The hexose transporter SWEET5 confers galactose sensitivity to Arabidopsis pollen via the galactokinase GALK

Galactose is an abundant and essential sugar used for the biosynthesis of many macromolecules in different organisms, including plants. Galactose metabolism is tightly and finely controlled since excess galactose and derivatives are inhibitory. In Arabidopsis, root growth and pollen germination were strongly inhibited upon excess galactose. However, the mechanism of galactose induced inhibition during pollen germination remains obscure. In this study, we characterized AtSWEET5 as a glucose and galactose plasma-membrane transporter localized in the pollen. SWEET5 protein level start to accumulate since tricellular stage of pollen development and peaked in mature pollen before rapidly declining after pollen was germinated. SWEET5 levels are responsible for the dosage-dependent sensitivity of galactose and GALK is essential for the inhibitory effects of galactose during pollen germination. The unexpected observation that GALK is required for efficient galactose uptake in pollen may reveal an unknown regulatory mechanism for galactose transporters. Overall, SWEET5 and GALK contribute to the maintenance of galactose metabolic homeostasis during pollen germination, and galactose transport is positively regulated by GALK. The study of SWEET5 upon galactose condition also suggests SWEET5 is a major low-affinity hexose transporter at the early stage of pollen germination.One-sentence summarySWEET5 mediates pollen galactose sensitivity via GALK that is required for efficient galactose uptake in pollen during pollen germination.

L-arabinose induces the formation of viable non-proliferating spheroplasts in Vibrio cholerae

Vibrio cholerae, the agent of the deadly human disease cholera, propagates as a curved rod-shaped bacterium in warm waters. It is sensitive to cold, but persists in cold waters under the form of viable but non-dividing coccoidal shaped cells. Additionally, V. cholerae is able to form non-proliferating spherical cells in response to cell wall damage. It was recently reported that L-arabinose, a component of the hemicellulose and pectin of terrestrial plants, stops the growth of V. cholerae. Here, we show that L-arabinose induces the formation of spheroplasts that lose the ability to divide and stop growing in volume over time. However, they remain viable and upon removal of L-arabinose they start expanding in volume, form branched structures and give rise to cells with a normal morphology after a few divisions. We further show that WigKR, a histidine kinase/response regulator pair implicated in the induction of a high expression of cell wall synthetic genes, prevents the lysis of the spheroplasts during growth restart. Finally, we show that the physiological perturbations result from the import and catabolic processing of L-arabinose by the V. cholerae homolog of the E. coli galactose transport and catabolic system. Taken together, our results suggest that the formation of non-growing spherical cells is a common response of Vibrios exposed to detrimental conditions. They also permit to define conditions preventing any physiological perturbation of V. cholerae when using L-arabinose to induce gene expression from the tightly regulated promoter of the Escherichia coli araBAD operon.ImportanceVibrios among other bacteria form transient cell wall deficient forms as a response to different stresses and revert to proliferating rods when permissive conditions have been restored. Such cellular forms have been associated to antimicrobial tolerance, chronic infections and environmental dispersion.The effect of L-Ara on V. cholerae could provide an easily tractable model to study the ability of Vibrios to form viable reversible spheroplasts. Indeed, the quick transition to spheroplasts and reversion to proliferating rods by addition or removal of L-Ara is ideal to understand the genetic program governing this physiological state and the spatial rearrangements of the cellular machineries during cell shape transitions.

Cross-utilization of β-galactosides and cellobiose in Geobacillus stearothermophilus

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-β-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-β-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

SLC35A5 Protein—A Golgi Complex Member with Putative Nucleotide Sugar Transport Activity

Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising nucleotide sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in nucleotide sugar transport.