Differential effects of mutations in tonB1 on intrinsic multidrug resistance and iron acquisition in Pseudomonas aeruginosa

Abstract

Loss of tonB1 adversely affects iron acquisition and intrinsic multidrug resistance in Pseudomonas aeruginosa. Several mutations in tonB1 compromised the protein's contribution to both processes, although TonB1 derivatives altered in residues C35, Q268, R287, Q292, R300, and R304 were compromised vis-à-vis their contribution to drug resistance only.

FIG. 1.

Alignment of P. aeruginosa TonB1 (TonB_Pa) and TonB_Ec, and identification of mutations constructed in TonB1. Identical (|) and conserved (.) residues are indicated. The putative transmembrane domain is in italics. Deletions are indicated by a solid bar above the TonB1 sequence. Point mutations are underlined, in bold text, and identified above the TonB1 sequence. The crossover points of TonB1-TonB_Ec (NPa series) and TonB_Ec-TonB1 (CPa series) chimeras are indicated by arrows. Numbers at the right represent the position of the right-most amino acid within the sequence of the respective TonB proteins. Alignment was carried out using the PALIGN program of the PCGene software package (Intelligenetics, Inc.).

FIG. 2.

Expression of plasmid-encoded TonB1 in the tonB1 deletion strain P. aeruginosa K1040. Cell extracts of K1040 harboring various mutant tonB1 genes (A and B) and tonB1-tonB_Ec chimeras (C) were immunoblotted and probed with antibodies to TonB1 (with the exception of the CPa and CEc series of chimeras, which were probed with monoclonal anti-TonB_Ec antibody 1C3). The nature of the mutation in the sequence of TonB1 is indicated above each lane in panels A and B, with the exceptions of lanes ΔC (deletion of D260 to A310), ΔN2 (deletion of S6 to P83), and ΔT (deletion of P82 to T111). Lanes: WT, wild-type TonB1; pMMB, pMMB206, vector control without a cloned tonB1 gene. Variations seen in the levels of the various mutant TonB1 proteins in panel B were within the natural variation seen for any given mutant TonB1 as observed over three separate experiments. Chimeras shown in panel C are described in the legend to Fig. 1. E.c.tonB, tonB_Ec.