Transcriptome perturbations induced by the autotrophic and cold condition. (A) The autotroph-responsive only (C1 and C2), temperature-responsive only (C3 and C4), combined condition-responsive (C6, C7, C8, and C9), and particular condition-specific (C5, C10, C11, C12, C13, and C14) differential gene expression patterns were all combined and are shown as a heat map. The heat map shows log2 expression values (“Exp.”) and log2 fold-changes (“FC”) at 20°C heterotrophic (“H20”), 20°C autotrophic (“A20”), 10°C heterotrophic (“H10”), and 10°C autotrophic (“A10”) conditions. Black bars represent clustered columns resulting from K-mean clustering. Two thousand sixty-eight DEG are listed in Supplemental Table S4. C1 and C2 were highly up-regulated or down-regulated under autotrophic conditions, respectively; C3 and C4 were highly down-regulated or up-regulated at low temperature, respectively; C5 showed down-regulation of gene expression under A20, but their gene expression was inversely altered by cold temperature; the expression of C6 was down-regulated either by autotrophic or cold temperature conditions; the expression of C7 was up-regulated both by the autotrophic and cold temperature conditions; C8 and C9 were found to be highly down-regulated and up-regulated by A10, respectively; C10 and C11 were found to be highly up-regulated and down-regulated by H10, respectively; C12 and C13–C14 were found to be highly down-regulated or up-regulated by A20, respectively. Clustering was performed based on gene expression-fold change. Twenty-one clusters were assigned to 14 clusters manually. Gray and red lines indicate log2 (fold-changes) and median of each group, respectively. See also Supplemental Table S4 for all transcript abundances. (B) Enriched KEGG pathways of the C1, C2, C3, C4, and C7 clusters. Bonferroni-corrected P < 0.05 was considered as the significance cutoff. KEGG pathways, including acetogenesis-related genes, are highlighted in bold blue. For clarity of illustration, KEGG enrichment significance is shown as −log10 (P-value). See also Supplemental Figure S2. (C) An example of RNA-seq profiles for the genes in the A group. RNA-seq reads of both strands are shown. The putative cobalt ABC transporter cluster (ABAKI_c2420–c24260), a putative lipid A ABC transporter (ABAKI_c24270–c24290), and the bifurcating lactate dehydrogenase gene cluster (ABAKI_c24310–c24350) are highlighted in indigo blue. (D) A violin plot shows the expression distribution for the genes in the C7 group. See also Supplemental Figure S3. (E) DEG involved in acetogenesis, as well as in the glycolysis and gluconeogenesis pathways. Low temperature triggers the expression of acetogenesis-related genes. Bold and gray arrows indicate positive and negative regulation, respectively, under the cold-heterotrophic growth condition (H10). The heat map shows log2 fold-changes. See also Supplemental Table S5. (F) Quantitative RT-PCR data of acsD (from the carbonyl branch of the WLP), metF (from the methyl branch of the WLP), hydA (from the FDH operon), and pyc genes. The graph shows fold-changes in the transcript abundance of A. bakii and A. woodii for the four different growth conditions. Each value is the mean of three replicates in independently repeated experiments, and error bars show ±SEM. The secA gene was used as the reference. (**) P ≤ 0.01 > 0.001; (***) P < 0.001 (Student's unpaired t-test).
