Trp Repressor Research Articles

TrpR-Like Protein PXO_00831, Regulated by the Sigma Factor RpoD, Is Involved in Motility, Oxidative Stress Tolerance, and Virulence in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a Gram-negative bacterium that causes bacterial leaf blight in rice. In this study, we identified a putative TrpR-like protein, PXO_TrpR (PXO_00831), in Xoo. This protein contains a tryptophan (Trp) repressor domain and is highly conserved in Xanthomonas. Auxotrophic assays and RT-qPCR confirmed that PXO_TrpR acts as a Trp repressor, negatively regulating the expression of Trp biosynthesis genes. Pathogenicity tests showed that PXO_trpR knockout in Xoo significantly reduced lesion development and disease symptoms in the leaves of susceptible rice. RNA-seq analysis and phenotypic tests revealed that the PXO_trpR mutant exhibited impaired cell motility and was more sensitive to H2O2 oxidative stress than the wild-type strain. Furthermore, we found that the sigma 70 factor RpoD controlled the transcription of PXO_trpR by directly binding to its promoter region. This study demonstrates the biological function and transcriptional mechanism of PXO_TrpR as a Trp repressor in Xoo and evaluates its novel pathogenic roles by regulating flagellar motility and the oxidative stress response.

The role of tryptophan in Chlamydia trachomatis persistence.

Chlamydia trachomatis (C. trachomatis) is the most common etiological agent of bacterial sexually transmitted infections (STIs) and a worldwide public health issue. The natural course with C. trachomatis infection varies widely between individuals. Some infections clear spontaneously, others can last for several months or some individuals can become reinfected, leading to severe pathological damage. Importantly, the underlying mechanisms of C. trachomatis infection are not fully understood. C. trachomatis has the ability to adapt to immune response and persist within host epithelial cells. Indoleamine-2,3-dioxygenase (IDO) induced by interferon-gamma (IFN-γ) degrades the intracellular tryptophan pool, to which C. trachomatis can respond by converting to a non-replicating but viable state. C. trachomatis expresses and encodes for the tryptophan synthase (TS) genes (trpA and trpB) and tryptophan repressor gene (trpR). Multiple genes interact to regulate tryptophan synthesis from exogenous indole, and persistent C. trachomatis can recover its infectivity by converting indole into tryptophan. In this review, we discuss the characteristics of chlamydial infections, biosynthesis and regulation of tryptophan, the relationship between tryptophan and C. trachomatis, and finally, the links between the tryptophan/IFN-γ axis and C. trachomatis persistence.

Engineering of a TrpR-Based Biosensor for Altered Dynamic Range and Ligand Preference.

Transcriptional factors play a crucial role in regulating cellular functions. Understanding and altering the dynamic behavior of the transcriptional factor-based biosensors will expand our knowledge in investigating biomolecular interactions and facilitating biosynthetic applications. In this study, we characterized and engineered a TrpR-based tryptophan repressor system in Escherichia coli. We found that the reconstructed TrpR1-PtrpO1 biosensor system exhibited low basal expression and narrow dynamic range in the presence of tryptophan or its analogue 5-hydroxytryptophan (5-HTP). Given the application potential of the biosensor, we introduced engineering approaches in multiple levels to optimize its dynamic behavior. First, the I57 and V58 residues in the ligand-binding pocket were rationally mutated in search of variants with altered ligand specificity. Two TrpR1 variants, V58E and V58K, successfully acquired ligand preference toward tryptophan and 5-HTP, respectively. The biosensor-induced expression levels were increased up to 10-fold with those variants. Furthermore, to pursue broader operational range, we tuned the regulator-operator binding affinity by mutating the binding box of TrpR1. Collectively, we demonstrated that the biosynthesis-significant biosensor TrpR1-PtrpO1 can be engineered to acquire extended dynamic ranges and improved ligand preference. The engineered biosensor variants with remarkable dynamic behavior can serve as key genetic elements in high-throughput screening and dynamic regulation in biosynthetic scenarios.

A structural feature of Dda helicase which enhances displacement of streptavidin and trp repressor from DNA.

Helicases are molecular motors with many activities. They use the energy from ATP hydrolysis to unwind double-stranded nucleic acids while translocating on the single-stranded DNA. In addition to unwinding, many helicases are able to remove proteins from nucleic acids. Bacteriophage T4 Dda is able to displace a variety of DNA binding proteins and streptavidin bound to biotinylated oligonucleotides. We have identified a subdomain of Dda that when deleted, results in a protein variant that has nearly wild type activity for unwinding double-stranded DNA but exhibits greatly reduced streptavidin displacement activity. Interestingly, this domain has little effect on displacement of either gp32 or BamHI bound to DNA but does affect displacement of trp repressor from DNA. With this variant, we have identified residues which enhance displacement of some proteins from DNA.

Crystal structures of Val58Ile tryptophan repressor in a domain-swapped array in the presence and absence ofL-tryptophan

The crystal structures of domain-swapped tryptophan repressor (TrpR) variant Val58Ile before and after soaking with the physiological ligand L-tryptophan (L-Trp) indicate that L-Trp occupies the same location in the domain-swapped form as in native dimeric TrpR and makes equivalent residue contacts. This result is unexpected because the ligand binding-site residues arise from three separate polypeptide chains in the domain-swapped form. This work represents the first published structure of a domain-swapped form of TrpR with L-Trp bound. The presented structures also show that the protein amino-terminus, whether or not it bears a disordered extension of about 20 residues, is accessible in the large solvent channels of the domain-swapped crystal form, as in the structures reported previously in this form for TrpR without N-terminal extensions. These findings inspire the exploration of L-Trp analogs and N-terminal modifications as labels to orient guest proteins that cannot otherwise be crystallized in the solvent channels of crystalline domain-swapped TrpR hosts for potential diffraction analysis.

Tryptophan Operon Diversity Reveals Evolutionary Trends among Geographically Disparate Chlamydia trachomatis Ocular and Urogenital Strains Affecting Tryptophan Repressor and Synthase Function.

ABSTRACTThe obligate intracellular pathogen Chlamydia trachomatis (Ct) is the leading cause of bacterial sexually transmitted infections and blindness globally. To date, Ct urogenital strains are considered tryptophan prototrophs, utilizing indole for tryptophan synthesis within a closed-conformation tetramer comprised of two α (TrpA)- and two β (TrpB)-subunits. In contrast, ocular strains are auxotrophs due to mutations in TrpA, relying on host tryptophan pools for survival. It has been speculated that there is strong selective pressure for urogenital strains to maintain a functional operon. Here, we performed genetic, phylogenetic, and novel functional modeling analyses of 595 geographically diverse Ct ocular, urethral, vaginal, and rectal strains with complete operon sequences. We found that ocular and urogenital, but not lymphogranuloma venereum, TrpA-coding sequences were under positive selection. However, vaginal and urethral strains exhibited greater nucleotide diversity and a higher ratio of nonsynonymous to synonymous substitutions [Pi(a)/Pi(s)] than ocular strains, suggesting a more rapid evolution of beneficial mutations. We also identified nonsynonymous amino acid changes for an ocular isolate with a urogenital backbone in the intergenic region between TrpR and TrpB at the exact binding site for YtgR—the only known iron-dependent transcription factor in Chlamydia—indicating that selective pressure has disabled the response to fluctuating iron levels. In silico effects on protein stability, ligand-binding affinity, and tryptophan repressor (TrpR) affinity for single-stranded DNA (ssDNA) measured by calculating free energy changes (ΔΔG) between Ct reference and mutant tryptophan operon proteins were also analyzed. We found that tryptophan synthase function was likely suboptimal compared to other bacterial tryptophan prototrophs and that a diversity of urogenital strain mutations rendered the synthase nonfunctional or inefficient. The novel mutations identified here affected active sites in an orthosteric manner but also hindered α- and β-subunit allosteric interactions from distant sites, reducing efficiency of the tryptophan synthase. Importantly, strains with mutant proteins were inclined toward energy conservation by exhibiting an altered affinity for their respective ligands compared to reference strains, indicating greater fitness. This is not surprising as l-tryptophan is one of the most energetically costly amino acids to synthesize. Mutations in the tryptophan repressor gene (trpR) among urogenital strains were similarly detrimental to function. Our findings indicate that urogenital strains are evolving more rapidly than previously recognized with mutations that impact tryptophan operon function in a manner that is energetically beneficial, providing a novel host-pathogen evolutionary mechanism for intracellular survival.

A Strategy Employing a TF-Splinting Duplex Nanoswitch to Achieve Single-Step, Enzyme-Free, Signal-On Detection of l-Tryptophan.

Transcription factor (TF)-based metabolite detection mainly depends on TF-regulated gene expression in cells. From TF activation to gene transcription and translation, the signal travels a relatively long way before it is received. Here, we propose a TF-splinting duplex DNA nanoswitch to detect metabolites. We show its feasibility using tryptophan repressor (TrpR) to detect l-tryptophan as a model. The assay has been optimized and characterized after obtaining a proof of concept, and the detection of l-tryptophan in complex biological samples is feasible. Unlike an equivalent gene expression approach, the whole process is a single-step, enzyme-free, and signal-on method. It can be completed within 20 min. This proposed TF-splinting duplex has the potential to be applied to the quick and convenient detection of other metabolites or even TFs.

Development of a regulatable low-temperature protein expression system using the psychrotrophic bacterium, Shewanella livingstonensis Ac10, as the host.

A low-temperature protein expression system is useful for the production of thermolabile proteins. We previously developed a system that enables constitutive protein production at low temperatures, using the psychrotrophic bacterium Shewanella livingstonensis Ac10 as the host. To increase the utility of this system, in the present study, we introduced a repressible promoter of the trp operon of this bacterium into the system. When ß-lactamase was produced under the control of this promoter at 18°C and 4°C, the yields were 75 and 33 mg/L-culture, respectively, in the absence of L-Trp, and the yields were decreased by 72% and 77%, respectively, in the presence of L-Trp. We also found that 3-indoleacrylic acid, a competitive inhibitor of the Escherichia coli trp repressor, increased the expression of the reporter gene. This repressible gene expression system would be useful for regulatable recombinant protein production at low temperatures.

Trp-ing upon new repressors.

Bioengineers have used directed evolution to generate a new family of synthetic transcription factors based on the tryptophan repressor. The evolved repressor family enables researchers to build new gene circuits for biomedical applications.

Directed evolution of a synthetic phylogeny of programmable Trp repressors.

As synthetic regulatory programs expandin sophistication, an ever increasing number of biological components with predictable phenotypes is required. Regulators are often 'part mined' from a diverse, but uncharacterized, array of genomic sequences, often leading to idiosyncratic behavior. Here, we generate an entire synthetic phylogeny from the canonical allosteric transcription factor TrpR. Iterative rounds of positive and negative compartmentalized partnered replication (CPR) led to the exponential amplification of variants that responded with high affinity and specificity to halogenated tryptophan analogs and novel operator sites. Fourteen repressor variants were evolved with unique regulatory profiles across five operators and three ligands. The logic of individual repressors can be modularly programmed by creating heterodimeric fusions, resulting in single proteins that display logic functions, such as 'NAND'. Despite the evolutionarily limited regulatory role of TrpR, vast functional spaces exist around this highly conserved protein scaffold and can be harnessed to create synthetic regulatory programs.

A novel strategy to analyze L-tryptophan through allosteric Trp repressor based on rolling circle amplification.

Rolling circle amplification (RCA) has been considered as a powerful tool for nucleic acids detection. Here, a novel repressor-RCA-based method for L-tryptophan (L-Trp) detection was developed. This method utilizes the specific interaction between the RCA circular template and the Trp repressor protein (TrpR) involved in trp operon of Escherichia coli (E. coli). In the absence of L-Trp, the TrpR protein could not bind to the RCA template, and the RCA process can be continued. When L-Trp is present, the activated TrpR will bind to the operon sequence on the RCA template and inhibit the RCA reaction. Thus, the concentration of L-Trp is correlated directly with the fluorescent RCA signals. We succeeded in detecting L-Trp in a single step in simple homogeneous reaction system. The detection limit was estimated to be 0.77 μM (S/N=3) with good linearity. The method can unambiguously distinguish L-Trp from other 19 standard amino acids and L-Trp analogs. This strategy is also promising for detecting many small molecules such as other amino acids and carbohydrates.

A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations

The ability to visualize and manipulate cell fate and gene expression in specific cell populations has made gene expression systems valuable tools in developmental biology studies. Here, we describe a new system that uses the E. coli tryptophan repressor and its upstream activation sequence (TrpR/tUAS) to drive gene expression in stable zebrafish transgenic lines and in mammalian cells. We show that TrpR/tUAS transgenes are not silenced in subsequent generations of zebrafish, which is a major improvement over some of the existing systems, such as Gal4/gUAS and the Q-system. TrpR transcriptional activity can be tuned by mutations in its DNA-binding domain, or silenced by Gal80 when fused to the Gal4 activation domain. In cases in which more than one cell population needs to be manipulated, TrpR/tUAS can be used in combination with other, existing systems.

Effect of mutation at the interface of Trp-repressor dimeric protein: a steered molecular dynamics simulation

The strength of key interfacial contacts that stabilize protein-protein interactions have been studied by computer simulation. Experimentally, changes in the interface are evaluated by generating specific mutations at one or more points of the protein structure. Here, such an evaluation is performed by means of steered molecular dynamics and use of a dimeric model of tryptophan repressor and in-silico mutants as a test case. Analysis of four particular cases shows that, in principle, it is possible to distinguish between wild-type and mutant forms by examination of the total energy and force-extension profiles. In particular, detailed atomic level structural analysis indicates that specific mutations at the interface of the dimeric model (positions 19 and 39) alter interactions that appear in the wild-type form of tryptophan repressor, reducing the energy and force required to separate both subunits.

Internal Dynamics of the Tryptophan Repressor (TrpR) and Two Functionally Distinct TrpR Variants, L75F-TrpR and A77V-TrpR, in Their l-Trp-Bound Forms

Backbone amide dynamics of the Escherichia coli tryptophan repressor protein (WT-TrpR) and two functionally distinct variants, L75F-TrpR and A77V-TrpR, in their holo (l-tryptophan corepressor-bound) form have been characterized using (15)N nuclear magnetic resonance (NMR) relaxation. The three proteins possess very similar structures, ruling out major conformational differences as the source of their functional differences, and suggest that changes in protein flexibility are at the origin of their distinct functional properties. Comparison of site specific (15)N-T(1), (15)N-T(2), (15)N-{(1)H} nuclear Overhauser effect, reduced spectral density, and generalized order (S(2)) parameters indicates that backbone dynamics in the three holo-repressors are overall very similar with a few notable and significant exceptions for backbone atoms residing within the proteins' DNA-binding domain. We find that flexibility is highly restricted for amides in core α-helices (i.e., helices A-C and F), and a comparable "stiffening" is observed for residues in the DNA recognition helix (helix E) of the helix D-turn-helix E (HTH) DNA-binding domain of the three holo-repressors. Unexpectedly, amides located in helix D and in adjacent turn regions remain flexible. These data support the concept that residual flexibility in TrpR is essential for repressor function, DNA binding, and molecular recognition of target operators. Comparison of the (15)N NMR relaxation parameters of the holo-TrpRs with those of the apo-TrpRs indicates that the single-point amino acid substitutions, L75F and A77V, perturb the flexibility of backbone amides of TrpR in very different ways and are most pronounced in the apo forms of the three repressors. Finally, we present these findings in the context of other DNA-binding proteins and the role of protein flexibility in molecular recognition.

Development of l-tryptophan production strains by defined genetic modification in Escherichia coli

Construction and improvement of industrial strains play a central role in the commercial development of microbial fermentation processes. L-tryptophan producers have usually been developed by classical random mutagenesis due to its complicated metabolic network and regulatory mechanism. However, in the present study, an L-tryptophan overproducing Escherichia coli strain was developed by defined genetic modification methodology. Feedback inhibitions of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroF) and anthranilate synthase (TrpED) were eliminated by site-directed mutagenesis. Expression of deregulated AroF and TrpED was achieved by using a temperature-inducible expression plasmid pSV. Transcriptional regulation of trp repressor was removed by deleting trpR. Pathway for L-Trp degradation was removed by deleting tnaA. L-phenylalanine and L-tyrosine biosynthesis pathways that compete with L-tryptophan biosynthesis were blocked by deleting their critical genes (pheA and tyrA). The final engineered E. coli can produce 13.3 g/l of L-tryptophan. Fermentation characteristics of the engineered strains were also analyzed.

Problem-solving test: Attenuation: A mechanism to regulate bacterial tryptophan biosynthesis

Terms to be familiar with before you start to solve the test: tryptophan, transcription unit, operon, trp repressor, corepressor, operator, promoter, palindrome, initiation, elongation, and termination of transcription, open reading frame, coupled transcription/translation, chromosome-polysome complex.

Backbone Amide Dynamics Studies of Apo-L75F-TrpR, a Temperature-Sensitive Mutant of the Tryptophan Repressor Protein (TrpR): Comparison with the 15N NMR Relaxation Profiles of Wild-Type and A77V Mutant Apo-TrpR Repressors

Backbone amide dynamics studies were conducted on a temperature-sensitive mutant (L75F-TrpR) of the tryptophan repressor protein (TrpR) of Escherichia coli in its apo (i.e., no l-tryptophan corepressor-bound) form. The (15)N NMR relaxation profiles of apo-L75F-TrpR were analyzed and compared to those of wild-type (WT) and super-repressor mutant (A77V) TrpR proteins, also in their apo forms. The (15)N NMR relaxation data ((15)N-T(1), (15)N-T(2), and heteronuclear (15)N-{(1)H}-nOe) recorded on all three aporepressors at a magnetic field strength of 600 MHz ((1)H Larmor frequency) were analyzed to extract dynamics parameters, including diffusion tensor ratios (D(∥)/D(⊥)), correlation times (τ(m)) for overall reorientations of the proteins in solution, reduced spectral density terms [J(eff)(0), J(0.87ω(H)), J(ω(N))], and generalized order parameters (S(2)), which report on protein internal motions on the picosecond to nanosecond and slower microsecond to millisecond chemical exchange time scales. Our results indicate that all three aporepressors exhibit comparable D(∥)/D(⊥) ratios and characteristic time constants, τ(m), for overall global reorientation, indicating that in solution, all three apoproteins display very similar overall shape, structure, and rotational diffusion properties. Comparison of (15)N NMR relaxation data, reduced spectral density profiles, and generalized S(2) order parameters indicated that these parameters are quite uniform for backbone amides positioned within the four (A-C and F) core α-helices of all three aporepressors. In contrast, small but noticeable differences in internal dynamics were observed for backbone amides located within the helix D-turn-helix E DNA-binding domain of the apo-TrpR proteins. The significance of these dynamics differences in terms of the biophysical characteristics and ligand binding properties of the three apo-TrpR proteins is discussed.

Backbone dynamics of Tryptophan repressor A77V mutant protein in holo-form

Backbone dynamics of Tryptophan repressor A77V mutant protein in holo-form

Effects of Macromolecular Crowding on Protein Conformational Changes

Many protein functions can be directly linked to conformational changes. Inside cells, the equilibria and transition rates between different conformations may be affected by macromolecular crowding. We have recently developed a new approach for modeling crowding effects, which enables an atomistic representation of “test” proteins. Here this approach is applied to study how crowding affects the equilibria and transition rates between open and closed conformations of seven proteins: yeast protein disulfide isomerase (yPDI), adenylate kinase (AdK), orotidine phosphate decarboxylase (ODCase), Trp repressor (TrpR), hemoglobin, DNA β-glucosyltransferase, and Ap4A hydrolase. For each protein, molecular dynamics simulations of the open and closed states are separately run. Representative open and closed conformations are then used to calculate the crowding-induced changes in chemical potential for the two states. The difference in chemical-potential change between the two states finally predicts the effects of crowding on the population ratio of the two states. Crowding is found to reduce the open population to various extents. In the presence of crowders with a 15 Å radius and occupying 35% of volume, the open-to-closed population ratios of yPDI, AdK, ODCase and TrpR are reduced by 79%, 78%, 62% and 55%, respectively. The reductions for the remaining three proteins are 20–44%. As expected, the four proteins experiencing the stronger crowding effects are those with larger conformational changes between open and closed states (e.g., as measured by the change in radius of gyration). Larger proteins also tend to experience stronger crowding effects than smaller ones [e.g., comparing yPDI (480 residues) and TrpR (98 residues)]. The potentials of mean force along the open-closed reaction coordinate of apo and ligand-bound ODCase are altered by crowding, suggesting that transition rates are also affected. These quantitative results and qualitative trends will serve as valuable guides for expected crowding effects on protein conformation changes inside cells.

Molecular Dynamics Free Energy Calculations to Assess the Possibility of Water Existence in Protein Nonpolar Cavities

Are protein nonpolar cavities filled with water molecules? Although many experimental and theoretical investigations have been done, particularly for the nonpolar cavity of IL-1β, the results are still conflicting. To study this problem from the thermodynamic point of view, we calculated hydration free energies of four protein nonpolar cavities by means of the molecular dynamics thermodynamic integration method. In addition to the IL-1β cavity (69 Å3), we selected the three largest nonpolar cavities of AvrPphB (81 Å3), Trp repressor (87 Å3), and hemoglobin (108 Å3) from the structural database, in view of the simulation result from another study that showed larger nonpolar cavities are more likely to be hydrated. The calculations were performed with flexible and rigid protein models. The calculated free energy changes were all positive; hydration of the nonpolar cavities was energetically unfavorable for all four cases. Because hydration of smaller cavities should happen more rarely, we conclude that existing protein nonpolar cavities are not likely to be hydrated. Although a possibility remains for much larger nonpolar cavities, such cases are not found experimentally. We present a hypothesis to explain this: hydrated nonpolar cavities are quite unstable and the conformation could not be maintained.