Functional diversification of ROK-family transcriptional regulators of sugar catabolism in the Thermotogae phylum

Nucleic Acids Research, Jan 2013

Large and functionally heterogeneous families of transcription factors have complex evolutionary histories. What shapes specificities toward effectors and DNA sites in paralogous regulators is a fundamental question in biology. Bacteria from the deep-branching lineage Thermotogae possess multiple paralogs of the repressor, open reading frame, kinase (ROK) family regulators that are characterized by carbohydrate-sensing domains shared with sugar kinases. We applied an integrated genomic approach to study functions and specificities of regulators from this family. A comparative analysis of 11 Thermotogae genomes revealed novel mechanisms of transcriptional regulation of the sugar utilization networks, DNA-binding motifs and specific functions. Reconstructed regulons for seven groups of ROK regulators were validated by DNA-binding assays using purified recombinant proteins from the model bacterium Thermotoga maritima. All tested regulators demonstrated specific binding to their predicted cognate DNA sites, and this binding was inhibited by specific effectors, mono- or disaccharides from their respective sugar catabolic pathways. By comparing ligand-binding domains of regulators with structurally characterized kinases from the ROK family, we elucidated signature amino acid residues determining sugar-ligand regulator specificity. Observed correlations between signature residues and the sugar-ligand specificities provide the framework for structure functional classification of the entire ROK family.

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Functional diversification of ROK-family transcriptional regulators of sugar catabolism in the Thermotogae phylum

Marat D. Kazanov 0 1 Xiaoqing Li 1 Mikhail S. Gelfand 0 Andrei L. Osterman 1 Dmitry A. Rodionov 0 1 0 A.A.Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences , Moscow 127994, Russia 1 Sanford-Burnham Medical Research Institute , La Jolla, CA 92037, USA Large and functionally heterogeneous families of transcription factors have complex evolutionary histories. What shapes specificities toward effectors and DNA sites in paralogous regulators is a fundamental question in biology. Bacteria from the deep-branching lineage Thermotogae possess multiple paralogs of the repressor, open reading frame, kinase (ROK) family regulators that are characterized by carbohydrate-sensing domains shared with sugar kinases. We applied an integrated genomic approach to study functions and specificities of regulators from this family. A comparative analysis of 11 Thermotogae genomes revealed novel mechanisms of transcriptional regulation of the sugar utilization networks, DNA-binding motifs and specific functions. Reconstructed regulons for seven groups of ROK regulators were validated by DNA-binding assays using purified recombinant proteins from the model bacterium Thermotoga maritima. All tested regulators demonstrated specific binding to their predicted cognate DNA sites, and this binding was inhibited by specific effectors, mono- or disaccharides from their respective sugar catabolic pathways. By comparing ligand-binding domains of regulators with structurally characterized kinases from the ROK family, we elucidated signature amino acid residues determining sugar-ligand regulator specificity. Observed correlations between signature residues and the sugar-ligand specificities provide the framework for structure functional classification of the entire ROK family. - DNA-binding transcription factors (TFs) in bacteria are classified in at least 50 protein families based on sequence similarity and domain composition (15). Prokaryotic TFs are usually composed of two domains: (i) a DNA-binding domain that provides the basic function in the recognition of specific DNA sequences and (ii) an effector-sensing domain that modulates the TF activity by monitoring cellular signals and binding specific ligands. The distribution of TFs by families varies among bacterial lineages, mainly due to massive lineage-specific expansions of specific TF families and frequent horizontal gene transfer of individual TFs (6). Gene duplication followed by functional diversification of the duplicated genes is a major driver of evolution. Evolution of diverse DNA-binding and ligand-binding activities in a given TF family is a widely observed phenomenon. However, our understanding of the evolutionary mechanisms driving the observed diversity in large and functionally heterogeneous families of TFs is very limited. Several families of transcriptional regulators in bacteria possess effector-sensing domains that are homologous to ligand-binding domains of non-regulatory proteins. For instance, the sugar-binding domains in regulators from the LacI and DeoR families share the fold with periplasmic binding proteins (PBPs) of sugar uptake ABC transporters (7) and enzymes from the sugar isomerase family (8), respectively. Structural similarity between the effector-sensing domains of these regulators and the ligand-binding domains of enzymes and transporters suggests that the respective protein families are evolutionarily related. Phylogenetic analysis of PBPs and LacI family regulators revealed that the acquisition of the DNA-binding domain occurred in the last common ancestor of bacteria, and that both functional groups have since undergone extensive gene duplication with parallel evolution of ligand specificity (7). Whether similar evolutionary scenarios could explain the origin of functional divergence of ligand specificities in other TF families remains an open question. To understand the mechanisms of diversification of ligand specificity, we selected the ROK (repressor, open reading frame, kinase) protein family that includes two functionally diverse groups of proteins: (i) catalytically active sugar kinases and (ii) sugar-responsive transcriptional repressors that possess an N-terminal DNA-binding fused to a C-terminal sugar-binding domain from the ROK family (9). The ROK protein family is characterized by the PF00480 domain that belongs to the Actin-ATPase clan in the Pfam database (10). The broad distribution of this family in bacterial genomes is illustrated by 9600 proteins in Pfam (on August 2012). Among these, nearly 1700 proteins (18%) are potential regulators that possess an N-terminal DNA-binding domain. Some taxonomic groups of bacteria, such as the deep-branching phylum Thermotogae analyzed in this work, demonstrate lineage-specific expansion of putative regulators from the ROK family. However, the specificity and functional role of most ROK regulators remain unknown. The only regulators in this family that hav (...truncated)


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Marat D. Kazanov, Xiaoqing Li, Mikhail S. Gelfand, Andrei L. Osterman, Dmitry A. Rodionov. Functional diversification of ROK-family transcriptional regulators of sugar catabolism in the Thermotogae phylum, Nucleic Acids Research, 2013, pp. 790-803, 41/2, DOI: 10.1093/nar/gks1184