Genome-Scale Analysis of Acetobacterium woodii Identifies Translational Regulation of Acetogenesis

ABSTRACT Acetogens synthesize acetyl-CoA via the CO2-fixing Wood-Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon and energy metabolisms in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. Data integration of genome-scale transcriptomic and translatomic analyses revealed that the acetogenesis genes, including those of the Wood-Ljungdahl pathway and energy metabolism, showed changes in translational efficiency under autotrophic growth conditions. In particular, genes encoding the Wood-Ljungdahl pathway are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas genes encoding the carbonyl branch present increased translation levels in comparison to those for the methyl branch under heterotrophic growth conditions. The translation efficiency of genes in the pathways is differentially regulated by 5′ untranslated regions and ribosome-binding sequences under different growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas-fermenting acetogenic bacteria for better productivity. IMPORTANCE Acetogens are capable of reducing CO2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Here, we dissect the translational regulation of carbon and energy metabolisms in the model acetogen Acetobacterium woodii under heterotrophic and autotrophic growth conditions. The latter may be experienced when acetogen is used as a cell factory that synthesizes products from CO2 during the gas fermentation process. We found that the methyl and carbonyl branches of the Wood-Ljungdahl pathway are activated at similar translation levels during autotrophic growth. Translation is mainly regulated by the 5′-untranslated-region structure and ribosome-binding-site sequence. This work reveals novel translational regulation for coping with autotrophic growth conditions and provides the systematic data set, including the transcriptome, translatome, and promoter/5′-untranslated-region bioparts.


Transcriptomic landscape of A. woodii under fructose and H2+CO2 conditions
Total RNA was extracted from the cell cultures at the mid-exponential phase and subjected to Illumina short-read sequencing. We obtained 1.2-4.7 million highquality sequencing reads, which were uniquely mapped to the reference genome sequence of A. woodii (CP002987) (1), corresponding to at least 42.8-fold coverage (Table S1). The correlation coefficient value between the biological replicates demonstrated an experimental reproducibility (Pearson's r = 0.98). Principal component analysis revealed a significant difference in gene expression between heterotrophic and autotrophic growth conditions, indicating a global alteration of cellular functions between the two growth conditions (Fig. S1A-B). RNA-Seq data were then normalised by DEseq2 (2) to estimate differentially expressed genes (DEG) between the two growth conditions with adjusted P-value (Padj < 0.01), revealing that total 1221 genes were determined as DEGs (Table S2). Expression levels of the selected genes including acetogenesis-related genes were also validated independently by qRT-PCR experiments (Pearson's r > 0.94; Fig. S1E).
Among the DEGs, 674 and 547 genes were up-regulated and downregulated, respectively, with a dynamic transcriptional expression range from 1.5 × 10 −3 to 2.8 × 10 3 -fold change in response to autotrophic growth conditions ( Fig. S2A and Table S2). To comprehend the functions of the DEGs, their functional categories were determined based on the clusters of orthologous groups (COGs). Most of the up-regulated genes belonged to energy production and conversion (C, 15.97%), amino acid transport and metabolism (E, 8.19%), carbohydrate transport and metabolism (G, 8.06%), coenzyme transport and metabolism (H, 5.28%), and translation, ribosomal structure and biogenesis (J, 5.69%), similar to Clostridium ljungdahlii (3) and Eubacterium limosum (4). In contrast, most of the down-regulated genes belonged to transcription (10.68%) and cell wall/membrane/envelope biogenesis (3.74%). Furthermore, we identified the DEGs associated with particular metabolic pathways using KEGG pathway enrichment analysis. In general, glycolysis, gluconeogenesis, pentose phosphate pathway, fructose and mannose metabolism, and fatty acid biosynthesis were significantly enriched (Bonferroni corrected P < 0.0017; Fig. S2B).
Importantly, A. woodii activates genes in the WL pathway under autotrophic growth conditions, where most genes such as those encoding the hydrogendependent carbon dioxide reductase (HDCR, Awo_c08190-Awo_c08260), the methyl-branch (Awo_c09260-Awo_c09310), and the carbonyl-branch (Awo_c10670-Awo_c10760) were significantly upregulated (fold changes > 2 and Padj < 0.01; Fig. S2C and Table S2). In particular, the highest difference between the two growth conditions was observed for the HDCR genes. The selenium-containing FDH (FdhF2) was highly upregulated (fold change > 5.2, Padj < 8.42 × 10 −20 ), whereas selenium-free FDH (FdhF1) and the small electron-transferring subunit HycB1 (Awo_c08190-Awo_c08200) were not expressed, indicating that A. woodii employed FdhF2 for CO2 reduction in the presence of selenium in the culture medium. Furthermore, the WL pathway is linked to the chemiosmotic energy conservation system comprising the Rnf complex (Awo_c22010-Awo_c22060) and F1FO ATP synthase (Awo_c02140-Awo_c02240) (1,5). In addition, the electronbifurcating hydrogenase is required to balance the reduction of the redox carriers NADH and ferredoxin, which are both required for the WL pathway. The Rnf complex has a dual function in redox balancing and energy conservation (6). All these genes were upregulated (fold change > 2, Padj < 0.01), implying their important roles during growth on H2+CO2 (Fig. S2C and Table S2). Collectively, the transcriptomic results suggest that the genes encoding acetogenesis as well as gluconeogenesis and pentose phosphate pathway were activated for biomass formation and CO2 reduction during autotrophic growth (3,4,7,8).

Energy conservation system
We observed that the expression of the genes encoding energy conservation associated enzymes were transcriptionally up-regulated under autotrophic growth conditions ( Fig. S2C and Table S2). In A. woodii, the WL pathway is linked with the energy conservation system comprising F1FO-ATP synthase and Rnf complex (1,5). For energy conservation, first, ferredoxin and NAD+ are reduced with two H2 by the electron-bifurcating hydrogenase (12). A. woodii genome contains multimeric [FeFe]hydrogenase genes (hydCEDBA1) comprising five genes (Awo_c26970-Awo_c27010), which were highly activated at both transcription (2.0-7.7 foldchanges, Padj < 2.26 × 10 −6 ) and translation (2.3-3.7 fold-changes, Padj < 2.24 × 10 −20 ) levels during acetogenesis with H2+CO2 ( Fig. S2C and Table S2). Collectively, electron-bifurcating hydrogenase and the hydrogenase of the HDCR complex, which catalyse hydrogen activation, were significantly up-regulated during H2-dependent autotrophic growth. Subsequently, reduced ferredoxin is used for reductive reactions of WL pathway and the establishment of sodium ion gradient via the Rnf complex (Awo_c22010-Awo_c22060). Expression of the Rnf complex was significantly upregulated at the transcription level (1.9-2.9 fold-changes, Padj < 2.26 × 10 −6 ) under autotrophic growth conditions, except rnfC1 (Awo_c22060), whereas these genes were retained at a similar (Padj < 0.27) or slightly lower stage (0.68-0.74 foldchanges, Padj < 8.12 × 10 −3 ) in RPF level ( Fig. S2C and Table S2). The generated sodium ion gradient is used for producing ATP via F1FO ATP synthase complexes. A. woodii has a particular membrane-bound F1FO ATP synthase (atpIBE1E2E3FHAGDC, Awo_c02140-Awo_c02240), which has more c-subunits than a typical bacterial ATP synthase (atpIBEFHAGDC) (1,13). The gene clusters of F1FO ATP synthase were significantly up-regulated at the transcription level (1.8-6.9 fold-changes and Padj < 1.72×10 −4 ) under autotrophic growth conditions, whereas the translation levels of these genes were retained at a similar or lower level (0.69-0.89 fold-changes; Table  S2). Interestingly, we observed that the transcription of ATP synthase operon commenced from the atpI gene body. TSS position of ATP synthase operon was in accordance with our dRNA-seq and rapid amplification of sequences from the 5′ends of mRNAs (5′RACE) validation ( Fig. S5C and Table S2). Although AtpI might be essential for hybrid rotor assembly in E. coli (14), the functional role was not exactly determined in A. woodii. Thus, most of the energy conservation related genes are highly activated at the transcription level during acetogenesis with H2+CO2. The autotrophic-specific Rnf expression was detected in E. limosum (4), C. ljungdahlii (15) and C. autoethanogenum (7); however, the Ribo-Seq analysis presented direct evidence that A. woodii regulates the expression of Rnf complex and ATP synthase at the translation level.

Central carbon metabolic pathway
The expression of several central carbon metabolic pathways was also regulated during autotrophic growth. Initially, the rate-limiting enzymes for gluconeogenesis, that is fructose-1,6-bisphosphatase (Awo_c08060) and pyruvate phosphate dikinase (PPDK) genes, were significantly up-regulated both at transcription (3.0-9.7 foldchanges, Padj < 4.14 × 10 −12 ) and translation (2.6-6.8 fold-changes, Padj < 3.84 × 10 −27 ) levels, suggesting that gluconeogenesis is enhanced under autotrophic growth conditions. In contrast, glycolysis-specific genes, such as 6-phosphofructokinase (PFK, Awo_c12790) and pyruvate kinase (PK) Awo_c12800 are significantly downregulated at the translation level (0.5-0.6 fold-changes, Padj < 1.08 × 10 −13 ) under autotrophic growth conditions, which is one of the key regulatory and rate-limiting steps of glycolysis. In the case of non-oxidative pentose phosphate pathway, all genes encoding trans-ketolase (TKT) and trans-aldolase (TAL) acting as a major enzyme in non-oxidative pentose phosphate pathway are highly up-regulated, and overall expression pattern of the pentose phosphate pathway is similar for both transcription and translation levels ( Fig. S2C and Table S2). Pyruvate:ferredoxin oxidoreductase (PFOR) is also up-regulated at both transcription (5.9-15.1 foldchanges and Padj < 4.60 × 10 −32 ) and translation (2.9-8.4 fold-changes, Padj < 2.35 × 10 −35 ) levels under autotrophic growth conditions. The PFOR is central to cellular carbon synthesis as it catalyses the reductive carboxylation of acetyl-CoA to pyruvate during autotrophic growth by the WL pathway (16). For the branched TCA cycle, we did not observe the up-regulated or considerable change in the transcript abundance between the two growth conditions (Fig. S2C).