Uptake of Pyocin S3 Occurs through the Outer Membrane Ferripyoverdine Type II Receptor of Pseudomonas aeruginosa

Pyocin S3 kills type II pyoverdine producers.Forty-nine isolates of P. aeruginosa (19 CF, 22 non-CF, and 8 reference strains for pyocin typing) were tested by the spot agar technique on glutamate minimal medium (12) for their susceptibilities to pyocins S1, S2, S3, and AP41. These pyocins were used after partial purification from lysates of recombinant E. coli strains containing the corresponding genes by ammonium sulfate precipitation and ion-exchange chromatography (9). The pyoverdine-containing culture supernatant of each strain grown in iron-poor Casamino Acids (CAA) medium was analyzed by the isoelectric-focusing technique in order to determine the type of pyoverdine produced, as evidenced by the pattern of fluorescent bands representing isoforms of pyoverdine (16, 18). As shown in Table 1, all strains susceptible to pyocin S3 produced and/or could take up type II pyoverdine (isolates which failed to produce pyoverdine were tested for their uptake of the three ⁵⁹Fe-ferripyoverdines, according to the method described in reference 4). In striking contrast, the pyocin S3-resistant isolates produced either type I or III pyoverdine. No correlation was found between strain susceptibility to other pyocins (S1, S2, AP41) and pyoverdine type production (results not shown). These observations were further confirmed by testing the susceptibilities to pyocin S3 of 40 other strains, which had already been typed for pyoverdine in a previous study (18) (Table1). As expected, the 15 strains producing type II pyoverdine were all killed by pyocin S3 while the isolates producing type I (n = 10) or type III (n = 10) pyoverdine were all pyocin S3 resistant (Table 1).

Competition between pyoverdine and pyocin S3.The killing activity of pyocin S3 against strain ATCC 27853 as determined by the spot test assay was clearly inhibited by type II pyoverdine (Fig.1). This antagonism was more important in the presence of ferripyoverdine than in the presence of pyoverdine. Conversely, the addition of either of the other two pyoverdines (type I and type III) increased the killing activity of S3 on Luria-Bertani medium (compared with that on water [results not shown]), probably by creating a higher iron deficiency (Fig. 1). Two explanations for this result come to mind: either there is a direct competition between the two ligands (type II pyoverdine and pyocin S3) for the same receptor or the cognate ferripyoverdine, being taken up, reduces the iron deficiency, which in turn results in a down regulation of the expression of the receptor gene. The second hypothesis, however, is unlikely, since it has been demonstrated that pyoverdine induces the production of its receptor (10).

• Open in new tab
• Download powerpoint

Fig. 1.

Plate showing the effects of different pyoverdines on the killing of P. aeruginosa ATCC 27853 by pyocin S3. To a lawn of P. aeruginosa ATCC 27853 on Luria-Bertani medium drops of purified pyocin S3 (20 μl, corresponding to 25 U ml⁻¹, defined as the reciprocal of the highest pyocin dilution inhibiting bacterial growth) were deposited at three places in the middle. Next, on each side of the pyocin drop, 20 μl of the following purified pyoverdines (2 μmol) was added: that from PAO1 (top), that from ATCC 27853 (middle), and that from clinical isolate PA6 (bottom). Only the cognate pyoverdine (ATCC 27853), recognized by the pyoverdine receptor, competes with pyocin S3, which uses the same receptor, inhibiting the killing.

Generation of Tn5 mutants resistant to pyocin S3.Pb60, a clinical isolate producing type II pyoverdine and susceptible to pyocin S3 (Table 1), was chosen because of its low resistance to kanamycin. Insertion mutants were obtained after conjugational transfer (21) of the suicide plasmid pSUP2021 carrying transposon Tn5 (27) and isolation on Pseudomonasagar medium (Institut Pasteur Productions, Paris, France) containing kanamycin at 400 μg/ml. Two mutants of 500 transconjugants were selected for their inability to grow in the presence of 1 mg of the iron(III) chelator ethylenediamine-di-( o -hydroxyphenylacetic acid) (EDDHA) per ml. One mutant turned out to be susceptible to pyocin S3 (Pb60S), but the other one (Pb60R) was resistant.

Interestingly, mutant Pb60S, although not producing pyoverdine, was still able to grow in the presence of 1 mg of EDDHA per ml when purified type II pyoverdine was added to the medium while Pb60R (which produced ten times less pyoverdine than the wild type) was not, suggesting that the latter had a defect in the uptake of pyoverdine. In agreement with these results, Pb60R grown in glutamate minimal medium was found to lack an 85-kDa iron-repressed outer membrane protein (IROMP) (results not shown) and to incorporate about 20-fold less⁵⁹Fe-pyoverdine than the wild type or Pb60S (Table2). The levels of pyochelin produced by Pb60 and its two mutants Pb60S and Pb60R were determined after extraction of the acidified growth medium supernatant by ethyl acetate (11) and were found to be nearly identical (results not shown). This result was corroborated by pyochelin-uptake experiments showing normal pyochelin-mediated iron uptake by the two mutants (results not shown). A second insertion mutagenesis was carried out with the type II pyoverdine strain ATCC 27853, which is susceptible to pyocin S3, and a mini-Tn5 with a tetracycline resistance gene (7) as previously described (1). This time, 7 of 1,500 mutants were directly selected for their resistance to pyocin S3. All were partially or totally deficient for pyoverdine production, did not grow in the presence of EDDHA supplemented with type II pyoverdine, and failed to take up type II⁵⁹Fe-pyoverdine (Table 2). Furthermore, these mutants all lacked the 85-kDa IROMP in their outer membranes (results for 4 mutants are shown in Fig. 2). The fact that mutants that do not produce the ferripyoverdine receptor are also defective in pyoverdine production has already been demonstrated (22, 28), indicating that the biosynthesis of pyoverdine and the production of the receptor are coregulated.

• Open in new tab
• Download powerpoint

Fig. 2.

Sodium dodecyl sulfate-polyacrylamide gel (7%) electrophoresis of 10-μg quantities of outer membrane proteins fromP. aeruginosa ATCC 27853 grown in CAA medium (lane 1) and in CAA medium plus FeCl₃ (50 μM) (lane 2), mutant ATCC 27853-5B (lane 3), mutant ATCC 27853-6A (lane 4), mutant ATCC 27853-9A1 (lane 5), and mutant ATCC 27853-16B1 (lane 6). Lane 7 contains the molecular mass markers (High molecular mass markers; Pharmacia-LKB, Uppsala, Sweden).

Conclusions.Altogether, our results strongly suggest that pyocin S3 utilizes the receptor for type II ferripyoverdine to enter a bacterial cell, because of (i) the perfect correlation between the ability of an isolate to utilize type II pyoverdine and its susceptibility to pyocin S3, (ii) the inhibition of killing by the addition of type II pyoverdine, and (iii) the fact that mutants resistant to killing by pyocin S3 are all deficient in pyoverdine production and uptake and lack an 85-kDa IROMP.

Work is now in progress to further characterize the Tn5mutants obtained and to clone the genes involved in the expression of the type II ferripyoverdine receptor.