THE ESCHERICHIA-COLI K-12 WILD TYPES W3110 AND MG1655 HAVE AND RPH FRAMESHIFT MUTATION THAT LEADS TO PYRIMIDINE STARVATION DUE TO LOW PYRE EXPRESSION LEVELS
THE ESCHERICHIA-COLI K-12 WILD TYPES W3110 AND MG1655 HAVE AND RPH FRAMESHIFT MUTATION THAT LEADS TO PYRIMIDINE STARVATION DUE TO LOW PYRE EXPRESSION LEVELS
复制标题
DOI：
10.1128/jb.175.11.3401-3407.1993
复制
发表时间：
1993-06-01
影响因子：
3.2
通讯作者：
JENSEN, KF
中科院分区：
文献类型：
--
作者：
JENSEN, KF
广泛使用的和密切相关的大肠杆菌“野生型”W3110和MG 1655，以及它们的共同祖先W1485，在基本培养基中缺乏嘧啶，因为乳清酸磷酸核糖基转移酶的含量不理想，这是由pyrE基因编码的。该结论基于以下发现：（i）菌株在不含嘧啶的培养基中的生长速度比在含尿嘧啶的培养基中慢10 - 15%;（ii）其天冬氨酸转氨甲酰酶水平高度去抑制，这是嘧啶饥饿条件的特征;（iii）其乳清酸磷酸核糖基转移酶水平低。在引入携带来自菌株HfrH的rph-pyrE操纵子的质粒后，生长速率不再被尿嘧啶刺激，并且天冬氨酸转氨甲酰酶的水平较低，并且与在其他E. coli K-12、E.大肠杆菌B和鼠伤寒沙门氏菌。为了鉴定导致这些表型的突变，将W3110的rph-pyrE操纵子克隆到来自Kohara噬菌体p51 da 2A 6的pBR 322中。DNA测序结果显示，W3110的rph基因末端附近缺失了一个GC碱基对。这一个碱基对的缺失导致了最后15个密码子的翻译移码，并使rph基因产物的大小相对于其他E.大肠杆菌菌株，如通过分析微细胞中的蛋白质合成所证实的。截短的蛋白质缺乏RNase PH活性，并且rph顺反子中的过早翻译停止解释了W3110中乳清酸磷酸核糖基转移酶的低水平，因为需要转录和翻译之间的紧密偶联来支持经过顺反子间pyrE衰减子的最佳转录水平。
The widely used and closely related Escherichia coli ''wild types'' W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene. This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low. After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E. coli K-12, E. coli B, and Salmonella typhimurium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda2A6. DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110. This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E. coli strains, as confirmed by analysis of protein synthesis in minicells. The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.