Use of Polymerase Chain Reaction for the Determination of About 2.5 kb fpvA and fpvB Gene Sequences in Pseudomonas aeruginosa Strains

Pseudomonas aeruginosa produces three different pyoverdines, types I-III (Cornelis et al., 1989), which are able to chelate iron and form ferripyoverdine complexes that are recognized and transported by different ferripyoverdine receptors present on the outer membrane. The ferripyoverdine receptor gene, fpvA of P. aeruginosa (PAO1) has been characterized previously (Poole et al., 1993). In addition, the other iron-repressible outer membrane receptor proteins for types II and III ferripyoverdine complexes were recently identified and characterized by cloning (De Chial et al., 2003). Following the observation that an fpvA mutant could demonstrate low ferripyoverdine uptake compared with wild type (Poole et al., 1991; Gensberg et al., 1992), an alternative ferripyoverdine receptor gene fpvB was identified and a fragment (562 bp) was amplified by polymerase chain reaction (Ghysels et al. 2004). In addition, the growth of several P. aeruginosa pyoverdine-negative mutants, found to inhabit the lungs of cystic fibrosis patients, were stimulated by existing pyoverdine types, providing additional confirmation for the existence of an alternative route for ferripyoverdine uptake (De Vos et al., 2001; Ghysels et al., 2004). PCR was developed in 1983 by Kary Mullis (Karry Mullis Nobel Lecture, December 8, 1993) and involves the selective amplification of specific regions of DNA for extensive use in molecular biology (Sambrook and Russell, 2001). Using primers designed in this study, the complete sequence of the ferripyoverdine receptor genes (fpvA and fpvB) from several P. aeruginosa clinical and environmental isolates were amplified and sequenced, allowing the identification of variant forms of these receptor genes.