Substrate size-dependent conformational changes of bacterial pectin-binding protein crucial for chemotaxis and assimilation.

Gram-negative Sphingomonas sp. strain A1 exhibits positive chemotaxis toward acidic polysaccharide pectin. SPH1118 has been identified as a pectin-binding protein involved in both pectin chemotaxis and assimilation. Here we show tertiary structures of SPH1118 with six different conformations as determined by X-ray crystallography. SPH1118 consisted of two domains with a large cleft between the domains and substrates bound to positively charged and aromatic residues in the cleft through hydrogen bond and stacking interactions. Substrate-free SPH1118 adopted three different conformations in the open form. On the other hand, the two domains were closed in substrate-bound form and the domain closure ratio was changed in response to the substrate size, suggesting that the conformational change upon binding to the substrate triggered the expression of pectin chemotaxis and assimilation. This study first clarified that the solute-binding protein with dual functions recognized the substrate through flexible conformational changes in response to the substrate size.

Polyunsaturated fatty acids-enriched lipid from reduced sugar alcohol mannitol by marine yeast Rhodosporidiobolus fluvialis Y2.

Brown macroalgae is a promising marine biomass for the production of bioethanol and biodiesel fuels. Here we investigate the biochemical processes used by marine oleaginous yeast for assimilating the major carbohydrate found in brown macroalgae. Briefly, yeast Rhodosporidiobolus fluvialis strain Y2 was isolated from seawater and grown in minimal medium containing reduced sugar alcohol mannitol as the sole carbon source with a salinity comparable to seawater. Conditions limiting nitrogen were used to facilitate lipid synthesis. R. fluvialis Y2 yielded 55.1% (w/w) and 39.1% (w/w) of lipids, per dry cell weight, from mannitol in the absence and presence of salinity, respectively. Furthermore, mannitol, as a sugar source, led to an increase in the composition of polyunsaturated fatty acids, linoleic acid (C18:2) and linolenic acid (C18:3), compared to glucose. This suggests that oxidation of mannitol leads to the activation of NADH-dependent fatty acid desaturases in R. fluvialis Y2. Such fatty acid composition may contribute to the cold-flow properties of biodiesel fuels. Our results identified a salt-tolerant oleaginous yeast species with unique metabolic traits, demonstrating a key role as a decomposer in the global carbon cycle through marine ecosystems. This is the first study on mannitol-induced synthesis of lipids enriched with polyunsaturated fatty acids by marine yeast.

Bacterial chemotaxis towards polysaccharide pectin by pectin-binding protein.

As opposed to typical bacteria exhibiting chemotaxis towards low-molecular-weight substances, such as amino acids and mono/oligosaccharides, gram-negative Sphingomonas sp. strain A1 shows chemotaxis towards alginate and pectin polysaccharides. To identify the mechanism of chemotaxis towards macromolecules, a genomic fragment was isolated from the wild-type strain A1 through complementation with the mutant strain A1-M5 lacking chemotaxis towards pectin. This fragment contained several genes including sph1118. Through whole-genome sequencing of strain A1-M5, sph1118 was found to harbour a mutation. In fact, sph1118 disruptant lost chemotaxis towards pectin, and this deficiency was recovered by complementation with wild-type sph1118. Interestingly, the gene disruptant also exhibited decreased pectin assimilation. Furthermore, the gene product SPH1118 was expressed in recombinant E. coli cells, purified and characterised. Differential scanning fluorimetry and UV absorption spectroscopy revealed that SPH1118 specifically binds to pectin with a dissociation constant of 8.5 µM. Using binding assay and primary structure analysis, SPH1118 was predicted to be a periplasmic pectin-binding protein associated with an ATP-binding cassette transporter. This is the first report on the identification and characterisation of a protein triggering chemotaxis towards the macromolecule pectin as well as its assimilation.