The occurrence of protein phosphorylation in Escherichia coli B, Bacillus megaterium, Bacillus sphaericus, Pseudomonas fluorescens and Arthrobacter S1-55, was investigated by means of both in vivo and in vitro experiments. 2. In each bacterial species the presence of several phosphorylated proteins was evidenced by gel electrophoresis and autoradiography after either labelling of growing cells with [32P]orthophosphate or incubating cellular extracts with radioactive ATP. 3. The analysis of the radioactive moiety of proteins showed that they contained phosphoserine, phosphothreonine and phosphotyrosine. These three phosphoamino acids were found in varying proportions depending both on the bacterial species and, within the same species, on the conditions used for labelling proteins, either in vivo or in vitro. 4. By measuring the effect of cyclic nucleotides on the extent of protein phosphorylation in cellular extracts, it was observed that, in all five bacterial species analyzed, neither cyclic AMP nor cyclic GMP was able to stimulate the activity of protein kinases. 5. All together these results bring evidence that protein phosphorylation catalyzed by protein kinases is a post-translational modification widespread among prokaryotes.
