Evidence for protein tyrosine kinase activity in Pseudomonas aeruginosa

Pseudomonas aeruginosa flagellin exhibits a single repeating subunit protein designated either as b-type (53 kDa) or a-type (45 to 52 kDa). Previous data demonstrated that b-type flagellins are phosphorylated at tyrosine in vivo. Data presented extends that finding to include a-type flagellins, as well. Thin layer electrophoresis of flagellar protein treated with alkaline phosphatase demonstrated a release of ³²Pi, indicative of a phosphomonoester linkage. Immunoblots employing anti-phosphotyrosine monoclonal antibody showed that unmodified phosphotyrosine existed in both flagellar types. Experiments were designed to demonstrate phosphorylation of flagellar protein in vitro. Evidence presented showed flagellin was labeled by γ³²P ATP, but not by α³²P ATP, when incubated with cell envelope fractions. Results suggested that autophosphorylation of a 42 kDa membrane protein occurred. No kinase activity was detected in cytoplasmic fractions. Confirmation of tyrosine kinase activity was shown by labeling of synthetic poly(Glu:Tyr) as a substrate with γ³²P ATP. Labeled phosphotyrosine was observed in partial acid hydrolysates of substrates. Using poly(Glu:Tyr) as a substrate, tyrosine kinase activity was shown to be heat sensitive and time dependent and was shown to be inhibited by sulfhydryl reagents. It appears that tyrosine kinase and flagellin phosphorylation occur in several Pseudomonas spp.