3.A.1.1.10
Alginate (MW 27,000 Da) (and Alginate oligosaccharides) uptake porter. ABC transport system, AlgQ1AlgM1AlgM2(AlgS)₂: AlgS, 363 aas, BAB03314; AlgQ1, 502 aas, 3VLW_A;  AlgM1, 324 aas, BAB03315.1; AlgM2, 293 aas, BAB03316.1. Sphingomonas species A1 is a 'pit-forming' bacterium that directly incorporates alginate into its cytoplasm through a pit-dependent transport system, termed a 'superchannel' (Murata et al., 2008). The pit is a novel organ acquired through the fluidity and reconstitution of cell surface molecules, and through cooperation with the transport machinery in the cells. It confers upon bacterial cells a more efficient way to secure and assimilate macromolecules (Murata et al., 2008).  The substrate-transport characteristics and quaternary structure of AlgM1M2SS with AlgQ1 have been determined (Maruyama et al. 2015). The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg²⁺. The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides (Maruyama et al. 2015).  This bacterium  is a Gram-negative rod, containing glycosphingolipids in the cell envelope, and is named Sphingomonas sp. strain A1 (Murata et al. 2022). The pit was dynamic, with repetitive opening and closing during growth on alginate, and directly included alginate concentrated around the pit, particularly by flagellins, alginate-binding proteins, localized on the cell surface. Alginate incorporated into the periplasm was subsequently transferred to the cytoplasm by cooperative interactions of periplasmic solute-binding proteins and an ATP-binding cassette transporter in the cytoplasmic membrane. The mechanisms of assembly, functions, and interactions between the above-mentioned molecules were clarified using structural biology. The pit was transplanted into other strains of sphingomonads, and the pitted recombinant cells were effectively applied to the production of bioethanol, bioremediation for dioxin removal (Murata et al. 2022). The outer membranes of Sphingomonas strains contain GSL and is different from that of other Gram-negative bacteria, which contain LPSs in their cell envelope. Because of this property, the cell surface of Sphingomonas strains is more hydrophobic than that of other Gram-negative bacteria and shows high affinity toward hydrophobic chemicals such as dioxin and polypropylene glycol. Strain A1 cells use polyuronates (alginate and pectin) and their depolymerization products as carbon sources for growth. Glucose and pyruvate can be utilized as carbon sources, but far less efficiently than polyuronates. Strain A1 cells grew well on alginate and oligoalginates with different M/G ratios at pH 6–7, 30 ℃ in aerobic conditions, with a doubling time of approximately 25 min. However, unlike almost all of the alginate-degrading bacteria analyzed to date, the cells of strain A1 contained most of their alginate lyases in the cytoplasm. This means that alginate in the medium has to enter the cells in order to make contact with alginate lyases (Murata et al. 2022). The morphological characteristics of the cell surface were examined with cells of strain A1 grown in the presence or absence of alginate. The following morphological observations were made (Murata et al. 2022): (i) cells grown on alginate were of two types that always coexisted in the medium: cells with or without a pit, and this feature was not observed in the absence of alginate. (ii) The surface of cells grown in the absence of alginate showed a pleat-like structure without a pit. (iii) Cells grown in the presence of alginate produced pits on their cell surface, and The pits contained even globular particles, some of which were insoluble forms (granules) of alginate. (iv) When the alginate-grown cells were treated with ruthenium red, an agent used to stain mucopolysaccharides, the pit periphery was strongly and specifically stained, suggesting that alginate was concentrated in the pit. (v) The thin section of cells grown on alginate showed a specific region where the cell surface sunk into the cells but no such structures were observed in cells grown in the absence of alginate. (vi) The average pit size was 0.02–0.1 µm in diameter (Murata et al. 2022). Thus, (a) the pit is formed only in the presence of alginate, (b) the pit functions as a concentrator of alginate, and (c) strain A1 cells have a pit-dependent alginate assimilation system, which differs from the alginate import and degradation pathway of other alginate-degrading microbes. There are six protein constituents in the ABC transporter: AlgQ1, Q2, M1 M2 and S (AlgS is present with two copies where Q1 and 2 are periplasmic binding proteins, M1 and M2 comprise the integral membrane transport channel, and S is the dimeric ATPase. Alginate accumulated in the pit is delivered into the periplasm and then transported to the cytoplasm by this ABC transporter. Alginate is finally degraded into constituent monosaccharides by alginate lyases present in the cytoplasm. The gene cluster encoding these proteins are AlgO (regulatory protein)-AlgS-AlgM1-AlgM2-AlgQ1-AlgQ2. There are 8 cell surface proteins, p1 - p8.  P1 - p4 are TonB-dependent outer membrane transporters; p5 and p6 are flagellin-like proteins with alterred central domains of ~150 aas and high affinity for alginate (Kd = 10^-9), and p7 and p8 are periplasmic alginate binding proteins (Murata et al. 2022).