Rhaese H J, Grade R, Dichtelmüller H
Eur J Biochem. 1976 Apr 15;64(1):205-13. doi: 10.1111/j.1432-1033.1976.tb10289.x.
Unusual highly phosphorylated nucleotides are found in sporulating cells of Bacillus subtilis. Adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp (highly phosphorylated nucleotide I), and adenosine 3(2')-dephosphate 5'-triphosphate, pppApp (highllls are starved for carbon and nitrogen sources. These nucleotides are correlated with sporulation because only ribosomes from sporulating but not vegetative cells are able to synthesize ppApp and pppApp in vitro. Two other nucleotides, adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp (highly phosphorylated nucleotide IV), and a nucleotide with a tentative structure of ppZpUp (highly phosphorylated nucleotide III), where Z is an undetermined sugar, also seem to be involved in regulation of sporulation, especially initiation of sporulation. Sporulation can be initiated even in the presence of amino acids, salts, vitamins etc. in logarithmically growing or stationary-phase cells when carbon sources, i.g. glucose, are used up or artifically removed from the medium. A drastic increase in spore titer is observed 4--5 h later. Also, carbon starvation causes accumulation of the highly phosphorylated nucleotides pppAppp and ppZpUp. On the other hand, sporulation is prevented under the same conditions when excess glucose is maintained in the medium. Correlated with this inhibition of sporulation is the inhibition of formation of highly phosphorylated nucleotides, pppAppp and ppZpUp. Since synthesis of these nucleotides is closely related to sporulation, we anticipate that these substances can cause initiation of development in B. subtilis. Further evidence for our hypothesis on initiation of sporulation by highly phosphorylated nucleotides is that phosphate starvation also causes sporulation with prior accumulation of pppAppp and ppZpUp. Apparently, as long as phosphate is present to synthesize phosphorylated metabolites of glucose, formation of highly phosphorylated nucleotides is repressed. Derepression occurs when either lack of glucose or phosphate or both prevents synthesis of phosphorylated metabolites of glucose allowing synthesis of highly phosphorylated nucleotides. These nucleotides, representing the signal 'lack of glucose or phosphate', then somehow cause changes in gene activity, initiating the complex process of sporulation. Whether or not pppAppp alone or together wtih ppZpUp or even further substances (nucleotides, proteins etc.) is necessary for the above described processes will be answered with the help of suitable mutants lacking the ability to synthesize either one or both regulatory nucleotides. Guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp, are not involved in regulation of devlopment as is shown by using a normally sporulating mutant of B. subtilis, unable to synthesize these nucleotides.
在枯草芽孢杆菌的芽孢形成细胞中发现了异常高度磷酸化的核苷酸。3'（2'）-二磷酸腺苷5'-二磷酸（ppApp，高度磷酸化核苷酸I）和3（2'）-去磷酸腺苷5'-三磷酸（pppApp，高度磷酸化核苷酸II）在芽孢形成细胞中积累，而在营养细胞中含量极低。这些细胞极度缺乏碳源和氮源。这些核苷酸与芽孢形成相关，因为只有来自芽孢形成细胞而非营养细胞的核糖体能够在体外合成ppApp和pppApp。另外两种核苷酸，3'（2'）-三磷酸腺苷5'-三磷酸（pppAppp，高度磷酸化核苷酸IV）和一种暂定结构为ppZpUp（高度磷酸化核苷酸III）的核苷酸（其中Z是一种未确定的糖），似乎也参与芽孢形成的调控，尤其是芽孢形成的起始过程。即使在对数生长期或稳定期细胞中存在氨基酸、盐、维生素等物质的情况下，当碳源（如葡萄糖）被耗尽或从培养基中人工去除时，芽孢形成仍可启动。4 - 5小时后可观察到孢子滴度急剧增加。此外，碳饥饿会导致高度磷酸化核苷酸pppAppp和ppZpUp的积累。另一方面，当培养基中维持过量葡萄糖时，在相同条件下芽孢形成会受到抑制。与这种芽孢形成的抑制相关的是高度磷酸化核苷酸pppAppp和ppZpUp形成的抑制。由于这些核苷酸的合成与芽孢形成密切相关，我们推测这些物质可引发枯草芽孢杆菌的发育起始。关于高度磷酸化核苷酸引发芽孢形成这一假说的进一步证据是，磷酸盐饥饿也会导致芽孢形成，并伴有pppAppp和ppZpUp的预先积累。显然，只要有磷酸盐用于合成葡萄糖的磷酸化代谢产物，高度磷酸化核苷酸的形成就会受到抑制。当缺乏葡萄糖或磷酸盐或两者都缺乏而阻止葡萄糖磷酸化代谢产物的合成，从而允许高度磷酸化核苷酸的合成时，就会发生去抑制。这些核苷酸代表“缺乏葡萄糖或磷酸盐”的信号，然后以某种方式引起基因活性的变化，启动复杂的芽孢形成过程。对于上述过程，单独的pppAppp或与ppZpUp甚至其他进一步的物质（核苷酸、蛋白质等）是否必要，将借助缺乏合成一种或两种调节核苷酸能力的合适突变体来解答。如使用枯草芽孢杆菌的一个正常芽孢形成突变体（无法合成这些核苷酸）所表明的，3'（2'）-二磷酸鸟苷5'-二磷酸（ppGpp）和3'（2'）-二磷酸鸟苷5'-三磷酸（pppGpp）不参与发育调控。
