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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.


Experimental validation of UxaR regulon in T. maritima. (A) Non-redundant set of UxaR binding sites identified in T. maritima (TM), T. neapolitana (Tnea), Thermotoga sp. RQ-2 (TRQ2), and T. petrophila (Tpet). (B) Sequence logo for UxaR binding sites. (C) Fluorescence polarization binding assay of UxaR with target DNA operators in T. maritima. Increasing protein concentrations were mixed with 27-bp fluorescence-labeled DNA fragments of gene regions containing UxaR-binding sites. As a negative control (N.C.), the T. maritima regulator Rex (TM0169) and DNA fragment of the TM0201 gene containing the Rex-binding site were used. (D) Gene expression fold change for T. maritima grown on pectin vs. ribose.
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Figure 7: Experimental validation of UxaR regulon in T. maritima. (A) Non-redundant set of UxaR binding sites identified in T. maritima (TM), T. neapolitana (Tnea), Thermotoga sp. RQ-2 (TRQ2), and T. petrophila (Tpet). (B) Sequence logo for UxaR binding sites. (C) Fluorescence polarization binding assay of UxaR with target DNA operators in T. maritima. Increasing protein concentrations were mixed with 27-bp fluorescence-labeled DNA fragments of gene regions containing UxaR-binding sites. As a negative control (N.C.), the T. maritima regulator Rex (TM0169) and DNA fragment of the TM0201 gene containing the Rex-binding site were used. (D) Gene expression fold change for T. maritima grown on pectin vs. ribose.

Mentions: The GntR-type transcriptional regulator UxaR was predicted to bind a conserved DNA motif in regulatory regions of four pectin and galacturonate utilization operons in T. maritima (Figures 7A,B). The UxaR regulon includes the extracytoplasmic pectate lyase PelA, the digalacturonate-specific transporter AguEFG, the cytoplasmic polygalacturonase PelB, and the extensive set of galacturonate catabolic enzymes (Rodionova et al., 2012a). We performed experimental validation of the predicted UxaR regulon by both in vitro and in vivo approaches. To assess specific binding of the purified recombinant UxaR protein to the predicted UxaR operators in T. maritima we used a fluorescence polarization assay (Figure 7C). The results show that UxaR specifically binds to the synthetic 27-nt DNA fragments containing UxaR binding sites. All tested DNA fragments demonstrated the concentration-dependent increase of fluorescence polarization, confirming specific interaction between the regulator and DNA fragments. The apparent Kd values for the UxaR protein interacting with the tested DNA fragments were in the range of 40–80 nM. We also tested the influence of potential sugar effectors on protein-DNA interaction, however, the addition of digalacturonate, galacturonate, glucuronate, or hexuronate catabolic pathway intermediates had no effect on the complex formation (data not shown).


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)

Experimental validation of UxaR regulon in T. maritima. (A) Non-redundant set of UxaR binding sites identified in T. maritima (TM), T. neapolitana (Tnea), Thermotoga sp. RQ-2 (TRQ2), and T. petrophila (Tpet). (B) Sequence logo for UxaR binding sites. (C) Fluorescence polarization binding assay of UxaR with target DNA operators in T. maritima. Increasing protein concentrations were mixed with 27-bp fluorescence-labeled DNA fragments of gene regions containing UxaR-binding sites. As a negative control (N.C.), the T. maritima regulator Rex (TM0169) and DNA fragment of the TM0201 gene containing the Rex-binding site were used. (D) Gene expression fold change for T. maritima grown on pectin vs. ribose.
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Related In: Results  -  Collection

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Figure 7: Experimental validation of UxaR regulon in T. maritima. (A) Non-redundant set of UxaR binding sites identified in T. maritima (TM), T. neapolitana (Tnea), Thermotoga sp. RQ-2 (TRQ2), and T. petrophila (Tpet). (B) Sequence logo for UxaR binding sites. (C) Fluorescence polarization binding assay of UxaR with target DNA operators in T. maritima. Increasing protein concentrations were mixed with 27-bp fluorescence-labeled DNA fragments of gene regions containing UxaR-binding sites. As a negative control (N.C.), the T. maritima regulator Rex (TM0169) and DNA fragment of the TM0201 gene containing the Rex-binding site were used. (D) Gene expression fold change for T. maritima grown on pectin vs. ribose.
Mentions: The GntR-type transcriptional regulator UxaR was predicted to bind a conserved DNA motif in regulatory regions of four pectin and galacturonate utilization operons in T. maritima (Figures 7A,B). The UxaR regulon includes the extracytoplasmic pectate lyase PelA, the digalacturonate-specific transporter AguEFG, the cytoplasmic polygalacturonase PelB, and the extensive set of galacturonate catabolic enzymes (Rodionova et al., 2012a). We performed experimental validation of the predicted UxaR regulon by both in vitro and in vivo approaches. To assess specific binding of the purified recombinant UxaR protein to the predicted UxaR operators in T. maritima we used a fluorescence polarization assay (Figure 7C). The results show that UxaR specifically binds to the synthetic 27-nt DNA fragments containing UxaR binding sites. All tested DNA fragments demonstrated the concentration-dependent increase of fluorescence polarization, confirming specific interaction between the regulator and DNA fragments. The apparent Kd values for the UxaR protein interacting with the tested DNA fragments were in the range of 40–80 nM. We also tested the influence of potential sugar effectors on protein-DNA interaction, however, the addition of digalacturonate, galacturonate, glucuronate, or hexuronate catabolic pathway intermediates had no effect on the complex formation (data not shown).

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.