Purification of glucose-inducible outer membrane protein OprB of Pseudomonas putida and reconstitution of glucose-specific pores

Abstract

A 43,000 molecular-weight, glucose-inducible, organic acid-repressible protein (OprB) was identified in the outer membrane of Pseudomonas putida. OprB was surface expressed in whole cells, had a high ,8-sheet content, and was heat modifiable, as demonstrated by 125I-labeling, circular dichroism spectroscopy, and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. OprB was extracted from outer membrane preparations by using 2% Lubrol PX with 10 mM EDTA and purified by DEAE-Sephacel ion exchange chromatography following ammonium sulfate precipitation. Reconstitution experiments with black lipid membranes showed that OprB formed small, cation-selective pores which bound glucose (Ks = 110 mM) and other carbohydrates. However, the binding site of OprB appeared to be distinct from that of the maltodextrin-specific porin LamB from Escherichia coli.Pseudomonas putida is a member of the Pseudomonas fluorescens branch (group I) of the family Pseudomonadaceae, a group of organisms that also includes P. aeruginosa (17). The outer membranes of P. putida and related pseudomonads contain a number of proteins which are homologous biochemically, immunologically and genetically to those described in P. aeruginosa (15). However, to date no functional studies of P. putida porins have been described.One interesting class of porins are those outer membrane proteins which are involved in high-affinity substrate transport and contain channels with saturable binding sites for specific substrates. The best studied examples of this class are the maltodextrin-specific porin LamB (5, 6, 21) and nucleotide-specific porin Tsx (18) from Escherichia coli and the phosphate-specific porin OprP (12) and imipenem/basic amino acid-specific porin OprD (26) of P. aeruginosa.LamB is part of a shock-sensitive maltose transport system which mediates the accumulation of maltose into the cell against 100,000-fold concentration gradients, and mutants of E. coli lacking LamB are impaired in maltose uptake (5, 7). LamB reconstituted into black lipid membranes has been shown to bind maltose and maltodextrins up to maltoheptose with Kd decreasing from 10 to 0.067 mM with increasing maltodextrin molecular weight (5, 6). On the basis of this data, a binding site that interacts with up to 5 glucose residues has been proposed (6). In contrast, the phosphate porin OprP appears to have a simple binding site comprising a ring of 3 lysine residues (12).A less well-characterized system for carbohydrate uptake has been described in the pseudomonads. P. aeruginosa and P. putida have common pathways for the dissimilation of glucose (1, 16). Glucose uptake follows either an oxidative pathway in which it is oxidized extracellularly to gluconate or 2-ketogluconate on the cytoplasmic membrane and subsequently transported (1, 10) in which it is transported directly by a high-affinity uptake system (Km = 7 jiM) and subsequently phosphorylated intracellularly (10, 16). High-affinity glucose uptake is induced by growth on glucose (10), and under these condition...

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose‐selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae

“…Subsequently, a binding site for glucose and several other carbohydrates was demonstrated in P. aeruginosa OprB and its homologue in Pseudomonas putida by Wylie et al. (1993) and Saravolac et al (1991), respectively. f?…”

“…Trias et al (1988), using the liposome-swelling assay, characterized OprB as a substrate-selective porin by demonstrating selectivity of OprB for glucose and xylose. Subsequently, a binding site for glucose and several other carbohydrates was demonstrated in P. aeruginosa OprB and its homologue in Pseudomonas putida by Wylie et al (1993) and Saravolac et al (1991), respectively. f?…”

“…aeruginosa and I? putida OprB appear closely related based on immunological and biochemical evidence (Saravolac et al, 1991 ; Wylie et al, 1993), the two porins show opposite ion selectivity (Wylie et al, 1993).Neither of the two currently identified OprB proteins have been sequenced. In this paper, we report the cloning, expression and nucleotide sequence of I?…”

“…aeruginosa and I? putida OprB appear closely related based on immunological and biochemical evidence (Saravolac et al, 1991 ;Wylie et al, 1993), the two porins show opposite ion selectivity (Wylie et al, 1993).…”

“…Single-channel-conductance measurements indicated that both P. aeruginosa and P. putida OprB form small constricted channels [25 pS (picosiemens) for P. aeruginosa OprB (Wylie et al, 1993) and 35 pS for I? putida OprB (Saravolac et al, 1991)l. Most nonspecific porins have single channel conductance values in the nS range.…”