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An Analysis of the Changes in Soluble Hydrogenase and Global Gene Expression in Cupriavidus Necator (Ralstonia Eutropha) H16 Grown in Heterotrophic Diauxic Batch Culture

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An Analysis of the Changes in Soluble Hydrogenase and Global Gene Expression in Cupriavidus Necator (Ralstonia Eutropha) H16 Grown in Heterotrophic Diauxic Batch Culture

Bat-Erdene Jugder et al. Microb Cell Fact.

Abstract

Background: Soluble hydrogenases (SH) are enzymes that catalyse the oxidation of molecular hydrogen. The SH enzyme from Cupriavidus necator H16 is relatively oxygen tolerant and makes an attractive target for potential application in biochemical hydrogen fuel cells. Expression of the enzyme can be mediated by derepression of the hox promoter system under heterotrophic conditions. However, the overall impact of hox derepression, from a transcriptomic perspective, has never been previously reported.

Results: Derepression of hydrogenase gene expression upon fructose depletion was confirmed in replicate experiments. Using qRT-PCR, hoxF was 4.6-fold up-regulated, hypF2 was up-regulated in the cells grown 2.2-fold and the regulatory gene hoxA was up-regulated by a mean factor of 4.5. A full transcriptomic evaluation revealed a substantial shift in the global pattern of gene expression. In addition to up-regulation of genes associated with hydrogenase expression, significant changes were observed in genes associated with energy transduction, amino acid metabolism, transcription and translation (and regulation thereof), genes associated with cell stress, lipid and cell wall biogenesis and other functions, including cell motility.

Conclusions: We report the first full transcriptome analysis of C. necator H16 grown heterotrophically on fructose and glycerol in diauxic batch culture, which permits expression of soluble hydrogenase under heterotrophic conditions. The data presented deepens our understanding of the changes in global gene expression patterns that occur during the switch to growth on glycerol and suggests that energy deficit is a key driver for induction of hydrogenase expression in this organism.

Figures

Figure 1
Figure 1
Heterotrophic growth of C. necator H16 cells during a 48 h bioreactor fermentation. A) Log10 OD600nm and soluble hydrogenase activity (by NAD+ reduction) with time; B) dO2 and pH variation with time. These graphs are based on three biological replicates and represent their mean values with standard deviation given.
Figure 2
Figure 2
Venn Diagram of Differentially Expressed Genes. (A) - 2606 genes up-regulated at 34 hours identified in edgeR and DESeq2 analyses. (B) - 2206 genes down-regulated at 34 hours identified in edgeR and DESeq2 analyses.
Figure 3
Figure 3
Clusters of Orthologous Groups (COGs) of the up and down regulated genes during derepression of hydrogenase genes. The numbers of up-regulated SDE genes belonged to each COG category are represented in black bars, whereas down-regulated genes are in grey bars.
Figure 4
Figure 4
Heatmap showing the expression data of the 50 most differently expressed genes. The data is ordered by absolute values of fold-changes of raw counts calculated from variance stabilizing transformation. 711HS1 and 711HS2, as well as 811HS1 and 811HS2 are technical replicates of two biological replicates of pre-induction samples, whereas 734HS1, 734HS2, 834HS1 and 834HS2 are technical and biological replicates of post-induction samples.
Figure 5
Figure 5
Overview of the hydrogenase operons of C. necator. The three operons, where four hydrogenases (MBH, SH, RH and AH) are located, are depicted with respective genes. Results of the transcriptome analyses are given in log2 fold changes on top of each gene detected.

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