Functional reconstitution, gene isolation and topology modelling of porins from Burkholderia pseudomallei and Burkholderia thailandensis.

Abstract

The sequences for Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis have been deposited in the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under the accession numbers AY312416 and AY312417 respectively. The intracellular pathogen Burkholderia pseudomallei is the causative agent of tropical melioidosis, and Burkholderia thailandensis is a closely-related Gram-negative bacterium that does not cause serious disease. Like other bacteria, the major outer membrane (OM) porins of Burkholderia strains, Bps Omp38 and Bth Omp38 may have roles in antibiotic resistance and immunity. We purified both proteins and found them to be immunologically related, SDS-resistant, heat-sensitive trimers with M (r) of approx. 110000. In functional liposome-swelling assays, both proteins showed similar permeabilities for small sugar molecules, compatible with a pore diameter of between 1.2 and 1.6 nm. Secondary structure analysis by FTIR (Fourier-transform infrared) spectroscopy revealed almost identical spectra with predominantly beta-sheet structures, typical of bacterial porins. MALDI-TOF (matrix-assisted laser-desorption ionization-time of flight) MS and ESI/MS (electrospray ionization MS) analysis of each protein showed extensive sequence similarities to the OpcP1 porin from Burkholderia cepacia (later found to be 76.5% identical). Based on information from the incomplete B. pseudomallei genome-sequencing project, the genes encoding Omp38 were identified and amplified by PCR from B. pseudomallei and B. thailandensis genomic DNA. The nucleotide sequences are 99.7% identical, and the predicted processed proteins are 100% identical. Topology prediction and molecular modelling suggest that this newly-isolated and cloned porin is a 16-stranded beta-barrel and the external loops of the protein could be important determinants of the immune response to infection.