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.

* Degenerate primer used to amplify and sequence fpvA gene in P. aeruginosa strain Br678. Table 2. List of external and internal primers designed and used in this study.
www.intechopen.com Table 3A. PCR mix for fpvB gene amplification using primers PA4168F and PA4168R (Ghysels et al., 2004) Table 3C. PCR mix for fpvA type I gene amplification using primers 1A-PF and 1A-PR Table 3D. PCR mix for fpvA gene amplification using primers FpVAF1, FpVAF2, FpVAR1, and FpVAR2 www.intechopen.com Gel electrophoresis. Amplified PCR products were run on an agarose gel (0.8%) at 100 V for 70 min. Subsequently, the gel was stained in ethidium bromide for 12 min. Stained gels were visualized and illuminated under UV light (260 nm). Sequencing of PCR products. Amplified DNA was purified using a Sigma Gen-Elute PCR clean up kit, and 100 ng/µl of purified DNA was sent for sequencing with 5 µM of each primer; these primers were used to sequence the external portions of the fpvB gene of nine P. aeruginosa strains as shown below: Table 4. Primers used to sequence the external portions of the fpvB gene of nine P. aeruginosa strains The resulting sequences were aligned with the sequence of the PAO1 fpvB gene using DNAmanager software. The alignment was performed for the PAO1 fpvB sequence plus all resulting forward sequences (for nine sequenced strains) and the PAO1 fpvB sequence plus all resulting reverse sequence (for nine sequenced strains). The purpose of this alignment was to facilitate the design of internal primers for the sequencing of the internal portion of the fpvB gene, and two sets of internal primers were designed for this purpose as shown below: -FpVBF1 (forward) from position (803-820) of aligned forward sequences; -FpVBF2 (reverse) from position (833-850) of aligned forward sequences; -FpVBR1 (forward) from position (1609-1628) of aligned reverse sequences; and -FpVBR2 (reverse) from position (1631-1648) of aligned reverse sequences. 100 ng/µl of purified DNA plus 5 µM of each primer was used for sequencing the internal portion of the gene. 2.5 kb fpvB gene sequence for nine strains. Using the CAP2 software program, the resulting external forward and reverse sequences, in addition to four internal sequences for each of the nine strains, were aligned; the resulting consensus sequence was approximately 2.5 kb. Amplification of the fpvA gene (2.5 kb) in P. aeruginosa strains. Amplification of the fpvA types I, IIa, IIb, and III genes in eight P. aeruginosa strains in which about 2.5 kb fpvB gene was previously amplified was performed using primers designed in this study (Table 2: List of primers) DNA preparation method. Sterile colonies of 12 bacterial samples (eight Woluwe River water strains that included fpvA types I-III plus four positive controls) were grown and templates were prepared for PCR as described above. Table 5. Additional information for strains used in this study.
www.intechopen.com Gel electrophoresis. Amplified PCR products were run and processed as described above. Sequencing of PCR products. Amplified DNA was purified as described above and 100 ng/µl of purified DNA was sequenced with 5 µM of each primer; these primers were used to sequence the external portions of the fpvA gene of three P. aeruginosa strains as shown below: Table 6. Three P. aeruginosa strains The resulting sequences were aligned with the complete sequence of the fpvA gene of three reference strains (PAO1, PA14, and 10-15) as described above. The alignment was done for the fpvA sequences of three reference strains plus all resulting forward sequences (for three sequenced strains) and the fpvA sequences of three reference strains plus all resulting reverse sequences (for three sequenced strains). The purpose of this alignment was to facilitate the design of internal primers for the sequencing of the internal portion of the fpvA gene, and two sets of internal primers were designed for this purpose as shown below: -Int1AF1 (forward) from position (800-818) of aligned forward sequences; -Int1AR1 (Reverse) from position (860-879) of aligned forward sequences; -Int1BF1 (forward) from position (1632-1650) of aligned reverse sequences; and -Int1BR1 (Reverse) from position (1718-1736) of aligned reverse sequences. 100 ng/µl of purified DNA plus 5 µM of each internal primer was used to sequence the internal portion of the fpvA gene. fpvA gene (2.5 kb) sequence for three strains. Using the CAP2 software program, the resulting external forward and reverse sequences, in addition to four internal sequences for each of the three strains, were aligned, and the resulting consensus sequence was approximately 2.5 kb. PCR mix for fpvA type IIa gene amplification using primers 2A-PF and 2A-PR was as in Table 3C, except that no MgCl 2 was used. PCR cycling conditions were as in Table 3B. Gel electrophoresis was performed as described above. Sequencing of PCR products. Amplified DNA was purified as described above and100 ng/µl of purified DNA was sequenced with 3 µM of each primer and these primers were used to sequence the external portions of the fpvA gene of P. aeruginosa strain W15 Aug 15 as shown below: The resulting sequence was aligned with the sequence of the fpvA gene of five reference strains (7NSK2, ATCC 27853, MSH, 2-164, and 1-60) as described above. The alignment was done for the fpvA gene sequences of five reference strains plus resulting forward sequences (for 1 sequenced strain) and the fpvA gene sequences of five reference strains plus resulting reverse sequences (for one sequenced strain). The purpose of this alignment was to facilitate the design of internal primers to enable the sequencing of the internal portion of the fpvA gene; the internal primers designed are shown below: 100 ng/µl of purified DNA plus 5µM each of internal primers were used for sequencing the internal portion of the gene. The same procedure was followed for type 11b except for the external primers that differed (ExtF-2A and ExtR-2A) and only internal primers shown in bold above were used. fpvA gene (2.5 kb) sequence for two strains. Using the CAP2 software program, the resulting external forward and reverse sequences in addition to the four internal sequences for each of the two strains (seven internal sequences in the case of W15Aug15) were aligned and the resulting consensus sequence was approximately 2.5 kb. PCR mix for fpvA type III gene amplification using primers EFT-II1A and ERT-II1A is as shown in Table 3C. PCR cycling conditions are as shown in Table 3B. Gel electrophoresis was performed as described above. Sequencing of PCR products. Amplified DNA was purified as described above and 100 ng/µl of purified DNA was sequenced with 3 µM of each primer; these primers were used to sequence the external portions of the fpvA gene of three P. aeruginosa strains as shown below: Table 8. Three P. aeruginosa strains.
The resulting sequences were aligned with the sequence of the fpvA gene of three reference strains (59.2O, ATCC 013, and 206-12) as described above. The alignment was done for the fpvA sequences of three reference strains plus all resulting forward sequences (for three sequenced strains) and the fpvA sequences of three reference strains plus all resulting reverse sequences (for three sequenced strains). FpVA-3PR (Reverse) from position (2542-2559). 100 ng/µl of purified DNA plus 3 µM (5µM in the case of int3AF3 and int3BR3) each of internal primers were used for sequencing the internal portion of the gene. Also, a new PCR amplification using primers int3-BF3 and FpVA-3PR was performed to enable sequencing of the end portion of the fpvA gene. fpvA gene (2.5kb) sequence for 3 strains. Using the CAP2 software program, the resulting external forward and reverse sequences, in addition to five internal sequences for each of the three strains, were aligned; the resulting consensus sequence was approximately 2.5 kb. Table 9. PCR amplification of fpvA gene (1.5 kb) in Pseudomonas strain Br678 Table 10. ClustalX alignment of fpvA sequences www.intechopen.com Design of primers. Two sets of degenerate primers were designed in this study following a ClustalX alignment of the fpvA sequences of 11 P. aeruginosa strains as shown above. DNA was purified and prepared for PCR as described above. PCR cycling conditions were as described above except that the annealing temperature was increased to 57°C. Gel electrophoresis. Gel electrophoresis of amplified DNA involved an application of 8 µL amplified PCR product and 2 µL loading dye on a 0.8% agarose gel in 1× TAE buffer and performed at 100 V for 65 min. Subsequently, the gel was stained in ethidium bromide for 12 min and illuminated under UV light. Sequencing of PCR products. Amplified DNA was purified and sequenced as described above with 5µM each of primer; these primers were used to sequence the amplified purified PCR product of strain Br678. fpvA gene (1.5 kb) sequence for strain Br678. Using the CAP2 software program, the resulting forward and reverse sequences were aligned, and the resulting consensus sequence was approximately 1.5 kb. The 1.5-kb fpvA sequence of Br678 was 96% identical and 97% similar to fpvA type II of P. aeruginosa isolate 2-164 at nucleotide and amino acid levels, respectively.

Amplification of fpvB gene (2.5 kb) in P. aeruginosa strain Br678
DNA was purified and prepared for PCR as described above. PCR cycling conditions were as described above in Table 3B. Gel electrophoresis was as described above. Sequencing of PCR products. Amplified DNA was purified and sequenced as described above with 5 µM of each primer ( PA4168F and PA4168R as above); these primers were used to sequence the external portions of the fpvB gene of strain Br678. In addition, the two sets of internal primers previously designed for the sequencing of the internal portion of the fpvB gene were used to sequence the internal portion of the fpvB gene in this strain as shown below: -FpVBF1 (forward) from position (803-820) of aligned forward sequences; -FpVBF2 (Reverse) from position (833-850) of aligned forward sequences; -FpVBR1 (forward) from position (1609-1628) of aligned reverse sequences; and -FpVBR2 (Reverse) from position (1631-1648) of aligned reverse sequences. 100 ng/µl of purified DNA of filter sterilized water plus 5µM of each internal primer was used to sequence the internal portion of the gene.

fpvB gene (2.5 kb) sequence for strain Br678
Using the CAP3 software program, the resulting external forward and reverse sequences, in addition to four internal sequences for strain Br678, were aligned, and the resulting consensus sequence was approximately 2.5 kb.

Results
The nucleotide sequences of both FpvA and FpvB determined in this study have been deposited in the GenBank database (Bodilis et al., 2009).     Table 13. Percent similarity of fpvA and fpvB at the nucleotide and amino acid levels (BLAST search against the NCBI database)

Discussion
Under iron limiting conditions, pyoverdine is produced by Pseudomonas aeruginosa, a human opportunistic pathogen, several studies track its occurrence as a noscomial pathogen indicating that antibiotic resistance is increasing in clinical isolates (which may be true for the strains I worked with). Pyoverdine is a metal chelating compound, P. aeruginosa, in the past has been studied to acquire plasmid (Mercer and Loutit, 1979), several transporting mechanisms have been extensively studied in this organism of which is the pyoverdine transport. Based on the different pyoverdine types, three siderovars which exists within the P. aeruginosa group have been detected by siderotyping, (Fuchs et al., 2001;Meyer et al., 1997;; this technique, however, is limited. The existence of pyoverdine-negative isolates of P. using PCR-specific primers designed for the amplification of genes in these organisms. PCR is not limited by pyoverdine production as is siderotyping and, as such, is considered reliable and less time-intensive than cloning. Since PCR-specific primers are designed to carry out amplification procedures, the problems with false positives may not likely arise, although this has not always been the case. P. aeruginosa secretes pyocyanin which has been documented to strip iron from transferrin (Cox, C. 1986), it also produces pyoverdine, which strips iron and contributes to the virulence of this organism, thus pyoverdine production is accompanied by virulence factor secretion (Meyer, 1996;Clarke et al., 2001;Beare et al., 2003). Pyoverdine growth stimulation assays have also been used to type P. aeruginosa, but situations have arisen whereby the observable growth had been stimulated by more than one pyoverdine (Pirnay et al., 2002;Meyer, 1992;Meyer et al., 1999;Stintzi et al., 2000). The outcome of such assays may only be predictions, and, therefore, not of use for molecular diagnosis (Pirnay et al. 2005). The fpvA gene sequence has been proposed to be diverse (Thupvong et al., 1999;Smith et al., 2005); with such diversity, it may be difficult to perform PCR on such a gene without problems of non-specific amplification. However, in this study, single bands arising as a result of fpvA or fpvB gene amplification were either purified directly or excised and purified from the gel. Results of sequencing following a BLAST search against the NCBI database revealed that these sequences were approximately 95-100% identical and similar at both the nucleotide and amino acid levels to those of reference strains used in this study. Homology trees showing percent relatedness of the fpvA and fpvB genes in P. aeruginosa test and reference strains were also constructed using the DNA manager software following an alignment of all nucleotide sequences for the individual test and reference strains. The purpose of this study was to use PCR for the determination of about 2.5 kb gene sequence of the ferripyoverdine receptor genes (fpvA types I, IIa, IIb, and III, and fpvB) in P. aeruginosa clinical and environmental strains, and this goal was achieved using a series of external and internal primers designed for both amplification and sequencing. This study has provided for the first time a means to determine the fpvA and fpvB gene sequences (~2.5 kb) in P. aeruginosa clinical and environmental isolates using experimental PCR.

Future perspective
Pseudomonas aeruginosa affects immunocompromised individuals like the AIDS patients undergoing antiretroviral therapy, in these individuals, it has been documented that P. aeruginosa causes a range of infections amongst which are urinary tract infections, respiratory infections, gastrointestinal infections, bone and joint infections and bacteremia, the case fatality rate in these patients is near 50% (Todar, K. 2004). Pyoverdine the siderophore secreted by P. aeruginosa is very important to it and siderophore biosynthesis has been documented to represent an attractive antibiotic target (Quadri, 2000). fpvA has also been proposed to drive diversity at the pyoverdine locus (Smith et al., 2005), looking at the strains I worked with, especially the clinical isolates (Isolated from burn wound ), the primers used for amplification were different from those used for the existing fpvA type II pyoverdine isolates and following amplification and sequencing, a variant form (different from the already existing ferripyoverdine receptor gene types) of the ferripyoverdine receptor genes is presented (strain Br678, fpvA 93% identical to the fpvA gene of other strains in the homology tree (see homology tree c); comparison based on the sequenced 1.5 kb sized fpvA gene, strain Mi162 fpvB 98% identical to the fpvB gene of other strains in the homology tree (see homology tree a); comparison based on the sequenced 2.5 kb sized fpvB gene), this may then justify a correlation between amino acid sequence diversity of immunogenic bacterial proteins and evasion of host immune defense mechanisms (Tummler and Cornelis, 2005). It would be interesting to study these strains in the future to pave way for the full understanding of underlying mechanism of antibiotic resistance. More research would be done in this regard hopefully.

Acknowledgement
I am grateful to Professor P. Cornelis and the Vrije Universiteit Brussels (VUB) for the provision of Academic Scholarship.