Phosphate-starvation-induced outer membrane proteins of members of the families Enterobacteriaceae and Pseudomonodaceae: demonstration of immunological cross-reactivity with an antiserum specific for porin protein P of Pseudomonas aeruginosa

Abstract

Bacteria from members of the families Enterobacteriaceae and Pseudomonadaceae were grown under phosphate-deficient (0.1 to 0.2 mM Pi) conditions and examined for the production of novel membrane proteins. Of the 17 strains examined, 12 expressed a phosphate-starvation-induced outer membrane protein which was heat modifiable in that after solubilization in sodium dodecyl sulfate at low temperature the protein ran on gels as a diffuse band of higher apparent molecular weight, presumably an oligomer form, which shifted to an apparent monomer form after solubilization at high temperature. These proteins fell into two classes based on their monomer molecular weights and the detergent conditions required to release the proteins from the peptidoglycan. The first class, expressed by species of the Pseudomonas fluorescens branch of the family Pseudomonadaceae, was similar to the phosphate-starvation-inducible, channel-forming protein P of Pseudomonas aeruginosa. The second class resembled the major enterobacterial porin proteins and the phosphateregulated PhoE protein of Escherichia coli. Using a protein P-trimer-specific polyclonal antiserum, we were able to demonstrate cross-reactivity of the oligomeric forms of both classes of these proteins on Western blots. However, this antiserum did not react with the monomeric forms of any of these proteins, including protein P monomers. With a protein P-monomer-specific antiserum, no reactivity was seen with any of the phosphatestarvation-inducible membrane proteins (in either oligomeric or monomeric form), with the exception of protein P monomers. These results suggest the presence of conserved antigenic determinants only in the native, functional proteins.The cell envelope of gram-negative bacteria consists of a cytoplasmic membrane, a peptidoglycan layer, and an outer membrane. The outer membrane, which comprises phospholipids, lipopolysaccharide, and protein (16), functions as a permeability barrier, allowing the passage of hydrophilic molecules below a defined molecular weight (the exclusion limit) (18). This permeation is mediated by a class of proteins, termed porins, which form water-filled diffusion channels across the outer membrane (9, 18). Porins generally exist as native functional trimers (1, 18) which are resistant to denaturation by sodium dodecyl sulfate (SDS) (11, 16) but can be dissociated (to nonfunctional monomers) upon heating at temperatures greater than 60°C (11, 16). Heatdissociated porin monomers derived from numerous members of the family Enterobacteriaceae, including the OmpF, OmpC, and PhoE porin monomers of Escherichia coli, have been demonstrated to cross-react immunologically (13,21). This implies that porin structure is, to some degree, conserved during evolution. However, the observation that porin trimers and porin monomers express different antigenic determinants (14) makes the relevance of these studies to native porins questionable. Nonetheless, the possibility that proteins displaying a similar function might be structurally related is intr...

Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP

“…2 all contained a lower-mobility band which migrated at the position of nondenatured OprP. Normally, nondenatured trimers do not react with monomer-specific antisera (31). The fact that these mutant trimers were able to bind the antibody indicated that the heat treatment affected the secondary structure of the protein without causing dissociation of the individual monomers.…”

“…Monomer-specific anti-OprP rabbit serum was made during the course of a previous study (31). Trimer-specific antiserum was made as previously described (31), with fast protein liquid chromatography-purified OprP.…”

“…Monomer-specific anti-OprP rabbit serum was made during the course of a previous study (31). Trimer-specific antiserum was made as previously described (31), with fast protein liquid chromatography-purified OprP. An anti-malarial epitope monoclonal antibody was obtained from R. Wirtz (Department of Entomology, Walter Reed Army Institute of Research, Washington, D.C.).…”

Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC

“…Purified porins are immunogenic in mice and rabbits (19,43) as trimers or monomers. Although antisera to trimers usually do not recognize monomers and vice versa (19,43,46), MAbs raised to monomers can sometimes recognize epitopes on trimers (2).…”

Discrete dissolved and particulate proteins in oceanic waters

“…These proteins are known collectively as pains. Porin P is an unique type of pain, because it is a phosphate-starvation-induced protein that facilitates the uptake of phosphate and polyphosphate (Hancock et al 1982(Hancock et al , 1992Hancock and Benz 1986;Poole and Hancock 1986). Pomeroy et al (1995) showed phosphate to be the primary limiting factor for bacterial production and microbial community respiration in the Gulf of Mexico.…”