Hecker M, Riethdorf S, Bauer C, Schroeter A, Borriss R
Department of General Microbiology, Ernst-Moritz-Arndt-University, DDR, Greifswald.
Mol Gen Genet. 1988 Dec;215(1):181-3. doi: 10.1007/BF00331323.
Amino acid starvation of cells of the Escherichia coli relA strain, CP79, which cannot accumulate guanosine tetraphosphate (ppGpp) in response to amino acid limitation, increased the pEG1 plasmid content about 5- to 7-fold in comparison with exponentially growing cells (pEG1:pBR322 with an insertion of Bacillus amyloliquefaciens DNA coding for beta-glucanase). In contrast, no pEG1 amplification occurred in E. coli CP78, the stringently controlled counterpart, after amino acid starvation. In order to verify these results, the plasmid DNA content was monitored by measuring the expression of pEG1-encoded beta-glucanase from B. amyloliquefaciens both before and after plasmid amplification. When amino acid starved CP79 cells were given an additional dose of amino acids, a more than 10-fold increase in pEG1-encoded beta-glucanase activity (per cell mass) was measured. This increase in enzyme activity correlates with pEG1 amplification during amino acid limitation. Under comparable conditions the activity of beta-glucanase was not increased in strain CP78, which did not amplify the plasmid. We suggest that the replication of pEG1 in amino acid starved E. coli cells is somehow under negative control by ppGpp. Moreover, we found the Bacillus beta-glucanase in E. coli relA cells to be excreted into the growth medium after starvation and overexpression.
大肠杆菌relA菌株CP79的细胞在氨基酸饥饿时无法响应氨基酸限制而积累鸟苷四磷酸（ppGpp），与指数生长期的细胞相比（pEG1：插入了编码β-葡聚糖酶的解淀粉芽孢杆菌DNA的pBR322），其pEG1质粒含量增加了约5至7倍。相比之下，在氨基酸饥饿后，严格调控的对应菌株大肠杆菌CP78中未发生pEG1扩增。为了验证这些结果，在质粒扩增前后，通过测量pEG1编码的解淀粉芽孢杆菌β-葡聚糖酶的表达来监测质粒DNA含量。当给氨基酸饥饿的CP79细胞额外添加一剂氨基酸时，测得pEG1编码的β-葡聚糖酶活性（每细胞质量）增加了10倍以上。这种酶活性的增加与氨基酸限制期间的pEG1扩增相关。在可比条件下，未扩增质粒的CP78菌株中β-葡聚糖酶的活性没有增加。我们认为，在氨基酸饥饿的大肠杆菌细胞中，pEG1的复制在某种程度上受到ppGpp的负调控。此外，我们发现，在饥饿和过表达后，大肠杆菌relA细胞中的芽孢杆菌β-葡聚糖酶会分泌到生长培养基中。
