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FEMS Microbiol Lett
2019 Sep 01;36617:. doi: 10.1093/femsle/fnz222.
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Identification and characterisation of two high-affinity glucose transporters from the spoilage yeast Brettanomyces bruxellensis.
Tiukova IA
,
Møller-Hansen I
,
Belew ZM
,
Darbani B
,
Boles E
,
Nour-Eldin HH
,
Linder T
,
Nielsen J
,
Borodina I
.
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The yeast Brettanomyces bruxellensis (syn. Dekkera bruxellensis) is an emerging and undesirable contaminant in industrial low-sugar ethanol fermentations that employ the yeast Saccharomyces cerevisiae. High-affinity glucose import in B. bruxellensis has been proposed to be the mechanism by which this yeast can outcompete S. cerevisiae. The present study describes the characterization of two B. bruxellensis genes (BHT1 and BHT3) believed to encode putative high-affinity glucose transporters. In vitro-generated transcripts of both genes as well as the S. cerevisiae HXT7 high-affinity glucose transporter were injected into Xenopus laevis oocytes and subsequent glucose uptake rates were assayed using 14C-labelled glucose. At 0.1 mM glucose, Bht1p was shown to transport glucose five times faster than Hxt7p. pH affected the rate of glucose transport by Bht1p and Bht3p, indicating an active glucose transport mechanism that involves proton symport. These results suggest a possible role for BHT1 and BHT3 in the competitive ability of B. bruxellensis.
Figure 1. Identification and characterization of hypothetical genes encoding putative glucose transporters in the genome of B. bruxellensis. (A), Genomic context of the BHT1-BHT3 gene cluster in the genome of B. bruxellensis UMY321. Gene names of adjacent genes were assigned based on sequence homology to previously described genes in S. cerevisiae. (B), Alignment of the 3’ termini of the B. bruxellensis hypothetical genes BHT1, BHT2 and BHT3. The proposed 2-bp deletion in the BHT2 sequence is indicated in yellow. Genomic coordinates of the source contig (GenBank accession FYBN01000006) are indicated. (C), Maximum likelihood tree of predicted glucose transporters in S. cerevisiae strain S288C (blue) and B. bruxellensis strain UMY321 (red) using 423 aligned amino acid positions. Node stability is indicated by the bootstrap value of 1000 replicate analyses. Bootstrap values below 500 are not shown. The S. cerevisiae glycerol transporter Stl1p and its B. bruxellensis ortholog (Zemancíková et al. 2018) were used as outgroups. GenBank protein accession numbers for S. cerevisiae transporters are displayed. For B. bruxellensis hypothetical genes, the genomic contig GenBank accession number and genomic coordinates used for conceptual translation are indicated (‘c’ signifies that the hypothetical gene is encoded on the reverse strand). The three B. bruxellensis putative glucose transporters BHT1-BHT3 are highlighted in bold font.
Figure 2. Concentration-dependent effects on glucose uptake in X. laevis oocytes injected with cRNA of yeast genes encoding glucose transporters. Assays were performed at pH 5 with the indicated glucose concentration. Injection of cRNA GFP was used as negative controls to account for endogenous glucose uptake. The assays were performed at least in triplicate. Error bars indicate one standard deviation.
Figure 3. pH-dependent effects on glucose uptake in X. laevis oocytes injected with cRNA of B. bruxellensisgenes encoding glucose transporters. Oocytes were incubated 1 hour in 0.1 mM glucose buffer at the indicated pH. Injection of cRNA GFP was used as negative controls to account for endogenous glucose uptake. The assays were performed at least in triplicate. Error bars indicate one standard deviation.
Figure 4. Growth profiles of a S. cerevisiae hxt0 strain expressing individual hexose transporter genes. Cultivations were carried out in SC medium containing either 20 g l–1(A) or 2 g l–1(B) glucose. Growth assays were performed in triplicate with error bars indicating one standard deviation.
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