Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter, HasDEF, for the heme acquisition protein HasA

Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluores cens no. 33; one is the recently reported AprDEF system and the other is Ha sDEF, which exports a heme acquisition protein, HasA, The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P .fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens, The P. fluorescens Has exporter secreted HasA proteins from P.fluorescens and P, aeruginosa but n ot S, marcescens HasA in Escherichia coli, whereas the Has exporter from S, marcescens allowed secretion of all three HasA proteins. The P. fluorescen s HasDEF system also promoted the secretion of the lipase and alkaline prot ease of P.fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of exp ort substrates, Chimeric HasA proteins containing both P. fluorescens and S , marcescens sequences were produced and tested for secretion through the H as exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P.fluorescens Has exporter.