Inaoka Takashi, Ochi Kozo
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
J Bacteriol. 2002 Jul;184(14):3923-30. doi: 10.1128/JB.184.14.3923-3930.2002.
We found that the ability to develop genetic competence of a certain relaxed (relA) aspartate-auxotrophic strain of Bacillus subtilis is significantly lower than that of the isogenic stringent (relA+) strain. Transcriptional fusion analysis utilizing a lacZ reporter gene indicated that the amount of the ComK protein, known as the key protein for competence development, is greatly reduced in the relaxed strain than in the stringent strain. We also found that the addition of decoyinine, a GMP synthetase inhibitor, induces expression of a competence gene (comG) in the relaxed strain, accompanied by a pronounced decrease in the level of intracellular GTP as measured by high-performance liquid chromatography. The transformation efficiency of the relaxed strain increased 100-fold when decoyinine was added at t0 (the transition point between exponential to stationary growth phase). Conversely, supplementation of guanosine together with decoyinine completely abolished the observed effect of adding decoyinine on competence development. Furthermore, the impaired ability of the relaxed strain for competence development was completely restored by disrupting the codY gene, which is known to negatively control comK expression. Our results indicate that the RelA protein plays an essential role in the induction of competence development at least under certain physiological conditions by reducing the level of intracellular GTP and overcoming CodY-mediated regulation.
我们发现,枯草芽孢杆菌的某一松弛型(relA)天冬氨酸营养缺陷型菌株形成遗传感受态的能力显著低于同基因的严谨型(relA+)菌株。利用lacZ报告基因进行的转录融合分析表明,作为感受态形成关键蛋白的ComK蛋白的量在松弛型菌株中比在严谨型菌株中大幅减少。我们还发现,添加鸟苷酸合成酶抑制剂脱氧精胍菌素可诱导松弛型菌株中感受态基因(comG)的表达,同时通过高效液相色谱法测定的细胞内GTP水平显著降低。当在t0(指数生长期到稳定生长期的转变点)添加脱氧精胍菌素时,松弛型菌株的转化效率提高了100倍。相反,将鸟苷与脱氧精胍菌素一起添加则完全消除了观察到的添加脱氧精胍菌素对感受态形成的影响。此外,通过破坏已知对comK表达起负调控作用的codY基因,松弛型菌株受损的感受态形成能力得以完全恢复。我们的结果表明,RelA蛋白至少在某些生理条件下通过降低细胞内GTP水平并克服CodY介导的调控,在诱导感受态形成中发挥着重要作用。