Indra Bervoets

The protein–DNA contacts in RutR·carAB operator complexes

Pyrimidine-specific regulation of the upstream carP1 promoter of the carbamoylphosphate synthase operon of Escherichia coli requires numerous trans-acting factors: the allosteric transcription regulator RutR, the nucleoid-associated protein integration host factor, and the trigger enzymes aminopeptidase A and PyrH (UMP-kinase). RutR, a TetR family member, binds far upstream of carP1. Here, we establish a high-resolution contact map of RutR•carP1 complexes for backbone and base-specific contacts, analyze DNA bending, determine the DNA sequence specificity of RutR binding by saturation mutagenesis, demonstrate that uracil but not thymine is the physiologically relevant ligand that inhibits the DNA binding capacity of RutR and build a model of the RutR·operator DNA complex based on the crystal structures of RutR and of the DNA-bound family member QacR. Finally, we test the validity of this model with site-directed mutagenesis of the helix–turn–helix DNA binding motif and in vitro binding studies with the cognate purified mutant RutR proteins.

A sigma factor toolbox for orthogonal gene expression in Escherichia coli

Abstract Synthetic genetic sensors and circuits enable programmable control over timing and conditions of gene expression and, as a result, are increasingly incorporated into the control of complex and multi-gene pathways. Size and complexity of genetic circuits are growing, but stay limited by a shortage of regulatory parts that can be used without interference. Therefore, orthogonal expression and regulation systems are needed to minimize undesired crosstalk and allow for dynamic control of separate modules. This work presents a set of orthogonal expression systems for use in Escherichia coli based on heterologous sigma factors from Bacillus subtilis that recognize specific promoter sequences. Up to four of the analyzed sigma factors can be combined to function orthogonally between each other and toward the host. Additionally, the toolbox is expanded by creating promoter libraries for three sigma factors without loss of their orthogonal nature. As this set covers a wide range of transcription initiation frequencies, it enables tuning of multiple outputs of the circuit in response to different sensory signals in an orthogonal manner. This sigma factor toolbox constitutes an interesting expansion of the synthetic biology toolbox and may contribute to the assembly of more complex synthetic genetic systems in the future.

Ligand binding specificity of RutR, a member of the TetR family of transcription regulators in Escherichia coli.

RutR is a member of the large family of TetR transcriptional regulators inEscherichia coli. It was originally discovered as the regulator of therutABCDEFG operon encoding a novel pathway for pyrimidine utilization, but its highest affinity target is the control region of thecarAB operon, encoding carbamoylphosphate synthase. Unlike most other TetR‐like regulators, RutR exerts both positive and negative effects on promoter activity. Furthermore, RutR exhibits a very narrow ligand binding specificity, unlike the broad effector specificity that characterizes some of the well‐studied multidrug resistance regulators of the family. Here we focus on ligand binding and ligand specificity of RutR. We construct single alanine substitution mutants of amino acid residues of the ligand‐binding pocket, study their effect onin vitro DNA binding in absence and presence of potential ligands, and analyse their effect on positive regulation of thecarP1 promoter and negative autoregulationin vivo. Although RutR structures have been determined previously, they were deposited in the Protein Data Bank without accompanying publications. All of them have uracil bound in the effector‐binding site, representing the inactive form of the regulator. We determined the crystal structure of an unliganded mutant RutR protein and provide a structural basis for the use of uracil as sole effector molecule and the exclusion of the very similar thymine from the ligand‐binding pocket.

A novel and versatile dual fluorescent reporter tool for the study of gene expression and regulation in multi- and single copy number

To unravel intricate mechanisms of gene regulation it is imperative to work in physiologically relevant conditions and therefore preferentially in single copy constructs, which are not always easy to manipulate. Such in vivo studies are generally based on enzymatic assays, microarrays, RNA-seq, qRT-PCR, or multicopy reporter gene systems, frequently with β-galactosidase, luciferase or a fluorescent protein as reporter. Each method has its advantages and shortcomings and may require validation. Enzyme assays are generally reliable but may be quite complex, time consuming, and require a (expensive) substrate. Microarrays and RNA-seq provide a genome wide view of gene expression but may rapidly become expensive and time consuming especially for detailed studies with large numbers of mutants, different growth conditions and multiple time points. Multicopy reporter gene systems are handy to generate numerous constructs but may not provide accurate information due to titration effects of trans-acting regulatory elements. Therefore and in spite of the existence of various reporter systems, there is still need for an efficient and user-friendly tool for detailed studies and high throughput screenings. Here we develop and validate a novel and versatile fluorescent reporter tool to study gene regulation in single copy mode that enables real-time measurement. This tool bears two independent fluorescent reporters that allow high throughput screening and standardization, and combines modern efficient cloning methods (multicopy, in vitro manipulation) with classical genetics (in vivo homologous recombination with a stable, self-transmissible episome) to generate multi- and single copy reporter systems. We validate the system with constitutive and differentially regulated promoters and show that the tool can equally be used with heterologous transcription factors. The flexibility and versatility of this dual reporter tool in combination with an easy conversion from a multicopy plasmid to a stable, single copy reporter system makes this system unique and attractive for a variety of applications. Examples are in vivo studies of DNA-binding transcription factors (single copy) or screening of promoter and RBS libraries (multicopy) for synthetic biology purposes.