Yumiko Mishima

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

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.

The N-terminal domain of Drosophila Gram-negative binding protein 3 (GNBP3) defines a novel family of fungal pattern recognition receptors.

Gram-negative binding protein 3 (GNBP3), a pattern recognition receptor that circulates in the hemolymph of Drosophila, is responsible for sensing fungal infection and triggering Toll pathway activation. Here, we report that GNBP3 N-terminal domain binds to fungi upon identifying long chains of β-1,3-glucans in the fungal cell wall as a major ligand. Interestingly, this domain fails to interact strongly with short oligosaccharides. The crystal structure of GNBP3-Nter reveals an immunoglobulin-like fold in which the glucan binding site is masked by a loop that is highly conserved among glucan-binding proteins identified in several insect orders. Structure-based mutagenesis experiments reveal an essential role for this occluding loop in discriminating between short and long polysaccharides. The displacement of the occluding loop is necessary for binding and could explain the specificity of the interaction with long chain structured polysaccharides. This represents a novel mechanism for β-glucan recognition.

Enzymatic and genetic bases on assimilation, depolymerization, and transport of heteropolysaccharides in bacteria

When microorganisms utilize macromolecules for their growth, they commonly produce extracellular depolymerization enzymes and then incorporate the depolymerized low-molecular-weight products. Assimilation of heteropolysaccharides (gellan and xanthan) by Bacillus sp. GL1 depends on this generally accepted mechanism. On the other hand, Sphingomonas sp. A1 represents an unexplored specific and interesting system for macromolecule assimilation. In the presence of heteropolysaccharide (alginate), the bacterium forms a mouthlike pit on its cell surface and directly incorporates the macromolecule using a novel ATP-binding cassette transporter (ABC transporter). In this review, we discuss enzymatic and genetic bases on the depolymerization and assimilation routes of heteropolysaccharides in bacteria, with particular emphasis on the novel incorporation system for macromolecules, characteristic post-translational modification processes of polysaccharide lyases and on the mouthlike pit structure on the bacterial cell surface.

Special cell surface structure, and novel macromolecule transport/depolymerization system of Sphingomonas sp A1.

A bacterium isolated from soil as an alginate lyase producer shows characteristic morphological and taxonomical properties consistent with being classified in the genus Sphingomonas. The bacterium utilizes high molecular weight (HMW)-alginate for growth by depolymerization of the polymer with intracellular alginate lyases, which are generated from a common precursor protein through autoregulated post-translational modifications. Electron microscopic observations of the cell surface and of thin sections of cells grown on HMW-alginate revealed dynamic changes in both cell surface and membrane structures. The most remarkable change is recognized in the formation of mouth-like pits which open and close depending on the presence or absence of HMW-alginate. Enzymatic and genetic analyses of HMW-alginate incorporation processes confirmed the presence of a pit-dependent and macromolecule-specific ABC transporter system in cells of Sphingomonas species A1. This is the first description of a bacterium with a pit on the cell surface and a pit-dependent endocytosic uptake system for macromolecules.

Crystallization and preliminary X-ray analysis of AlgS, a bacterial ATP-binding cassette (ABC) protein specific to macromolecule import

Sphingomonas sp. A1 possesses a macromolecule (alginate; average molecular size 25 700 Da) uptake system mediated by a novel pit-dependent ABC transporter. In this system, AlgS (363 amino-acid residues; 40 kDa) functions as an ATPase and provides energy for the translocation of high molecular-weight alginate across the cytoplasmic membrane. Hexahistidine-tagged AlgS of Sphingomonas sp. A1 was overexpressed in Escherichia coli and crystallized by means of the hanging-drop vapour-diffusion method with ammonium dihydrogen monophosphate as the precipitant. Preliminary X-ray analysis of the resultant crystals was performed; they belonged to the monoclinic space group P2(1) and had unit-cell parameters a = 57.4, b = 92.7, c = 65.8 A, beta = 102.3 degrees. X-ray diffraction data to 3.2 A have been collected from the native crystal.

Super-channel in Bacteria: Function and Structure of a Macromolecule Import System Mediated by a Pit-dependent ABC Transporter

In a soil isolate, Sphingomonas sp. A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter. The transporter is different from other ABC transporters so far analyzed in that its function is dependent on the pit, a mouth-like organ formed on the cell surface only when the cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells. The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated as the “Super-channel”, and in this review, we discuss the three-dimensional structure and specific function of the “Super-channel” for macromolecule import found for the first time in a bacterium.

Structural analysis of β-glucosidase mutants derived from a hyperthermophilic tetrameric structure.

Substitutive mutations that convert a tetrameric β-glucosidase into a dimeric state lead to improvement of its crystal quality.