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 widely used and closely related Escherichia coli ''wild types'' W3 110 and MG1655, as well as their common ancestor W1485, starve for pyr imidine in minimal medium because of a suboptimal content of orotate p hosphoribosyltransferase, which is encoded by the pyrE gene. This conc lusion was based on the findings that (i) the strains grew 10 to 15% m ore slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepress ed, as is characteristic for pyrimidine starvation conditions; and (ii i) their levels of orotate phosphoribosyltransferase were low. After i ntroduction 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 obser ved for other strains of E. coli K-12, E. coli B, and Salmonella typhi murium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriopha ge 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 res ults in a frame shift of translation over the last 15 codons and reduc es 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 ana lysis of protein synthesis in minicells. The truncated protein lacks R Nase PH activity, and the premature translation stop in the rph cistro n explains the low levels of orotate phosphoribosyltransferase in W311 0, since close coupling between transcription and translation is neede d to support optimal levels of transcription past the intercistronic p yrE attenuator.