Identification of the rodA gene product of Escherichia coli

Abstract

Plasmids that carry the Escherichia coli cell shape gene rodA directed the synthesis of a cytoplasmic membrane protein (M(r), 31,000 [31K protein]) in minicells, maxicells, and an in vitro-coupled transcription-translation system. The 31K protein was identified as the rodA gene product, because it was not synthesized from the vector plasmids or from a plasmid in which the rodA gene was inactivated by insertion of Tn1000. Furthermore, a purified 1.6-kilobase KpnI-BamHI DNA fragment that contained the intact rodA gene directed the synthesis of only the 31K protein in an in vitro system. The apparent molecular weight of the protein was identical whether synthesized in vivo or in vitro, indicating that the rodA gene product is not made as a preprotein. The direction of transcription of rodA was from the KpnI site towards the BamHI site. The 31K protein was unusual in that it could only be detected when cell membranes were solubilized at low temperature (e.g. 37°C) before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Apparently the rodA gene product aggregates after being boiled in sodium dodecyl sulfate and fails to enter a polyacrylamide gel.