Conversion of OprO into an OprP-like Channel by Exchanging Key Residues in the Channel Constriction.

Under phosphate-limiting conditions, the channels OprP and OprO are induced and expressed in the outer membrane of Pseudomonas aeruginosa. Despite their large homology, the phosphate-specific OprP and the diphosphate-specific OprO pores show structural differences in their binding sites situated in the constriction region. Previously, it was shown that the mutation of amino acids in OprP (Y62F and Y114D) led to an exchange in substrate specificity similar to OprO. To support the role of these key amino acids in the substrate sorting of these specific channels, the reverse mutants for OprO (F62Y, D114Y, and F62Y/D114Y) were created in this study. The phosphate and diphosphate binding of the generated channels was studied in planar lipid bilayers. Our results show that mutations of key residues indeed reverse the substrate specificity of OprO to OprP and support the view that just a few strategically positioned amino acids are mainly responsible for its substrate specificity.

Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa.

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Applied-field MD simulations and electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well.

Draft Genome Sequence of Dietzia maris DSM 43672, a Gram-Positive Bacterium of the Mycolata Group.

Here, we report the draft genome sequence of Dietzia maris, known previously as Rhodococcus maris It is 3,505,372 bp in size with a G+C content of 73%. The draft genome sequence will improve our understanding of Dietzia maris related to other mycolata species and constitutes a basic tool for exploring the cell wall proteins.