds to the intracellular pools of amino acids. Direct CodY-mediated repression explains a long-standing puzzle, that is, why exoproteases are not produced when cells are growing exponentially in a medium containing abundant quantities of proteins or their degradation products. Indirect regulation of aprE and nprE through CodY-mediated repression of the scoC gene, encoding another pleiotropic repressor, serves to maintain a significant level of repression of exoprotease genes when CodY loses activity.
AprE和NprE是枯草芽孢杆菌中的两种主要胞外蛋白酶，其表达直接受多种多效转录因子调控，包括AbrB、DegU、ScoC和SinR。在富含复杂营养成分的培养基中生长的细胞中，aprE和nprE基因仅在指数生长后期强烈表达；编码已知调控因子的基因突变会影响指数生长后期基因表达水平，但在指数生长期不允许高水平表达。通过DNA结合试验以及表达和突变分析，我们发现这两种胞外蛋白酶的基因也受到全局转录调节因子CodY的强烈直接负调控。然而，由于CodY也抑制scoC，在codY缺失突变体中，由于scoC的过表达，几乎看不到aprE和nprE的去抑制。因此，CodY通过限制另一种阻遏物的合成，也是这些基因的间接正调控因子。此外，在激活CodY的条件下生长的细胞中，scoC缺失突变对aprE或nprE表达影响很小；只有在不存在另一种调节因子时，才能看到scoC或codY缺失突变的完全效应。然而，即使是codY scoC双突变体在富含复杂营养成分的培养基中指数生长期也未显示出高水平的aprE和nprE基因表达。只有abrB中的第三个突变允许这种表达。因此，在存在过量营养物的生长过程中，三种阻遏物可共同作用降低胞外蛋白酶基因的表达。
枯草芽孢杆菌主要的胞外蛋白酶AprE和NprE是重要的代谢酶，其基因受到多种转录因子的复杂调控。我们在此表明，aprE和nprE基因的表达也受到CodY的直接和间接控制，CodY是一种响应细胞内氨基酸池的全局转录调节因子。CodY介导的直接抑制解释了一个长期存在的谜题，即为什么当细胞在含有大量蛋白质或其降解产物的培养基中指数生长时不产生胞外蛋白酶。通过CodY介导对编码另一种多效阻遏物的scoC基因的抑制来间接调节aprE和nprE，当CodY失去活性时，有助于维持胞外蛋白酶基因的显著抑制水平。
