Burkholderia (formerly Pseudomonas) cepacia porin is an oligomer composed of two component proteins

Abstract

The 81 kDa protein (designated OpcPO) which forms a diffusion pore in the outer membrane of Burkholderia (formerly Pseudomonas) cepacia has a unique characteristic in that when the purified protein is heated it yields a major 36 kDa protein (designated OpcP1) and a minor 27 kDa protein (designated OpcP2). Moreover, incubation of OpcPO in citrate buffer at pH 3 9produced an unusual dissociation into 72 kDa and 27 kDa proteins. For the characterization of OpcPO and its derivatives, OpcPl and OpcP2 from purified OpcPO were isolated by preparative SDS-PAGE. Reconstitution of OpcPO using purified preparations of OpcPl and OpcP2 indicated that these derivatives were not proteolytic fragments of OpcPO. Moreover, immunoblot assays with murine polyclonal antisera specific for OpcPl and OpcP2 yielded the following results: (i) OpcPl and OpcP2 are immunologically distinguishable proteins; (ii) the unusual dissociation of OpcPO in citrate buffer at pH 3-0 resulted in the release of OpcP2 from opcPo# and the resulting 72 kDa protein was probably an oligomer of OpcP1; (iii) purified OpcPl itself produced two additional 53 kDa and 72 kDa proteins spontaneously following elution from the bottom of the SDS-PAGE gel. From these findings, it was concluded that OpcPO is formed by the non-covalent association of OpcP2 with an oligomer of OpcPl that has the ability to self-assemble.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…When the major porin species was isolated, it dissociated into a major 36-kDa species and a minor 27-kDa species on being heated in SDS. The authors speculate that the active porin is a trimer of the former, a conclusion that seems to be consistent with a later study from another laboratory (229). It produced also a low conductance (0.23 nS) channel in 1 M KCl (483).…”

Outer membrane proteins of Methylococcus capsulatus (Bath)

“…The two proteins differed in their Nterminal sequences, and the unheated high-molecularweight MopC/MopD complex contained a mixture of both N-terminal sequences, indicating that MopC is not a proteolytic derivative of MopD. Most porins exist as trimers of identical monomers (Schulz 1996), while porins composed of heterologous monomers have, to our knowledge, been described only for the OpcO porin of Burkholderia cepacia (Parr et al 1987;Gotoh et al 1994). Denaturation of the oligomeric form of the M. capsulatus porin resulted repeatedly in denatured monomeric MopC and MopD, and at present it cannot be concluded whether MopC, MopD, or both are required for poreactivity in the M. capsulatus outer membrane.…”

Serum IgG response to an outer membrane porin protein ofBurkholderia cepaciain patients with cystic fibrosis

“…A single protein was chosen because it is a more defined antigenic preparation than a system based on whole cells or whole outer membrane. The 80‐kDa OMP was selected as a B. cepacia ‐specific antigen after initial SDS‐PAGE studies and on the basis of previous immunoblotting studies [9, 11, 12]. The 80‐kDa OMP is composed of either 36‐kDa subunits alone or 36‐kDa subunits in combination with 27‐kDa subunits.…”

“…To improve recovery of this porin protein Gotoh et al [11]used total membranes (cell envelopes) and lithium dodecyl sulphate (LDS) instead of SDS to prevent crystallisation during chromatography. Using this protein Gotoh et al [11], detected by SDS‐PAGE a 140‐kDa protein in addition to the 80‐kDa protein and the subunits, the 36‐ and 27‐kDa proteins. They purified the 36‐kDa and 27‐kDa subunits separately from the 80‐kDa protein.…”

“…To avoid possible cross‐reaction with P. aeruginosa antigens we have used a B. cepacia ‐specific 80‐kDa outer membrane protein. The 80‐kDa outer membrane protein is thought to be a porin molecule and has been previously purified and characterised [10, 11]. In a previous study of serum IgG response to B cepacia outer membrane, the 80‐kDa outer membrane protein appeared to be B. cepacia ‐specific [12].…”