Timing of cell division in Escherichia coli

我们先前发现，cfc突变使DNA复制和细胞分裂解偶联，并提高了细胞分裂的频率。我们进一步分析了cfc基因的结构和作用。Cfc突变体在达到cfc^+细胞分裂的大小之前就会分裂，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量。Cfcb1在编码α水解酶的apah中有一个IS2插入。携带APAH+的多拷贝质粒抑制了两株突变株的CFCs特性，CFCA细胞内Ap4A水平是其亲本的15倍，CFCB是其亲本的100倍。用野生型细胞进行的实验表明，高水平的Ap4A会导致细胞早期分裂，而低水平的Ap4A会导致细胞延迟分裂。我们从Cfc~+和Cfc~-菌株中纯化了GlyS-6xHis标记蛋白，并分析了其对Ap4A合成的催化活性和GlyS催化tRNA氨酰化的动力学常数。突变型GlyS合成的Ap4A比野生型GlyS多，但对ADP的降解活性低于野生型GlyS。CFCA GlyS对甘氨酸化的催化活性(Km/Kcat)是野生型的20~100倍。因此，我认为Ap4A是一个诱导细胞分裂的信号。高水平的Ap4A负责启动细胞分裂。我们还鉴定了新的Ap4A结合蛋白A(ABPA)，并对其N端氨基酸序列进行了分析。ABPA条件性零突变导致细胞分裂延迟，过量产生ABPA诱导的CFCs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.