Studies of phosphate transport in Escherichia coli. I. Reexamination of the effect of osmotic and cold shock on phosphate uptake and some attempts to restore uptake with phosphate binding protein

Abstract

1. The first stage of osmotic shock caused a slight reduction in the primary uptake of inorganic orthophosphate (Pi) in AB3311 cells of Escherichia coli which normally exhibit a biphasic type of phosphate uptake. The second stage of osmotic shock resulted in a marked reduction (a total of 80-85%) in the primary uptake phase and a lesser reduction (50%) in the secondary uptake. When osmotically shocked cells are allowed to recover in a phosphate-free, but otherwise complete medium sufficient repair occurs in the shocked cells to overcome growth lags and to restore the above losses in phosphate uptake almost to normal after 90-180 min of recovery. 2. Extensive investigation was made of the more mild cold shock procedure which involves the rapid disperion of Escherichia coli cells into 80 vol. of water at 2 degrees C. The most consistent cold shock effects, as evidenced by reductions in phosphate uptake, were obtained in cells after they were washed in appropriate buffered salts media, suspended in a minimal volume of water and shocked from 37 degrees C rather than 24 degrees C. Less severe shocks were obtained after washing in imidazole/salts/glucose or Tris/salts/glucose media than in NaCl/Tris. 3. A number of attempts were made to restore by the addition of phosphate binding protein the reduction in phosphate uptake of E. coli AB3311 cells caused by a variety of cold shocks. In no instance was good restoration of phosphate uptake achieved. Qualitatively, it appeared that a better restoration of uptake occurred in unstarved and starved cells washed in imidazole/salts/glucose where the cold shock effect was less severe.