Department of Biological Sciences, Hunter College of the City University of New York (CUNY), 695 Park Ave., New York, NY 10021, USA.
J Bacteriol. 2011 Feb;193(3):723-33. doi: 10.1128/JB.00708-10. Epub 2010 Nov 19.
The in vivo expression levels of four rRNA promoter pairs (rrnp(1)p(2)) of Bacillus subtilis were determined by employing single-copy lacZ fusions integrated at the amyE locus. The rrnO, rrnJ, rrnD, and rrnB promoters displayed unique growth rate regulation and stringent responses. Both lacZ activity and mRNA levels were highest for rrnO under all growth conditions tested, while rrnJ, rrnB, and rrnD showed decreasing levels of activity. During amino acid starvation induced by serine hydroxamate (SHX), only the strong rrnO and rrnJ promoters demonstrated stringent responses. Under the growth conditions used, the rrn promoters showed responses similar to the responses to carbon source limitation induced by α-methyl glucoside (α-MG). The ratio of P2 to P1 transcripts, determined by primer extension analysis, was high for the strong rrnO and rrnJ promoters, while only P2 transcripts were detected for the weak rrnD and rrnB promoters. Cloned P1 or P2 promoter fragments of rrnO or rrnJ were differentially regulated. In wild-type (relA(+)) and suppressor [relA(S)] strains under the conditions tested, only P2 responded to carbon source limitation by a decrease in RNA synthesis, correlating with an increase in (p)ppGpp levels and a decrease in the GTP concentration. The weak P1 promoter elements remain relaxed in the three genetic backgrounds [relA(+), relA, relA(S)] in the presence of α-MG. During amino acid starvation, P2 was stringently regulated in relA(+) and relA(S) cells, while only rrnJp(1) was also regulated, but to a lesser extent. Both the relA(+) and relA(S) strains showed (p)ppGpp accumulation after α-MG treatment but not after SHX treatment. These data reveal the complex nature of B. subtilis rrn promoter regulation in response to stress, and they suggest that the P2 promoters may play a more prominent role in the stringent response.
采用整合在 amyE 基因座上的单拷贝 lacZ 融合，测定枯草芽孢杆菌的四个 rRNA 启动子对（rrnp(1)p(2)）的体内表达水平。rrnO、rrnJ、rrnD 和 rrnB 启动子表现出独特的生长速率调节和严格反应。在所有测试的生长条件下，rrnO 的 lacZ 活性和 mRNA 水平最高，而 rrnJ、rrnB 和 rrnD 的活性水平则降低。在丝氨酸羟胺（SHX）诱导的氨基酸饥饿期间，只有强 rrnO 和 rrnJ 启动子表现出严格反应。在使用的生长条件下，rrn 启动子表现出与α-甲基葡萄糖苷（α-MG）诱导的碳源限制反应相似的反应。通过引物延伸分析确定的 P2 与 P1 转录物的比值对于强 rrnO 和 rrnJ 启动子较高，而对于弱 rrnD 和 rrnB 启动子仅检测到 P2 转录物。rrnO 或 rrnJ 的克隆 P1 或 P2 启动子片段表现出不同的调节。在测试条件下的野生型（relA(+))和抑制子[relA(S)]菌株中，只有 P2 通过 RNA 合成减少来响应碳源限制，这与（p）ppGpp 水平增加和 GTP 浓度降低相关。在存在 α-MG 的情况下，弱的 P1 启动子元件在三种遗传背景[relA(+), relA, relA(S)]中仍然保持松弛。在氨基酸饥饿期间，P2 在 relA(+) 和 relA(S)细胞中受到严格调节，而仅 rrnJp(1)也受到调节，但程度较小。α-MG 处理后，relA(+) 和 relA(S) 菌株均显示（p）ppGpp 积累，但 SHX 处理后则没有。这些数据揭示了枯草芽孢杆菌 rrn 启动子在应激反应中的复杂调节性质，并表明 P2 启动子可能在严格反应中发挥更突出的作用。
