A Novel Bacterial ATP-Binding Cassette Transporter System That Allows Uptake of Macromolecules

Abstract

A gram-negative bacterium, Sphingomonas sp. strain A1, isolated as a producer of alginate lyase, has a characteristic cell envelope structure and forms a mouth-like pit on its surface. The pit is produced only when the cells have to incorporate and assimilate alginate. An alginate uptake-deficient mutant was derived from cells of strain A1. One open reading frame, algS (1,089 bp), exhibiting homology to the bacterial ATP-binding domain of an ABC transporter, was cloned as a fragment complementing the mutation. algS was followed by two open reading frames, algM1 (972 bp) and algM2 (879 bp), which exhibit homology with the transmembrane permeases of ABC transporters. Disruption of algS of strain A1 resulted in the failure to incorporate alginate and to form a pit. Hexahistidine-tagged AlgS protein (AlgS His6 ) overexpressed in Escherichia coli and purified by Ni 2؉ affinity column chromatography showed ATPase activity. Based on these results, we propose the occurrence of a novel pit-dependent ABC transporter system that allows the uptake of macromolecules.Alginate is a heteropolysaccharide consisting of ␣-L-mannuronate and its 5Ј-epimer, ␣-L-guluronate, and is produced by brown seaweeds and certain bacteria. Other than its utilization in the medical, chemical, and food areas, this biopolymer has been widely associated with chronic Pseudomonas aeruginosa infection in the lungs of cystic fibrosis patients (5). A bacterium, Sphingomonas sp. strain A1, was isolated from soil as a potent producer of alginate lyase catalyzing the depolymerization of alginate (24). The cells of strain A1 are covered by many large plaits (Fig. 1a), and a mouth-like pit (0.02 to 0.1 m in diameter) is formed on their surface when they are grown in a medium containing alginate as the sole carbon source (10, 11) ( Fig. 1b and c). The pit formed in the presence of alginate disappears when the cells are transferred to a medium without alginate (10, 11). In the presence of alginate, a specific region of the cell surface and its neighborhood is intensely stained with an agent that interacts with mucopolysaccarides (alginate), and thin sections of cells show an irregular site where the cell membrane sinks deeply into the cytosol (10, 11).For the utilization of polysaccharides and other macromolecules, microbes usually degrade them by means of extracellular enzymes and then take the depolymerized low-molecular-weight (LMW) products up through their membranes. However, alginate lyase is exclusively localized in the cytoplasm, and the periplasmic and membrane fractions contain no alginate lyase activity (25). Furthermore, no extracellular alginate-depolymerizing activities have been detected in concentrated culture fluid of strain A1 (24, 25), when assayed by measuring the changes in viscosity and absorbance at 235 nm (for alginate lyase) or analysis of the depolymerization profile by thin-layer chromatography (data not shown). The amino acid sequence of alginate lyase purified from strain A1 was 53 amino acids shorter in the N-terminal region than t...

Superchannel of Bacteria: Biological Significance and New Horizons

“…29) This is true of the ABC transporter responsible for alginate import in strain A1; i.e., the ABC transporter consists of four subunits [AlgM1 (37 kDa), AlgM2 (33 kDa), and two molecules of AlgS (40 kDa)]. 5) As is often observed in bacteria, the genes for ABC transporters (AlgS, AlgM1, and AlgM2) and alginate-binding proteins (AlgQ1 and AlgQ2) are assembled into a cluster. AlgM1 and AlgM2 are homologous to the permease domain of a bacterial ABC transporter, and AlgS exhibits ATPase activity in the dimeric form, indicating that AlgM1 and AlgM2 func-tion as permeases for alginate transport using the energy derived from ATP hydrolysis catalyzed by the AlgS homodimer.…”

“…5,23) AlgQ1 and AlgQ2 resemble each other in primary structure and bind specifically to alginate with K d values of 2:3 Â 10 À7 M and 1:5 Â 10 À7 M respectively. 23) Both values are comparable with those of other periplasmic binding proteins.…”

“…AlgM1 and AlgM2 are homologous to the permease domain of a bacterial ABC transporter, and AlgS exhibits ATPase activity in the dimeric form, indicating that AlgM1 and AlgM2 func-tion as permeases for alginate transport using the energy derived from ATP hydrolysis catalyzed by the AlgS homodimer. 5) The three-dimensional structure of the ABC transporter has been constructed through homology modeling with Monte Carlo minimizations. AlgM1 and AlgM2 contain seven transmembrane -helices each.…”

“…Mutants with disruption of the ABC transporter genes form no apparent pit on their cell surfaces and fail to incorporate alginate. 5) The activity of AlgS is regulated (activated) through a conformational change in AlgM1 and AlgM2, which is induced on contact with alginate-bound AlgQ1 or AlgQ2 with an AlgM1/AlgM2 heterodimer. 24) Therefore, the pit on the cell surface, the binding proteins in the periplasm, and the ABC transporter in the inner membrane are reciprocally related and constitute the superchannel for the import of alginate, confirming the existence of a novel macromolecule import system in bacteria.…”

“…The pit functions as a concentrator of alginate, and the polymer targeted there is transported into the periplasm. The alginate introduced into the periplasm is captured by periplasmic alginate-binding proteins, transferred to an ATP-binding cassette (ABC) transporter (importer) in the inner membrane, 5) and then transported into the cytoplasm, where it is depolymerized into its constituent monosaccharides through reactions catalyzed by exoand endotype alginate lyases (Fig. 2).…”