Use of sigma factor M from Bacillus subtilis in the development of an orthogonal expression system in Escherichia coli

Background: Technological advances in synthetic biology, systems biology, and metabolic engineering have boosted applications of industrial biotechnology for an increasing number of complex and high added-value molecules. In general, the transfer of multi- gene or poorly understood heterologous pathways into the production host leads to imbalances due to lack of adequate regulatory mechanisms. Hence, fine-tuning expression of synthesis pathways in specific conditions is mandatory. Objectives: Here we develop a new genetic circuit for regulated expression specifically in stationary phase due to clear advantages during this period (reduction of toxicity, competition). Methods: This circuit consists of a heterologous sigma factor () recognizing specific promoter sequences, which are not recognised by the native factors of E. coli and is expressed upon entering the stationary phase. First, several factors of B. subtilis were tested for their orthogonality in E. coli on the level of promoter recognition, by using a red-fluorescent reporter system. Secondly, the potential of factors of B. subtilis to work together with the E. coli core RNA polymerase was tested, by expressing these proteins together with their promoters. Based on the results a specific factor will be chosen for further optimalisation and the corresponding gene can be cloned in the S factor operon of E. coli, which is most abundantly expressed in stationary conditions. Conclusions: Combining all these elements should allow us to create an orthogonal genetic circuit that is able to transcribe specific genes under stationary phase with a limited influence on the host cell’s metabolism

The Lrp Family of Transcription Regulators in Archaea

Archaea possess a eukaryotic-type basal transcription apparatus that is regulated by bacteria-like transcription regulators. A universal and abundant family of transcription regulators are the bacterial/archaeal Lrp-like regulators. The Lrp family is one of the best studied regulator families in archaea, illustrated by investigations of proteins from the archaeal model organisms: Sulfolobus, Pyrococcus, Methanocaldococcus, and Halobacterium. These regulators are extremely versatile in their DNA-binding properties, response to effector molecules, and molecular regulatory mechanisms. Besides being involved in the regulation of the amino acid metabolism, they also regulate central metabolic processes. It appears that these regulatory proteins are also involved in large regulatory networks, because of hierarchical regulations and the possible combinatorial use of different Lrp-like proteins. Here, we discuss the recent developments in our understanding of this important class of regulators

A sigma factor toolbox for orthogonal gene expression in Escherichia coli

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

Analysis of the DNA-binding sequence specificity of the archaeal transcriptional regulator Ss-LrpB from Sulfolobus solfataricus by systematic mutagenesis and high resolution contact probing

To determine the sequence specificity of dimeric Ss-LrpB, a high resolution contact map was constructed and a saturation mutagenesis conducted on one half of the palindromic consensus box. Premodification binding interference indicates that Ss-LrpB establishes most of its tightest contacts with a single strand of two major groove segments and interacts with the minor groove at the center of the box. The requirement for bending is reflected in the preference for an A+T rich center and confirmed with C·G and C·I substitutions. The saturation mutagenesis indicates that major groove contacts with C·G at position 5 and its symmetrical counterpart are most critical for the specificity and strength of the interaction. Conservation at the remaining positions improved the binding. Hydrogen bonding to the O(6) and N(7) acceptor atoms of the G(5′) residue play a major role in complex formation. Unlike many other DNA-binding proteins Ss-LrpB does not establish hydrophobic interactions with the methyls of thymine residues. The binding energies determined from the saturation mutagenesis were used to construct a sequence logo, which pin-points the overwhelming importance of C·G at position 5. The knowledge of the DNA-binding specificity will constitute a precious tool for the search of new physiologically relevant binding sites for Ss-LrpB in the genome

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