The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels

Abstract

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 lambda 2A6. 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.

Keywords

Affiliated Institutions

• University of Copenhagen DK

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