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

Allosteric Regulatory Mechanisms of Bacterial RSH-enzymes

Long RSHs are enzymes that synthetize and hydrolyse a secondary messenger molecule, (p)ppGpp. This molecule acts as a global regulator targeting metabolic processes to ensure bacterial survival upon environmental challenges. Because (p)ppGpp is linked with bacterial virulence and resistance to antibiotics, a fundamental understanding of its regulation is key to develop novel antimicrobial treatments. Long RSH have two N-terminal domains, one for hydrolysis of (p)ppGpp (HD-domain) and one for synthesis of (p)ppGpp (SYNTH-domain), and four regulatory domains at the C-terminus that control catalysis by the N-terminal domains. A combination of structure determination and biochemical assays were used to study the long RSHs at the molecular level and determine the bases of the allosteric mechanisms that control the activities of the enzymes. The data demonstrate an allosteric conformational switch between the N-terminal catalytic domains of these enzymes that prevent futile catalytic cycles. Indeed, the data shows binding of substrates to one catalytic domain allosterically inhibits substrate-binding and catalysis by the opposing catalytic domain. Furthermore, feedback activation of (p)ppGpp-synthesis, the molecular basis of which was unknown, in context of amino-acid starvation was studied. The allosteric positive feedback regulation of (p)ppGpp-synthesis was shown to exploit the conformational switch between the two catalytic states to modulate both activities. An allosteric binding-pocket was located at the linker-region between the two catalytic domains at which (p)ppGpp-synthesis is auto-induced allosterically while (p)ppGpp-hydrolysis is inhibited. Lastly, the conformational landscape involved in the regulation of these enzymes was explored using Nanobodies to discover a previously undetected regulatory hotspot. Based on the conformational switch as well, this regulatory hotspot modulates catalytic activity allosterically. The newly discovered hotspot might be important to the autoregulation of long RSHs by its four regulatory domains that has been poorly understood for decades. This study uncovered three fundamental allosteric regulatory layers of the long RSHs that were previously unknown or poorly understood. It is the first step in the development of novel antimicrobial treatments that target (p)ppGpp-homeostasis, which is becoming more relevant as antibiotic resistance is occurring more frequently and is one of the most pressing problems to public health today.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

The Rel stringent factor from Thermus thermophilus: Crystallization and X-ray analysis

The stringent response, controlled by (p)ppGpp, enables bacteria to trigger a strong phenotypic resetting that is crucial to cope with adverse environmental changes and is required for stress survival and virulence. In the bacterial cell, (p)ppGpp levels are regulated by the concerted opposing activities of RSH (RelA/SpoT homologue) enzymes that can transfer a pyrophosphate group of ATP to the 3′ position of GDP (or GTP) or remove the 3′ pyrophosphate moiety from (p)ppGpp. Bifunctional Rel enzymes are notoriously difficult to crystallize owing to poor stability and a propensity for aggregation, usually leading to a loss of biological activity after purification. Here, the production, biochemical analysis and crystallization of the bifunctional catalytic region of the Rel stringent factor from Thermus thermophilus (Rel Tt NTD) in the resting state and bound to nucleotides are described. Rel Tt and Rel Tt NTD are monomers in solution that are stabilized by the binding of Mn2+ and mellitic acid. Rel Tt NTD crystallizes in space group P4122, with unit-cell parameters a = b = 88.4, c = 182.7 Å, at 4°C and in space group P41212, with unit-cell parameters a = b = 105.7, c = 241.4 Å, at 20°C.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'closed' conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity

(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity

Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate

Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools

While alarmone nucleotides guanosine-3′,5′-bisdiphosphate (ppGpp) and guanosine-5′-triphosphate-3′-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3′,5′-bisdiphosphate) and pppApp (adenosine-5′-triphosphate-3′-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3′-azido-3′-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate