Timing of cell division in Escherichia coli

我们以前发现，CFC突变解除了DNA复制和细胞分裂的耦合，并提高了细胞分裂的频率。我们进一步分析了cfc基因的结构和功能。cfc突变体在达到cfc^+细胞分裂的大小之前就分裂了，并产生许多小细胞每个细胞都有一个类核。这些突变影响细胞分裂的时间，但不影响细胞周期的其他过程，如细胞周期的长度和染色体复制的起始质量。CfcA在编码甘氨酰-tRNA合成酶α-亚基的glySa中有一个错义突变，cfcB 1在编码Ap 4A水解酶的apaH中有一个IS 2插入。两种cfc突变体的Cfc特性都受到携带apaH^+的多拷贝质粒的抑制，cfcA中Ap 4A的细胞内水平比它们的亲本高15倍，cfcB高100倍。使用野生型细胞的实验表明，高水平的Ap 4A导致早期细胞分裂，低水平的Ap 4A导致延迟细胞分裂。我们从cfc^+和cfc^-菌株中纯化了GlyS-6xHis标记的蛋白，并分析了GlyS催化合成Ap 4A的体外催化活性和tRNA氨酰化的动力学常数。突变型GlyS比野生型GlyS合成更多的Ap 4A，但表现出比野生型GlyS更低的Ap 4A降解为ADP的活性。cfcA中GlyS的表达量是野生型的20 ~ 100倍。因此，我得出结论，Ap 4A是诱导细胞分裂的信号。高水平的Ap 4A负责启动细胞分裂。通过glyS突变，获得了一个新的Ap 4A结合蛋白A（AbpA），并对其N端氨基酸序列进行了分析。abpA的条件性无效突变诱导细胞分裂延迟，abpA的过量产生诱导Cfc表型。

We found previously that cfc mutation uncouples DNA replication and cell division, and elevates the frequency of cell division. We further analyzed the structure and the role of the cfc genes. The cfc mutants divide before they reach the size at which cfc^+ cells divide, and produce many small cells-each with a single nucleoid. The mutations affect the timing of cell division, but not other processes of cell cycle, such as the length of a cell cycle and the initiation mass for chromosome replication. CfcA has a missence mutation in glySa which encodes the α-subunit of glycyl-tRNA synthetase, and cfcB1 has an IS2 insertion in apaH which encodes Ap4A hydrolase. The Cfc properties of both cfc mutants were suppressed by a multicopy plasmid carrying apaH^+, and the intracellular level of Ap4A in cfcA was 15-fold higher, and cfcB was 100-fold higher than their parent. Experiments using a wild-type cell showed that a high level of Ap4A caused early cell division, and a low level of Ap4A caused delayed cell division. we have purified the GlyS-6xHis tagged proteins from cfc^+ and cfc^- strains and analysed the catalytic activity in vitro for Ap4A synthesis and kinetic constants of tRNA aminoacylation catalyzed by GlyS.Mutant type GlyS synthesized more Ap4A than wild type GlyS but showed lower degradation activity of Ap4A to ADP than wild type GlyS.Catalytic activity for glycylation (Km/Kcat) of GlyS from cfcA is 20〜100 times higher than that from wild type. Therefore, I conclude that Ap4A is a signal for induction of cell division. High level of Ap4A is responsible for the initiation of cell division. The glyS mutation allows efficient synthesis of Ap4A.We also identified novel Ap4A binding protein A (AbpA) and analyzed N terminal sequence of amino acid. Conditional null mutation of abpA induced delayed cell division, and overproduction of abpA induced Cfc phenotype.