Abstract:The LysR protein PcaQ regulates the expression of genes encoding products relevant to the degradation of the aromatic acid protocatechuate (3,4-dihydroxybenzoate), and we have previously defined a PcaQ DNA-binding site located upstream of the target pcaDCHGB operon in Sinorhizobium meliloti. In this work, we show that PcaQ also regulates the expression of the S. meliloti smb20568-smb20787-smb20786-smb20785-smb20784 gene cluster, which is predicted to encode an ABC transport system. ABC transport systems have n… Show more
“…This altered profile suggests that the ligand-binding pocket is larger and can accommodate Tri-hydroxylated rings more easily. These observations are consistent with previous studies that mapped the “solute-binding protein-dependent transportome” and the function of the associated transcriptional regulator 37 of the ABC transport system containing the SMb20568 protein. The authors noted that expression of genes encoding key components of this transport system is induced by addition of aromatic compounds (DHB, PHB, and quinic acid) to the growth media.…”
In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein–ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure–function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino-acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequencebased methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria.
“…This altered profile suggests that the ligand-binding pocket is larger and can accommodate Tri-hydroxylated rings more easily. These observations are consistent with previous studies that mapped the “solute-binding protein-dependent transportome” and the function of the associated transcriptional regulator 37 of the ABC transport system containing the SMb20568 protein. The authors noted that expression of genes encoding key components of this transport system is induced by addition of aromatic compounds (DHB, PHB, and quinic acid) to the growth media.…”
In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein–ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure–function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino-acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequencebased methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria.
“…Only through this cooperative binding the TFs can recognize IR2, the less conserved of the TFBSs. This kind of binding for members of the LysR TF family has been demonstrated by footprinting assays [18, 29, 34, 40, 42, 43, 54, 57] and site directed mutagenesis analysis [21, 29, 34, 40, 53–56]. As a consequence of this binding, the hypersensitive DNA regions located around -50 bp upstream the TSSs markedly decrease.…”
Section: Discussionmentioning
confidence: 95%
“…Relevant changes in the nucleotides for TF recognition in our 5′-CTATA-n 9 -TATAG-3′ consensus resulted in decreased transcriptional activation of the target pcaMNVWX operon in the presence of its inducer [56]. These mutations involved the A➔G changes at the underlined nucleotides of the sequence 5′- A T A -n 10 -T A T-3′, generating the sequences 5′-GTA-n 10 -TAT-3′, 5′-ATG- n 10 -TAT-3′ and 5′-ATA- n 10 -TGT-3′ [56]. Fig.…”
Section: Discussionmentioning
confidence: 99%
“…Considering the results of our PProCoM analysis of the TFBSs of six representative members of this family in Gammaproteobacteria, we defined a new and extended version of this motif: 5′-CTATA-n 9 -TATAG-3′. Additionally, examples of the sequence consensus of the TFBSs of other members of the LysR-type family that have been experimentally verified are also shown and include the distal TFBSs of CatR of the Gammaproteobacteria Pseudomonas putida [53], OccR of the Alphaproteobacteria Agrobacterium tumefaciens [53, 54] and PcaQ of the Alphaproteobacteria Sinorhizobium meliloti [56]. Dots within the inter-motif sequences were used to align the conserved nucleotides of the consensus sequences…”
BackgroundThe goal of most programs developed to find transcription factor binding sites (TFBSs) is the identification of discrete sequence motifs that are significantly over-represented in a given set of sequences where a transcription factor (TF) is expected to bind. These programs assume that the nucleotide conservation of a specific motif is indicative of a selective pressure required for the recognition of a TF for its corresponding TFBS. Despite their extensive use, the accuracies reached with these programs remain low. In many cases, true TFBSs are excluded from the identification process, especially when they correspond to low-affinity but important binding sites of regulatory systems.ResultsWe developed a computational protocol based on molecular and structural criteria to perform biologically meaningful and accurate phylogenetic footprinting analyses. Our protocol considers fundamental aspects of the TF-DNA binding process, such as: i) the active homodimeric conformations of TFs that impose symmetric structures on the TFBSs, ii) the cooperative binding of TFs, iii) the effects of the presence or absence of co-inducers, iv) the proximity between two TFBSs or one TFBS and a promoter that leads to very long spurious motifs, v) the presence of AT-rich sequences not recognized by the TF but that are required for DNA flexibility, and vi) the dynamic order in which the different binding events take place to determine a regulatory response (i.e., activation or repression). In our protocol, the abovementioned criteria were used to analyze a profile of consensus motifs generated from canonical Phylogenetic Footprinting Analyses using a set of analysis windows of incremental sizes. To evaluate the performance of our protocol, we analyzed six members of the LysR-type TF family in Gammaproteobacteria.ConclusionsThe identification of TFBSs based exclusively on the significance of the over-representation of motifs in a set of sequences might lead to inaccurate results. The consideration of different molecular and structural properties of the regulatory systems benefits the identification of TFBSs and enables the development of elaborate, biologically meaningful and precise regulatory models that offer a more integrated view of the dynamics of the regulatory process of transcription.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3025-3) contains supplementary material, which is available to authorized users.
“…Acknowledging the vast numbers of transcriptional activators found among LTTR members 22 21,[33][34][35][36] . In this proof-of-principle study, we selected the four candidates based on a minimal set of information, including knowledge about operator sequences, experimental evidence for ligand-inducible control of target operons, and their mode of action in the native chassis (i.e., activation; Supplementary Fig.…”
Section: A Design For Engineering Lttr-based Biosensors In Yeastmentioning
TitleEngineering prokaryotic transcriptional activators as metabolite biosensors in yeast io-based production of chemicals and fuels is an attractive avenue to reduce dependence on petroleum. For bio-based production, biocatalysts must often be genetically modified to increase production. However, the current efficiency of genomeengineering methods and parts prospecting allows for unprecedented genotype diversity that vastly outstrips our ability to screen for best cell performance 1,2 .To meet current demand, bioengineers have started to develop genetically encoded devices and systems that enable screening and selection of better-performing biocatalysts in higher throughput. Genetic devices including oscillators, amplifiers and recorders, which have been developed based on fine-tuned relationships between input and output signals, are promising tools for programming and controlling gene expression in living cells [3][4][5] . These devices sense extracellular or intracellular perturbations and actuate cellular decision-making processes akin to logic gates in electrical circuits. Hence, from a diverse set of inputs, molecular gating components such as RNA aptamers and allosterically regulated transcription factors have been engineered to control outputs for applications such as high-throughput screening, actuation on cellular metabolism and evolution-based selection of optimal cell performance [6][7][8] .A key component in many of the reported devices is a ligandinducible transcriptional regulator. Transcriptional regulators are straightforward and powerful components, with many uses in genetic designs. Owing to their modular structure, transcriptional regulators have proven to be versatile platforms for genetically encoded Boolean logic functions 9,10 . In particular, gene switches based on ligand-binding transcriptional repressors bind to genomic targets in the absence of their cognate ligand and thereby repress gene expression of the downstream gene(s), whereas binding between ligand and repressor causes the release of the repressor from the DNA and thereby a derepression 11 . In such 'NOT' gates, simple steric hindrance of RNA polymerase progression, as in the case of the tetracycline-responsive gene switch TetR, have for decades been used for conditional control of gene expression in both prokaryotic and eukaryotic chassis 12,13 . Transcriptional repressors and other artificial transcriptional regulators can be further engineered, for example, via the addition of nuclear localization signals, destabilization domains and transcriptional activation regions, to repurpose conditional repressors into activators [13][14][15] . Though conceptually intriguing and practically relevant, the repurposing of logic gates can suffer from the inherent need for extensive engineering 9,16,17 .Though most ligand-inducible genetic devices adopted for eukaryotes historically have been founded on transcriptional repressors, a hitherto untapped resource for use in genetic designs is ligand-inducible transcriptional activators. Bac...
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