Pyrophosphate is a source of phosphoryl groups for Escherichia coli protein phosphorylation.

Pyrophosphate can serve as a source of phosphoryl groups for the phosphorylation of E. coli proteins. The main target of such phosphorylation is a 49-kDa protein which is covalently modified at serine. The same phosphorylation pattern is obtained in the presence of ATP, which suggests that pyrophosphate can substitute for ATP for bacterial protein phosphorylation.

Evidence of protein-tyrosine kinase activity in the bacterium Acinetobacter calcoaceticus.

Protein phosphorylation was investigated in the bacterium Acinetobacter calcoaceticus both in vivo and in vitro. In cells grown with [32P]orthophosphate, several radioactive phosphoproteins were detected by gel electrophoresis and autoradiography. These proteins were shown to contain phosphoserine, phosphothreonine, and a relatively large proportion of phosphotyrosine residues. Incubation of cellular extracts with [gamma-32P] ATP also resulted in the phosphorylation of several proteins. At least four of them, namely an 81-kDa protein, were modified at tyrosine. No protein labeling occurred when extracts were incubated with [gamma-32P] ATP or [14C]ATP. Moreover, phosphoproteins were insensitive to snake venom phosphodiesterase. All together these results indicate that A. calcoaceticus harbors different protein kinases including a protein-tyrosine kinase activity. Further analysis of this activity showed that it has little, if any, functional similarity with eukaryotic protein-tyrosine kinases.

Occurrence of protein phosphorylation in various bacterial species.

1. 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.

A peptide substrate for Escherichia coli protein kinase activity in vitro.

The phosphorylation in vitro of a series of exogenous peptides by E. coli protein kinases was studied. One of the substrates assayed, the hexapeptide Arg-Gly-Tyr-Ser-Leu-Gly, was found to be significantly phosphorylated at its serine residue. This finding provides the first example of an exogenous substrate utilizable by bacterial protein kinases. The kinetic parameters of the corresponding reaction were determined and the effect of various cations were analyzed. Magnesium, cobalt, manganese and zinc ions were all found to be activators, although to a varying extent. The results were discussed in terms of substrate specificity of bacterial protein kinases.

Cyclic AMP independence of Escherichia coli protein phosphorylation.

The effect of cyclic AMP on protein phosphorylation was analyzed comparatively in two strains of E.coli differing in their capacity to synthesize this nucleotide, one of them lacking the adenylate cyclase activity. The results obtained from both in vivo and in vitro experiments concurred in showing that the bacterial protein kinase activity is cAMP-independent.