IRON RELEASE FROM TRANSFERRIN BY PYOVERDIN AND ELASTASE FROM PSEUDOMONAS-AERUGINOSA

Pseudomonas aeruginosa produces the siderophores pyoverdin and pyochel in as well as receptors for siderophores in response to iron deprivati on. Previously, it has been shown in vitro that at neutral pH purified pyoverdin acquires iron from transferrin only in the presence of P. a eruginosa elastase (LasB); which proteolytically degrades transferrin. We constructed a LasB-negative mutant, PAO1E, by insertional mutagene sis to investigate whether this mutant differs in growth from the pare ntal strain PAO1 in an iron depleted medium supplemented with transfer rin or human serum. PAO1 and PAO1E did not differ in growth with 1.25 mu M Fe-2-transferrin as the only iron source. Urea gel electrophoresi s indicated iron release from intact transferrin during the logarithmi c growth phase of PAO1 and PAO1E. A total of 333 mu M LasB was synthes ized from PAO1 after onset of stationary-phase growth. Quantification of pyoverdin by spectroscopy revealed that up to 900 mu M pyoverdin wa s produced during growth of the strains in medium supplemented with Fe -2-transferrin or 10% human serum. Incubation of Fe-2-transferrin and purified pyoverdin in concentrations similar to those found in the cul ture supernatant resulted in release of iron from transferrin after 10 h at 37 degrees C. However, LasB significantly enhanced the rate cons tant for iron acquisition of pyoverdin from transferrin. We conclude t hat P. aeruginosa can use transferrin as an iron source without furthe r need of LasB or pH changes. This is further supported by experiments with P. aeruginosa K437, which has a defective iron uptake system, an d its LasB-negative mutant, K437E, Though K437 and K437E did not diffe r in growth with Fe-2-transferrin as the only iron source, their growt h was significantly reduced relative to that of PAO1 and PAO1E.