Article Figures & Data
Figures
- FIG 1
Schematic of pentose catabolism and the genomic structure of the involved genes. (A) Arabinose and xylose metabolism via the pentose phosphate pathway (PPP) (green arrows) and the phosphoketolase pathway (PKP) (red arrows). (B) Map of arabinose and xylose metabolism-associated genes: xylose associated (yellow arrows), arabinose associated (blue arrows), PPP genes (green arrows), and phosphoketolase (red arrow). The pattern within each arrow illustrates operon grouping. Turquoise bars indicate AraR binding sites, purple bars indicate XylR binding sites, and gray bars indicate CcpA binding sites (CRE). Striped bars indicate color-respective repressor sites proposed in this article. Asterisk indicates gene previously identified as an XylR, but most likely incorrectly annotated. Data from references 9, 10, 23, and 28 were used and expanded upon to construct these schematics.
- FIG 2
Arabinose supplementation alters growth rate, global sugar consumption, and acid production in C. acetobutylicum. (A) Growth curves of cultures after supplemental sugar addition. Error bars are standard deviations. * indicates two-tailed Student’s t test <0.01 for (+)Ara compared to (+)Glu, (+)Xyl, or (+)None. (B) Global sugar consumption in each culture was monitored after supplemental sugar addition by quantifying the amount of each sugar in the medium at each time point. Error bars are standard deviations. (C) Change in xylose concentration normalized to average OD600 for each hour after supplemental sugar addition. (D) Acetate concentrations in culture medium were monitored every hour after supplemental sugar addition, and reported values are the average from all experiments. (E) Butyrate concentrations in culture medium were monitored every hour after supplemental sugar addition, and reported values are the average from all experiments.
- FIG 3
Comparison of differentially expressed genes. (A) Cultures actively growing on xylose were supplemented with an additional sugar (glucose or arabinose), and samples were taken for RNA-Seq at 15, 30, and 60 min after supplementation. Heat map and hierarchical clustering results of differentially regulated genes from RNA-Seq samples. The columns on the right indicate the presence of CcpA, AraR, or XylR binding sites in coding or promoter regions of the genes. CA_C0149 (hypothetical) and CA_C1343 (xfp) are noted. Samples on the x axis are labeled by the supplemental sugar and the time point (i.e., Glu-T15 = glucose supplementation culture 15 min after supplemental sugar addition). (B) Venn diagram showing overlap between genes differentially regulated after arabinose addition or glucose addition with genes containing CcpA binding sites at 60 min after supplemental sugar addition. (C) Temporal response of CA_C0149 after addition of supplemental sugars. Each point represents a biological replicate. The x axis marks time after carbohydrate supplementation, and the y axis marks the CA_C0149 expression level in terms of reads per kilobase of transcript per million mapped reads (RPKM). Each panel displays results for each different carbohydrate supplementation: glucose (Glu), arabinose (Ara), xylose (Xyl), or no carbohydrate (None).
- FIG 4
Temporal response of differentially expressed genes after addition of glucose or arabinose expressed as log2 of fold change relative to preaddition levels. (A) Genes with identified AraR sites but lacking XylR and CcpA sites. (B) Genes with CcpA binding sites, but not AraR or XylR binding sites. (C) Genes with identified CcpA and XylR sites but lacking AraR binding sites. (D) Genes with identified CcpA and AraR binding sites but lacking identified XylR sites. (E) Genes with identified CcpA, AraR, and XylR binding sites. Each line represents a single gene, and the color of the line corresponds to the supplemental sugar added. The other two possibilities, AraR/XylR and XylR, contained no differentially expressed genes and were not represented in this figure.
- FIG 5
Putative Crh (CA_C0149) alignment and mRNA expression levels. (A) Alignment of C. acetobutylicum HPr (HPR_CAC) and putative Crh (CRH_CAC0149) with Crh proteins from several Bacillus species using the CLUSTALW (ver. 1.8) Multiple Sequence Alignment tool. CRH_BSU = B. subtilis Crh, CRH_BLI = B. licheniformis Crh, CRH_BHA = B. halodurans Crh, CRH_BCL = B. clausii Crh. The blue box highlights the conserved N-terminal histidine residue of HPr required for phosphotransfer to the PTS that is not present in the Crh proteins. The red box highlights the conserved C-terminal serine residue that when phosphorylated promotes activation of CCR through interaction with CcpA. (B) Fold expression of CA_C0149 in the indicated carbohydrate source relative to glucose expression in C. acetobutylicum obtained from a previous transcriptomic study (19).
- FIG 6
Proposed role of putative Crh and mechanism of catabolite repression by arabinose in C. acetobutylicum. (A) Schematic of central metabolic pathways and activation of CcpA-mediated CCR via phosphorylated HPr or Crh. Initial arabinose metabolic steps are shown in red, and xylose metabolic steps are shown in green. The increased metabolic rates during growth on arabinose compared to xylose are indicated by arrow thickness. Increased levels of FBP during growth on arabinose or glucose compared to xylose could activate HPrK, leading to phosphorylation of HPr and/or Crh. (B) Schematic showing interactions of repressor proteins during different nutritional states with genes having different regulatory schemes. Xylose to xylose: XylR binds xylose and XylR genes are derepressed, AraR is active and repressing AraR genes, CcpA is not active. Xylose to glucose: CcpA is activated, lack of intracellular arabinose and xylose causes repression via AraR and XylR. Xylose to arabinose 15 min: AraR binds arabinose resulting in derepression by AraR, XylR binds xylose, and XylR genes are derepressed, CcpA is not activated due to insufficient FBP levels to activate HPrK. Xylose to arabinose 60 min: AraR is derepressed due to arabinose binding, decreased import of xylose causes repression of XylR genes, and CcpA acts a repressor due to increased FBP levels as a result of increased metabolic rate following phosphoketolase activation.
Supplemental Material
FIG S1
Temporal response of differentially expressed genes after addition of xylose or no carbohydrate expressed as log2 of fold change relative to preaddition levels. (A) Genes with identified AraR sites but lacking XylR and CcpA sites. (B) Genes with CcpA binding sites, but not AraR or XylR binding sites. (C) Genes with identified CcpA and XylR sites but lacking AraR binding sites. (D) Genes with identified CcpA and AraR binding sites but lacking identified XylR sites. (E) Genes with identified CcpA, AraR, and XylR binding sites. Each line represents a single gene and the color of the line corresponds to the supplemental sugar added. The other two possibilities, AraR/XylR and XylR, contained no differentially expressed genes and were not represented in this figure. Download FIG S1, TIF file, 1.0 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
