Article Figures & Data
Figures
- Fig. 1.
Western immunoblot of whole-cell extracts (A) and outer membrane proteins (B) of P. aeruginosa K691 (lanes 1) and K691 harboring plasmids pPVR2 (lanes 2), pLK127-2 (lanes 3), pLK21S (lanes 4), pLK39S-1 (lanes 5), pLK16-1 (lanes 6), pLK161-3 (lanes 7), and pLK141-1 (lanes 8) probed with an FpvA-specific antiserum. Cells were cultured overnight in iron-deficient minimal medium and used to prepare whole-cell or outer membrane protein extracts which were subsequently electrophoresed on SDS-polyacrylamide gels and immunoblotted. In a typical experiment, 2.5 μl of outer membrane and 10 to 20 μl of whole-cell protein extracts were loaded onto SDS-polyacrylamide gels.
- Fig. 2.
Pyoverdine-mediated iron uptake by P. aeruginosa K691 harboring pAK1900 (■), pPVR2 (•), pLK127-2 (▴), pLK141-1 (▾), and pLK21S-1 (○). The data are representative of three separate experiments carried out in duplicate and are reported for cells at an A600 of 1.0.
Tables
- Table 1.
FpvA insertion derivativesa
Plasmid Restriction endonucleaseb Insertion site Growth in EDDHA-containing medium (A600 after 10 h )e 55Fe-pyoverdine binding (pmol ± SD )f Nucleotidec Amino acidd pLK141-1 RsaI 1134 Y350 0.29 0.54 ± 0.21 pLK127-2 MstI (FspI) 1288 A402 0.17 0.42 ± 0.26 pLK161-3 MstI (FspI) 1434 R451 0.95 6.19 ± 1.72 pLK16-1 RsaI 1502 G473 NDg ND pLK21S-1 NruI 1645 R521 0.95 8.2 ± 0.59 pLK39S-1 NruI 1757 R558 0.95 9.97 ± 1.66 pPVR2 0.99 11.80 ± 2.13 pAK1900 0.24 0.25 ± 0.21 ↵a 54 bp of DNA was inserted into a variety of sites within the fpvA gene present on plasmid pPVR2, as described in the text. Insertion sites were determined by sequencing and restriction analysis.
↵b Indicates the enzyme which was used to cleave pPVR2 in the process of inserting 54 bp into fpvA and, thus, the restriction site at which the insert is found in the indicated plasmids.
↵c Indicates the nucleotide position of the 54-bp insert within the fpvA coding region, where the A of the ATG start codon is bp 1.
↵d Indicates the site of insertion of the 54-bp-encoded 18 amino acids within the mature FpvA protein, assuming that amino acids 1 to 28 of the precursor are cleaved by signal peptidase and that the mature protein begins with a Q residue. The indicated amino acid was actually lost as a result of the insertion.
↵e P. aeruginosa K691 harboring the indicated plasmids was inoculated into iron-deficient minimal medium supplemented with EDDHA (150 μg/ml) at an A600 of 0.05 to 0.10 and incubated at 37°C. Culture density (A600) was monitored hourly for 10 h, and the final A600 value obtained is reported. The data are representative of three separate experiments carried out in duplicate.
↵f Outer membranes (100 μg) prepared fromP. aeruginosa K691 carrying the indicated plasmids and cultured under iron-limiting conditions were incubated in the presence of 55FeCl3-pyoverdine and harvested on membrane filters. Binding of 55Fe was then assessed as described previously (21). Values reported are means of three determinations ± standard deviations and have been corrected for background levels of 55Fe associating with membrane filters in the absence of added outer membranes.
↵g ND, not determined.
