Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties

Abstract

Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning P-strands, probably forming a P-barrel.Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCI and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.Pseudomonas aeruginosa is an opportunistic human pathogen which infects particularly persons who are suffering from cystic fibrosis. The initial infection occurs mostly with nonmucoid bacteria, which convert to mucoid strains in an early stage of infection (13,26). Mucoid bacteria produce copious amounts of alginate, which seems to be the most important virulence factor (12). The initial steps of the alginate biosynthesis, leading to the putative precursor GDP-mannuronic acid, are well-known (21). However, there is a lack of information about the final steps of the biosynthesis, i.e., the polymerization and export of alginate. Recently, the genes involved in acetylation (algF) and degradation (algL) of alginate were cloned and mapped in the alginate biosynthesis gene cluster (34 min) (10, 30). In addition, the gene product of algG (34 min) was purified and identified as an epimerase located in the periplasm that introduces guluronic acid residues into the alginate (9). We identified a 54-kDa protein (AlgE) in the outer membrane (OM) of P. aeruginosa which appears in mucoid strains only (15,26). This protein was purified and the N-terminal amino acid sequence was determined (14). At the same time...

Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

“…Bacterial alginates are to various extents also acetylated in the O-2 and/or O-3 position (54) by periplasmic enzymes (19). After these modifications, the alginate polymer is exported out of the cell, probably via an outer membrane channel encoded in the alginate biosynthetic cluster (46,47). The algU mucABCD cluster, which is a E -type cooperative regulatory unit, controls expression of the alginate biosynthesis genes in P. aeruginosa (45) and A. vinelandii (20,35,39).…”

The Azotobacter vinelandii gene algJ encodes an outer-membrane protein presumably involved in export of alginate

“…As a probe, digoxigenin-labelled RNA corresponding to the entire coding region of the algE gene from P. aeruginosa was used. The labelling reaction was performed with the previously described plasmid pTR7-2 using a T7 RNA polymerase digoxigeninlabelling kit (Boehringer Mannheim) (Rehm et al, 1994b). The labelling procedure was conducted according to the user's guide (Boehringer Mannheim).…”

“…As a probe, digoxigenin-1 1 -dUTP-labelled RNA, representing the entire coding region of algE from P. aerzlginosa, was applied. This labelling was done using plasmid pTR7-2, which enabled overexpression of algE under the control of the T7 promoter $10 (Rehm et al, 1994b;Tabor & Richardson, 1985). All of the alginateproducing bacteria analysed in this study showed, under stringent conditions, specific hybridization signals (Fig.…”

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

“…AlgL is an alginate lyase that can degrade alginate polymers (Schiller et al, 1993). AlgE has been shown to be an outer-membrane porin (Rehm et al, 1994).…”