Expression profiling of B.subtils SMY wild-type, (p)ppGpp0, ΔcodY (p)ppGpp0 and guaB- (p)ppGpp0 cells upon amino acid starvation (RHX) or guanosine (Guo) treatment.

The nucleotide (p)ppGpp is crucial for viability during amino acid limitation in bacteria, yet how it accomplishes this remains unknown. We found that the absence of (p)ppGpp in Bacillus subtilis cells leads to multiple amino acid auxotrophy, and that (p)ppGpp allows for prototrophy by reducing GTP levels. We provide evidence that reduction of GTP levels relieves the requirements for branched-chain amino acids primarily by preventing hyperactivity of the GTP-dependent transcriptional regulator CodY, but that GTP levels can also play an important role in regulating transcription of many amino acid biosynthesis genes independently of CodY. Thus, CodY-dependent and independent regulation of transcription by GTP levels plays overlapping yet distinct physiological roles in allowing amino acid prototrophy. Finally, supplementing these required amino acids does not protect against cell death upon nutrient downshift, but allows for sustained growth following this transition. We conclude that regulation of GTP levels by (p)ppGpp allows cells to adapt to conditions of amino acid limitation by first allowing survival during shifting nutrient conditions, and then allowing amino acid prototrophy by transcriptionally regulating amino acid biosynthesis. This strategy may be used to ensure viability during amino acid limitation in evolutionarily divergent bacteria. Twelve-condition experiment: wt, wt+RHX, wt+Guo, (p)ppGpp0, (p)ppGpp0+RHX, (p)ppGpp0+Guo, ΔcodY (p)ppGpp0, ΔcodY (p)ppGpp0+RHX, ΔcodY (p)ppGpp0+Guo, guaB- (p)ppGpp0, guaB- (p)ppGpp0+RHX, guaB- (p)ppGpp0+Guo. Biological replicates: 3 for each sample. Reference: a mixture of wt RNA from different growth phases and wt backgrounds.

核苷酸(p)ppGpp是细菌在氨基酸限制条件下维持存活的关键分子，但其具体作用机制仍未阐明。我们在枯草芽孢杆菌（Bacillus subtilis）中发现，缺失(p)ppGpp会导致多重氨基酸营养缺陷型，而(p)ppGpp可通过降低三磷酸鸟苷（GTP）水平恢复氨基酸原养型。本研究证实，降低GTP水平主要通过抑制GTP依赖型转录调控因子CodY的过度激活，解除细胞对支链氨基酸的需求；此外，GTP水平还可在不依赖CodY的前提下，对众多氨基酸生物合成基因的转录发挥重要调控作用。由此可见，GTP水平通过CodY依赖与非依赖途径调控转录，在实现氨基酸原养型的过程中兼具重叠与独特的生理功能。最后，补充上述必需氨基酸无法阻止营养水平下调引发的细胞死亡，但可使细胞在该过渡阶段后维持持续生长。综上，(p)ppGpp通过调控GTP水平，使细菌能够先在营养条件转变过程中维持存活，再通过转录调控氨基酸生物合成基因实现氨基酸原养型，从而适应氨基酸限制环境。该策略或可在进化亲缘关系较远的各类细菌中，保障其在氨基酸限制条件下的存活能力。
十二组实验条件如下：野生型（wild type, wt）、wt+RHX、wt+鸟苷（Guo）、(p)ppGpp缺失株（(p)ppGpp⁰）、(p)ppGpp⁰+RHX、(p)ppGpp⁰+Guo、codY基因缺失(p)ppGpp⁰株（ΔcodY (p)ppGpp⁰）、ΔcodY (p)ppGpp⁰+RHX、ΔcodY (p)ppGpp⁰+Guo、guaB基因缺陷(p)ppGpp⁰株（guaB⁻ (p)ppGpp⁰）、guaB⁻ (p)ppGpp⁰+RHX、guaB⁻ (p)ppGpp⁰+Guo。
生物学重复：每个样本设置3次重复。
参考样本：取自不同生长阶段、不同遗传背景的野生型菌株的RNA混合物。