The Crystal Structure of OprG from Pseudomonas aeruginosa, a Potential Channel for Transport of Hydrophobic Molecules across the Outer Membrane

Background: The outer membrane (OM) of Gram-negative bacteria provides a barrier to the passage of hydrophobic and hydrophilic compounds into the cell. The OM has embedded proteins that serve important functions in signal transduction and in the transport of molecules into the periplasm. The OmpW family of OM proteins, of which P. aeruginosa OprG is a member, is widespread in Gram-negative bacteria. The biological functions of OprG and other OmpW family members are still unclear. Methodology/Principal Findings: In order to obtain more information about possible functions of OmpW family members we have solved the X-ray crystal structure of P. aeruginosa OprG at 2.4 A ˚ resolution. OprG forms an eightstranded b-barrel with a hydrophobic channel that leads from the extracellular surface to a lateral opening in the barrel wall. The OprG barrel is closed off from the periplasm by interacting polar and charged residues on opposite sides of the barrel wall. Conclusions/Significance: The crystal structure, together with recent biochemical data, suggests that OprG and other OmpW family members form channels that mediate the diffusion of small hydrophobic molecules across the OM by a latera

Mutations in gyrA gene of quinolone-resistant Salmonella serotypes isolated from humans and animals.

The quinolone resistance-determining regions (QRDRs) of the gyrA genes of quinolone-resistant clinical and veterinary salmonella isolates were sequenced. Substitutions analogous to a substitution of a Ser to a Phe at position 83 (Ser83-->Phe) and Asp87-->Gly or Tyr in Escherichia coli were found, as was a single novel mutation outside of the QRDR resulting in Ala119-->Glu. The data suggest that gyrA mutations are associated with quinolone resistance in veterinary and clinical salmonella isolates and that the limits of the QRDR may require revision

The pyocin Sa receptor of Pseudomonas aeruginosa is associated with ferripyoverdin uptake.

We have used Tn5 mutagenesis to obtain a mutant resistant to pyocin Sa. When grown in iron-deficient succinate medium this mutant lacked an 85-kDa iron-regulated outer membrane protein (IROMP), and expression of a 75-kDa IROMP was increased compared with that in the parent strain. The mutant was deficient in pyoverdin biosynthesis and showed a 95% decrease in transport of ferripyoverdin purified from the parent strain, suggesting that the 85-kDa IROMP is the specific receptor for ferripyoverdin and pyocin Sa. The mutant compensated for the deficiency in pyoverdin biosynthesis and transport by exhibiting a fourfold increase in ferripyochelin transport. The low-level transport of ferripyoverdin in the Sa-resistant mutant, which extended to heterologous pyoverdins from other strains, suggests that Pseudomonas aeruginosa has a second ferripyoverdin uptake system of lower affinity and broader specificity

Iron-free pyoverdin binds to its outer membrane receptor FpvA in Pseudomonas aeruginosa: a new mechanism for membrane iron transport.

Under iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin, which, after complexing iron, is transported back into the cell via its outer membrane receptor FpvA. Previous studies demonstrated co-purification of FpvA with iron-free PaA and reported similar binding affinities of iron-free pyoverdin and ferric-pyoverdin to purified FpvA. The fluorescence resonance energy transfer between iron-free PaA and the FpvA receptor here reveals the existence of an FpvA-pyoverdin complex in P. aeruginosa in vivo, suggesting that the pyoverdin-loaded FpvA is the normal state of the receptor in the absence of iron. Using tritiated ferric-pyoverdin, it is shown that iron-free PaA binds to the outer membrane but is not taken up into the cell, and that in vitro and, presumably, in vivo ferric-pyoverdin displaces the bound iron-free pyoverdin on FpvA-PaA to form FpvA-PaA-Fe complexes. In vivo, the kinetics of formation of this FpvA-PaA-Fe complex are more than two orders of magnitude faster than in vitro and depend on the presence of TonB. In P. aeruginosa, two tonB genes have been identified (tonB1 and tonB2). TonB1 is directly involved in ferric-pyoverdin uptake, and TonB2 seems to be able partially to replace TonB1 in its role in iron acquisition. However, no effect of TonB1 or TonB2 on the apparent affinity of free pyoverdin to FpvA was observed, and a 17-fold difference was measured between the affinities of the two forms of pyoverdin (PaA and PaA-Fe) to FpvA in the absence of TonB1 or TonB2. The mechanism of iron uptake in P. aeruginosa via the pyoverdin pathway is discussed in view of these new findings