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Transcriptional regulation of the carbohydrate utilization network in Thermotoga maritima.

Rodionov DA, Rodionova IA, Li X, Ravcheev DA, Tarasova Y, Portnoy VA, Zengler K, Osterman AL - Front Microbiol (2013)

Bottom Line: The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons.Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima.In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.

View Article: PubMed Central - PubMed

Affiliation: Sanford-Burnham Medical Research Institute La Jolla, CA, USA ; A. A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences Moscow, Russia.

ABSTRACT
Hyperthermophilic bacteria from the Thermotogales lineage can produce hydrogen by fermenting a wide range of carbohydrates. Previous experimental studies identified a large fraction of genes committed to carbohydrate degradation and utilization in the model bacterium Thermotoga maritima. Knowledge of these genes enabled comprehensive reconstruction of biochemical pathways comprising the carbohydrate utilization network. However, transcriptional factors (TFs) and regulatory mechanisms driving this network remained largely unknown. Here, we used an integrated approach based on comparative analysis of genomic and transcriptomic data for the reconstruction of the carbohydrate utilization regulatory networks in 11 Thermotogales genomes. We identified DNA-binding motifs and regulons for 19 orthologous TFs in the Thermotogales. The inferred regulatory network in T. maritima contains 181 genes encoding TFs, sugar catabolic enzymes and ABC-family transporters. In contrast to many previously described bacteria, a transcriptional regulation strategy of Thermotoga does not employ global regulatory factors. The reconstructed regulatory network in T. maritima was validated by gene expression profiling on a panel of mono- and disaccharides and by in vitro DNA-binding assays. The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons. Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima. In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.

No MeSH data available.


Differential expression of trehalose and glucose utilization genes in T. maritima. (A) Gene expression fold changes for the treTR and gluR genes for the cells grown on glucose (Glu) and trehalose (Tre) vs. ribose (Rib), as determined by whole-genome microarrays. (B) Gene expression fold changes for the treE, gluE, and gluR genes as determined by RT-PCR. The bglA gene was used as a negative control. Normalized transcript levels with respect to the TM0688 (gap) gene are provided in Figure S1 in Supplementary Material. The mean of biological duplicate measurements was used. (C) Genomic clusters of genes involved in the trehalose (red) and glucose (blue) utilizations.
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Figure 6: Differential expression of trehalose and glucose utilization genes in T. maritima. (A) Gene expression fold changes for the treTR and gluR genes for the cells grown on glucose (Glu) and trehalose (Tre) vs. ribose (Rib), as determined by whole-genome microarrays. (B) Gene expression fold changes for the treE, gluE, and gluR genes as determined by RT-PCR. The bglA gene was used as a negative control. Normalized transcript levels with respect to the TM0688 (gap) gene are provided in Figure S1 in Supplementary Material. The mean of biological duplicate measurements was used. (C) Genomic clusters of genes involved in the trehalose (red) and glucose (blue) utilizations.

Mentions: The TreR-regulated treTR operon encoding a putative trehalose utilization enzyme and the trehalose-responsive repressor was highly upregulated in T. maritima cells grown on trehalose (Figure 6A). Although the DNA microarray hybridization confirmed the in vivo induction of treTR by trehalose, similar analysis of the treEFG and gluEFK genes was not possible due to the absence of the respective oligonucleotide probes. The T. maritima MSB8 isolate that was originally sequenced in 1999 [TIGR genomovar, (Nelson et al., 1999)] and that was used for microarray gene probe design lacks the 8870-bp DNA region between the genes TM1846 (cbpA) and TM1847 (gluR). This DNA region is present in two other T. maritima MSB8 isolates that have recently been resequenced [genomovars DSM 3109 (Boucher and Noll, 2011) and ATCC (Latif et al., 2013)]. The new region includes seven genes including bglA, a member of the CelR-regulated cbpA-bglA operon, and the treEFG and gluEFK genes that are regulated by TreR and GluR (Kazanov et al., 2013) (Figure 6C). To validate sugar-specific induction of these two operons in vivo, we performed real-time reverse transcription PCR (RT-PCR) with probes designed for treE, gluE, gluR, and bglA (used as a negative control) (Figure 6B). All three genes tested had demonstrated elevated expression levels in T. maritima cells grown on either glucose or trehalose compared to the cells grown on ribose. The highest fold changes in gene expression were observed for treE and gluE in the glucose-grown cells, whereas in the trehalose-grown cells only the treE gene was highly upregulated. These results confirm the dual control of the trehalose transporter treEFG by both TreR and GluR regulators in response to trehalose and glucose, respectively. Also, the RT-PCR results confirm that the glucose transporter gluEFGR operon is induced by glucose using the glucose-responsive repressor GluR.


Transcriptional regulation of the carbohydrate utilization network in Thermotoga maritima.

Rodionov DA, Rodionova IA, Li X, Ravcheev DA, Tarasova Y, Portnoy VA, Zengler K, Osterman AL - Front Microbiol (2013)

Differential expression of trehalose and glucose utilization genes in T. maritima. (A) Gene expression fold changes for the treTR and gluR genes for the cells grown on glucose (Glu) and trehalose (Tre) vs. ribose (Rib), as determined by whole-genome microarrays. (B) Gene expression fold changes for the treE, gluE, and gluR genes as determined by RT-PCR. The bglA gene was used as a negative control. Normalized transcript levels with respect to the TM0688 (gap) gene are provided in Figure S1 in Supplementary Material. The mean of biological duplicate measurements was used. (C) Genomic clusters of genes involved in the trehalose (red) and glucose (blue) utilizations.
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Figure 6: Differential expression of trehalose and glucose utilization genes in T. maritima. (A) Gene expression fold changes for the treTR and gluR genes for the cells grown on glucose (Glu) and trehalose (Tre) vs. ribose (Rib), as determined by whole-genome microarrays. (B) Gene expression fold changes for the treE, gluE, and gluR genes as determined by RT-PCR. The bglA gene was used as a negative control. Normalized transcript levels with respect to the TM0688 (gap) gene are provided in Figure S1 in Supplementary Material. The mean of biological duplicate measurements was used. (C) Genomic clusters of genes involved in the trehalose (red) and glucose (blue) utilizations.
Mentions: The TreR-regulated treTR operon encoding a putative trehalose utilization enzyme and the trehalose-responsive repressor was highly upregulated in T. maritima cells grown on trehalose (Figure 6A). Although the DNA microarray hybridization confirmed the in vivo induction of treTR by trehalose, similar analysis of the treEFG and gluEFK genes was not possible due to the absence of the respective oligonucleotide probes. The T. maritima MSB8 isolate that was originally sequenced in 1999 [TIGR genomovar, (Nelson et al., 1999)] and that was used for microarray gene probe design lacks the 8870-bp DNA region between the genes TM1846 (cbpA) and TM1847 (gluR). This DNA region is present in two other T. maritima MSB8 isolates that have recently been resequenced [genomovars DSM 3109 (Boucher and Noll, 2011) and ATCC (Latif et al., 2013)]. The new region includes seven genes including bglA, a member of the CelR-regulated cbpA-bglA operon, and the treEFG and gluEFK genes that are regulated by TreR and GluR (Kazanov et al., 2013) (Figure 6C). To validate sugar-specific induction of these two operons in vivo, we performed real-time reverse transcription PCR (RT-PCR) with probes designed for treE, gluE, gluR, and bglA (used as a negative control) (Figure 6B). All three genes tested had demonstrated elevated expression levels in T. maritima cells grown on either glucose or trehalose compared to the cells grown on ribose. The highest fold changes in gene expression were observed for treE and gluE in the glucose-grown cells, whereas in the trehalose-grown cells only the treE gene was highly upregulated. These results confirm the dual control of the trehalose transporter treEFG by both TreR and GluR regulators in response to trehalose and glucose, respectively. Also, the RT-PCR results confirm that the glucose transporter gluEFGR operon is induced by glucose using the glucose-responsive repressor GluR.

Bottom Line: The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons.Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima.In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.

View Article: PubMed Central - PubMed

Affiliation: Sanford-Burnham Medical Research Institute La Jolla, CA, USA ; A. A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences Moscow, Russia.

ABSTRACT
Hyperthermophilic bacteria from the Thermotogales lineage can produce hydrogen by fermenting a wide range of carbohydrates. Previous experimental studies identified a large fraction of genes committed to carbohydrate degradation and utilization in the model bacterium Thermotoga maritima. Knowledge of these genes enabled comprehensive reconstruction of biochemical pathways comprising the carbohydrate utilization network. However, transcriptional factors (TFs) and regulatory mechanisms driving this network remained largely unknown. Here, we used an integrated approach based on comparative analysis of genomic and transcriptomic data for the reconstruction of the carbohydrate utilization regulatory networks in 11 Thermotogales genomes. We identified DNA-binding motifs and regulons for 19 orthologous TFs in the Thermotogales. The inferred regulatory network in T. maritima contains 181 genes encoding TFs, sugar catabolic enzymes and ABC-family transporters. In contrast to many previously described bacteria, a transcriptional regulation strategy of Thermotoga does not employ global regulatory factors. The reconstructed regulatory network in T. maritima was validated by gene expression profiling on a panel of mono- and disaccharides and by in vitro DNA-binding assays. The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons. Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima. In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.

No MeSH data available.