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Abstract
Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.
Highlights
Cellulose is the most abundant polysaccharide on earth
The cellulose synthase machinery in G. hansenii was observed as a Terminal Complex (TC) arranged in linear rows by freeze-fracture transmission electron microscopy and immunogold labeling [17, 18]
For G. hansenii, this linkage must depend on structural features of the synthase (AcsAB) and accessory proteins (CcpAx, AcsD) required for translocation of cellulose across the periplasmic space and through the outer membrane (AcsC) (Fig 12)
Summary
Cellulose is the most abundant polysaccharide on earth. Cellulose microfibrils are composed of long polymeric chains of β-1,4 linked D-glucose. The first cellulose synthase gene to be identified was acsA of G. hansenii, and a number of studies have shown that the synthase works optimally when supplemented with the products of the genes acsB, acsC and acsD [20,21,22]. AcsA, the catalytic subunit, is an 83 kDa polypeptide localized in the cytoplasmic membrane that is able to transfer glucose to the growing glucan chain [23,24,25,26]. AcsB is a periplasmic protein and by homology to BcsB of R. sphaeroides it is believed to be anchored to the cytoplasmic membrane with a single C-terminal transmembrane helix (TMH) [32]. AcsC is believed to facilitate the transport of glucan chains outside the cell where they form microfibrils. AcsD is a soluble protein present in the periplasmic space [37], and its crystal structure revealed a homomeric octamer that forms a 2x4-ring structure with an apparent capacity to hold four β-glucan chains in close proximity [36]
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