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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A non-canonical fungal peroxisome PTS-1 signal, SYM, and its evolutionary aspects

Abstract Proteins localized to peroxisomes, particularly those expressed under specific conditions or in low abundance, are often undetected by routine proteomics methods due to detection sensitivity limits. In silico identification and experimental validation of peroxisomal targeting signals (PTSs) offer a reliable alternative. We demonstrate that SYM, a non-canonical plant PTS-1 signal, functions similarly inAspergillus nidulans, as GFP tagged with a SYM C-terminal tripeptide localizes to peroxisomes. One of two nativeA. nidulansproteins with C-terminal SYM tripeptide shows weak peroxisomal localization alongside cytoplasmic presence, indicating that only a subset of proteins with non-canonical signals access peroxisomes.In silicoanalysis of 1,010 fungal genomes identified diverse SYM-proteins with variable functions, suggesting that non-canonical PTS-1 signals may evolve spontaneously. Two-thirds of SYM-proteins are predicted to localize to specific intracellular compartments other than the peroxisome. We propose that despite their predicted localization, these proteins possessing SYM as a non-canonical peroxisomal signal might also have peroxisomal presence. Among SYM-proteins, pectinesterases, known plant pathogen virulence factors, were frequent. Notably, 25% of fungal pectinesterases harbor non-canonical PTS-1 signals, suggesting that partial peroxisomal localization of pectinesterases has evolved convergently. This suggests that partial peroxisomal localization may enhance protein functional flexibility, contributing to the organism’s adaptability.

Science & Technology - Other Topics↗
DOE OSTI2019

Time and frequency resolved transient-absorption and stimulated-Raman signals of stochastic light

Covariance spectroscopy signals based on the transmission of broadband stochastic probe light undergoing a nonlinear optical process with matter are studied. The resulting signal depends on intensity correlation functions of the probe pulse. Application is made to transient absorption and stimulated Raman signals, where an ultraviolet pump and a delayed stochastically modulated infrared or optical Raman probe are used for monitoring vibrational excitations. We show that the stochastic properties of light can be exploited to circumvent the limitations on the joint temporal and spectral resolution of the signal. We study a model system with a time-dependent frequency and show that the dynamical information can be fully extracted from the covariance signal. Finally, this information is heavily suppressed upon averaging of noisy signals and practically inaccessible in conventional stimulated Raman measurements.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Conformation-specific synthetic intrabodies modulate mTOR signaling with subcellular spatial resolution

Subcellular compartmentalization is integral to the spatial regulation of mechanistic target of rapamycin (mTOR) signaling. However, the biological outputs associated with location-specific mTOR signaling events are poorly understood and challenging to decouple. Here, we engineered synthetic intracellular antibodies (intrabodies) that are capable of modulating mTOR signaling with genetically programmable spatial resolution. Epitope-directed phage display was exploited to generate high affinity synthetic antibody fragments (Fabs) against the FKBP12–Rapamycin binding site of mTOR (mTOR FRB ). We determined high-resolution crystal structures of two unique Fabs that discriminate distinct conformational states of mTOR FRB through recognition of its substrate recruitment interface. By leveraging these conformation-specific binders as intracellular probes, we uncovered the structural basis for an allosteric mechanism governing mTOR complex 1 (mTORC1) stability mediated by subtle structural adjustments within mTOR FRB . Furthermore, our results demonstrated that synthetic binders emulate natural substrates by employing divergent yet complementary hydrophobic residues at defined positions, underscoring the broad molecular recognition capability of mTOR FRB . Intracellular signaling studies showed differential time-dependent inhibition of S6 kinase 1 and Akt phosphorylation by genetically encoded intrabodies, thus supporting a mechanism of inhibition analogous to the natural product rapamycin. Finally, we implemented a feasible approach to selectively modulate mTOR signaling in the nucleus through spatially programmed intrabody expression. These findings establish intrabodies as versatile tools for dissecting the conformational regulation of mTORC1 and should be useful to explore how location-specific mTOR signaling influences disease progression.

Science & Technology - Other Topics↗
DOE OSTI2017

Halo-independent determination of the unmodulated WIMP signal in DAMA: the isotropic case

We present a halo-independent determination of the unmodulated signal corresponding to the DAMA modulation if interpreted as due to dark matter weakly interacting massive particles (WIMPs). First we show how a modulated signal gives information on the WIMP velocity distribution function in the Galactic rest frame from which the unmodulated signal descends. Then we describe a mathematically-sound profile likelihood analysis in which the likelihood is profiled over a continuum of nuisance parameters (namely, the WIMP velocity distribution). As a first application of the method, which is very general and valid for any class of velocity distributions, we restrict the analysis to velocity distributions that are isotropic in the Galactic frame. In this way we obtain halo-independent maximum-likelihood estimates and confidence intervals for the DAMA unmodulated signal. We find that the estimated unmodulated signal is in line with expectations for a WIMP-induced modulation and is compatible with the DAMA background+signal rate. Specifically, for the isotropic case we find that the modulated amplitude ranges between a few percent and about 25% of the unmodulated amplitude, depending on the WIMP mass.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2025

Electrical signaling in fungi: past and present challenges

Abstract Electrical signaling is a fundamental mechanism for integrating environmental stimuli and coordinating responses in living organisms. While extensively studied in animals and plants, the role of electrical signaling in fungi remains a largely underexplored field. Early studies suggested that filamentous fungi generate action potential-like signals and electrical currents at hyphal tips, yet their function in intracellular communication remained unclear. Renewed interest in fungal electrical activity has fueled developments such as the hypothesis that mycorrhizal networks facilitate electrical communication between plants and the emerging field of fungal-based electronic materials. Given their continuous plasma membrane, specialized septal pores, and insulating cell wall structures, filamentous fungi possess architectural features that could support electrical signaling over long distances. However, studying electrical phenomena in fungal networks presents unique challenges due to the microscopic dimensions of hyphae, the structural complexity of highly modular mycelial networks, and the limitations of traditional electrophysiological methods. This review synthesizes current evidence for electrical signaling in filamentous fungi, evaluates methodological approaches, and highlights experimental challenges. By addressing these challenges and identifying best practices, we aim to advance research in this field and provide a foundation for future studies exploring the role of electrical signaling in fungal biology.

Buffi, Matteo↗
DOE OSTI2017

Structure of human POFUT1, its requirement in ligand-independent oncogenic Notch signaling, and functional effects of Dowling-Degos mutations

Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1 -/- background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Evaluating the QSO contribution to the 21-cm signal from the Cosmic Dawn

The upcoming radio interferometer Square Kilometre Array (SKA) is expected to directly detect the redshifted 21-cm signal from the neutral hydrogen present during the Cosmic Dawn. Temperature fluctuations from X-ray heating of the neutral intergalactic medium can dominate the fluctuations in the 21-cm signal from this time. This heating depends on the abundance, clustering, and properties of the X-ray sources present, which remain highly uncertain. We present a suite of three new large-volume, 349\,Mpc a side, fully numerical radiative transfer simulations including QSO-like sources, extending the work previously presented in Ross et al. (2017). The results show that our QSOs have a modest contribution to the heating budget, yet significantly impact the 21-cm signal. Initially, the power spectrum is boosted on large scales by heating from the biased QSO-like sources, before decreasing on all scales. Fluctuations from images of the 21-cm signal with resolutions corresponding to SKA1-Low at the appropriate redshifts are well above the expected noise for deep integrations, indicating that imaging could be feasible for all the X-ray source models considered. The most notable contribution of the QSOs is a dramatic increase in non-Gaussianity of the signal, as measured by the skewness and kurtosis of the 21-cm probability distribution functions. Furthermore, in the case of late Lyman-$\alpha$ saturation, this non-Gaussianity could be dramatically decreased particularly when heating occurs earlier. We conclude that increased non-Gaussianity is a promising signature of rare X-ray sources at this time, provided that Lyman-$\alpha$ saturation occurs before heating dominates the 21-cm signal.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2025

Modeling the differential rate for signal interactions in coincidence with noise fluctuations or large rate backgrounds

The characteristic energy of a relic dark matter interaction with a detector scales strongly with the putative dark matter mass. Consequently, experimental search sensitivity at the lightest masses will always come from interactions whose size is similar to noise fluctuations and low energy backgrounds in the detector. In this paper, we correctly calculate the net change in measured differential event rate due to low rate signal interactions that overlap in time with noise and backgrounds, accounting for both periods of time when the signal is coincident with noise/backgrounds and for the decreased amount of time in which only noise/backgrounds occur and we show that the introduction of random simulated signal events into the continuous raw data stream (a form of “salting”) provides a correct and practical implementation to estimate this net linear signal sensitivity. Unfortunately this does not apply to the situation with non negligible pileups as we show through explicit examples. Consequently, this exclusion technique should be complemented by less sensitive techniques that can separately exclude large rate, high pileup signal parameter space. An analysis threshold above the Gaussian-like noise component of the measured differential rate spectrum should also likely produce conservative limits. Though previous light mass dark matter searches did not correctly account for decreased background only live time effects in their signal sensitivity, conservative analysis choices likely protected their published limits from being nonconservative.

Li, Xinran↗
DOE OSTI2020

The enigmatic Galactic Center excess: Spurious point sources and signal mismodeling

The Galactic Center GeV excess (GCE) has garnered great interest as a possible signal of either dark matter annihilation or some novel astrophysical phenomenon, such as a new population of gamma-ray emitting pulsars. In a companion paper, we showed that in a 10° radius region of interest (ROI) surrounding the Galactic Center, apparent evidence for GCE point sources (PSs) from non-Poissonian template fitting (NPTF) is actually an artifact of unmodeled north-south asymmetry of the GCE. In this work, we develop a simplified analytic description of how signal mismodeling can drive an apparent preference for a PS population, and demonstrate how the behavior pointed out in the companion paper also appears in simpler simulated datasets that contain no PS signals at all. We explore the generality of this behavior in the real gamma-ray data, and discuss the implications for past and future studies using NPTF techniques. While the drop in PS preference once north-south asymmetry is included is not ubiquitous in larger ROIs, we show that any overly-rigid signal model is expected to yield a spurious PS signal that can appear very convincing: as well as apparent significance comparable to what one would expect from a true PS population, the signal can exhibit stability against a range of variations in the analysis, and a source count function that is very consistent with previous apparent NPTF-based detections of a GCE PS population. This contrasts with previously studied forms of systematic mismodeling which are unlikely to mimic a PS population in the same way. Finally, in the light of this observation, and its explicit realization in the region where the GCE is brightest, we argue that a dominantly smooth origin for the GCE is not in tension with existing NPTF analyses.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2025

Signal-preserving CMB component separation with machine learning

Analysis of microwave sky signals, such as the cosmic microwave background, often requires component separation using multifrequency methods, whereby different signals are isolated according to their different frequency behaviors. Many so-called blind methods, such as the internal linear combination (ILC), make minimal assumptions about the spatial distribution of the signal or contaminants, and only assume knowledge of the frequency dependence of the signal. The ILC produces a minimum-variance linear combination of the measured frequency maps. In the case of Gaussian, statistically isotropic fields, this is the optimal linear combination, as the variance is the only statistic of interest. However, in many cases the signal we wish to isolate, or the foregrounds we wish to remove, are non-Gaussian and/or statistically anisotropic (in particular for the case of Galactic foregrounds). In such cases, it is possible that machine learning (ML) techniques can be used to exploit the non-Gaussian features of the foregrounds and thereby improve component separation. However, many ML techniques require the use of complex, difficult-to-interpret operations on the data. We propose a hybrid method whereby we train an ML model using only combinations of the data that , and combine the resulting ML-predicted foreground estimate with the ILC solution to reduce the error from the ILC. We demonstrate our methods on simulations of extragalactic temperature and Galactic polarization foregrounds and show that our ML model can exploit non-Gaussian features, such as point sources and spatially varying spectral indices, to produce lower-variance maps than ILC—e.g., reducing the variance of the B-mode residual by factors of up to 5—while preserving the signal of interest in an unbiased manner. Moreover, we often find improved performance even when applying our ML technique to foreground models on which it was not trained. Published by the American Physical Society 2025

McCarthy, Fiona (ORCID:0000000253893565)↗
DOE OSTI2015

Enhancement of Second-Order Nonlinear-Optical Signals by Optical Stimulation

Second-order nonlinear optical interactions such as sum- and difference-frequency generation are widely used for bioimaging and as selective probes of interfacial environments. However, inefficient nonlinear optical conversion often leads to poor signal-to-noise ratio and long signal acquisition times. We demonstrate the dramatic enhancement of weak second-order nonlinear optical signals via stimulated sum- and difference-frequency generation. We present a conceptual framework to quantitatively describe the interaction and show that the process is highly sensitive to the relative optical phase of the stimulating field. To emphasize the utility of the technique, we demonstrate stimulated enhancement of second harmonic generation (SHG) from bovine collagen-I fibrils. Using a stimulating pulse fluence of only $3\text{ }\text{ }\mathrm{nJ}/{\mathrm{cm}}^{2}$, we obtain an SHG enhancement >${10}^{4}$ relative to the spontaneous signal. The stimulation enhancement is greatest in situations where spontaneous signals are the weakest—such as low laser power, small sample volume, and weak nonlinear susceptibility—emphasizing the potential for this technique to improve signal-to-noise ratios in biological imaging and interfacial spectroscopy.

42 ENGINEERING↗
DOE OSTI2023

C-terminal conformational changes in SCF-D3/MAX2 ubiquitin ligase are required for KAI2-mediated signaling

Karrikins (KARs) are bioactive molecules derived from burning vegetation. Plants perceive KARs through the hydrolase KARRIKIN INSENSITIVE 2 (KAI2) that interacts with the F-box protein ubiquitin ligase MORE AXILLARY GROWTH 2 (MAX2). MAX2 also plays a role in the perception and signal activation by Strigolactone (SL), a phytohormone controlling various developmental processes in plants. SL also acts as a rhizosphere signal to activate arbuscular mycorrhiza fungi that can be exploited by parasitic plants. kai2 knockouts exhibit distinct developmental defects and therefore KAI2 is hypothesized to perceive an unidentified endogenous ligand provisionally termed KAI2-Ligand (KL). Upon KAR/KL perception, the protein complex of KAI2-MAX2 targets SUPPRESSOR OF MAX2-1/2 (SMAX1)/SMXL2 for proteasomal degradation. Despite the identification of the key components KAI2, MAX2, and SMAX1 in KAR/KL signaling, their mode of interaction and regulation remains elusive. Recently, the regulatory function of the conformational switch of MAX2 C-terminal helix (CTH) in SL signaling has been demonstrated however its role in KAR/KL signaling remained unknown. Here we address the function of MAX2-CTH dynamics both in vitro and in planta and show that the central role of CTH is conserved between SL and KAR/KL signaling pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

More chemical detection through less sampling: amplifying chemical signals in hyperspectral data cubes through compressive sensing

Compressive sensing (CS) is a method of sampling which permits some classes of signals to be reconstructed with high accuracy even when they were sampled at sub-Nyquist rates. In this paper we explore a phenomenon in which bandwise CS sampling of a hyperspectral data cube followed by reconstruction can actually result in amplification of chemical signals contained in the cube. Perhaps most surprisingly, chemical signal amplification generally seems to increase as the level of sampling decreases. In some examples, the chemical signal is significantly stronger in a data cube reconstructed from 10% CS sampling than it is in the raw, 100% sampled data cube. We explore this phenomenon in two real-world datasets including the Physical Sciences Inc. Fabry-Perot interferometer sensor multispectral dataset and the Johns Hopkins Applied Physics Lab FTIR-based longwave infrared sensor hyperspectral dataset. Each of these datasets contains the release of a chemical simulant, such as glacial acetic acid, triethyl phospate, and sulfur hexafluoride, and in all cases we use the adaptive coherence estimator (ACE) to detect a target signal in the hyperspectral data cube. We end the paper by suggesting some theoretical justifications for why chemical signals would be amplified in CS sampled and reconstructed hyperspectral data cubes and discuss some practical implications.

Hyperspectral imagery, Compressive Sensing, single↗
DOE OSTI2014

TH-C-17A-01: Imaging Sensor Comparison for Real-Time Cherenkov Signal Detection From Tissue for Treatment Verification

Purpose: To identify the optimum imaging sensor for a clinical system that would provide real-time imaging of the surface beam profile on patients as novel visual information to radiation therapy technologists, and more rapidly collect clinical data for large-scale studies of Cherenkov applications in radiotherapy. Methods: Four camera types, CMOS, CCD, ICCD and EMICCD, were tested to determine proficiency in the detection of Cherenkov signal in the clinical radiotherapy setting, and subsequent maximum supportable frame rate. Where possible, time-gating between the trigger signal from the LINAC and the intensifiers was implemented to detect signal with room lighting conditions comparable to patient treatment scenarios. A solid water phantom was imaged by the EM-ICCD and ICCD to evaluate the minimum number of accumulations-on-chip required for adequate Cherenkov detection, defined as >200% electron counts per pixel over background signal. Additionally, an ICCD and EM-ICCD were used clinically to image patients undergoing whole-breast radiation therapy, to understand the impact of the resolution limitation of the EM-ICCD. Results: The intensifier-coupled cameras performed best at imaging Cherenkov signal, even with room lights on, which is essential for patient comfort. The tested EM-ICCD was able to support single-shot imaging and frame rates of 30 fps, however, the current maximum resolution of 512 × 512 pixels was restricting. The ICCD used in current clinical trials was limited to 4.7 fps at a 1024 × 1024 resolution. An intensifier with higher quantum efficiency at the entrance photocathode in the red wavelengths (30% QE vs current 7%) promises 16 fps at the same resolution at lower cost than the EM-ICCD. Conclusion: The ICCD with the better red wavelength QE intensifier was determined to be the best suited commercial-off-the-shelf camera to detect real-time Cherenkov signal and provide the best potential for real-time display of radiation dose on the skin during treatment. Funding is from grants from the NIH numbers R01CA109558 and R21EB017559.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Evaluation of intrinsic respiratory signal determination methods for 4D CBCT adapted for mice

Purpose: 4D CT imaging in mice is important in a variety of areas including studies of lung function and tumor motion. A necessary step in 4D imaging is obtaining a respiratory signal, which can be done through an external system or intrinsically through the projection images. A number of methods have been developed that can successfully determine the respiratory signal from cone-beam projection images of humans, however only a few have been utilized in a preclinical setting and most of these rely on step-and-shoot style imaging. The purpose of this work is to assess and make adaptions of several successful methods developed for humans for an image-guided preclinical radiation therapy system. Methods: Respiratory signals were determined from the projection images of free-breathing mice scanned on the X-RAD system using four methods: the so-called Amsterdam shroud method, a method based on the phase of the Fourier transform, a pixel intensity method, and a center of mass method. The Amsterdam shroud method was modified so the sharp inspiration peaks associated with anesthetized mouse breathing could be detected. Respiratory signals were used to sort projections into phase bins and 4D images were reconstructed. Error and standard deviation in the assignment of phase bins for the four methods compared to a manual method considered to be ground truth were calculated for a range of region of interest (ROI) sizes. Qualitative comparisons were additionally made between the 4D images obtained using each of the methods and the manual method. Results: 4D images were successfully created for all mice with each of the respiratory signal extraction methods. Only minimal qualitative differences were noted between each of the methods and the manual method. The average error (and standard deviation) in phase bin assignment was 0.24 ± 0.08 (0.49 ± 0.11) phase bins for the Fourier transform method, 0.09 ± 0.03 (0.31 ± 0.08) phase bins for the modified Amsterdam shroud method, 0.09 ± 0.02 (0.33 ± 0.07) phase bins for the intensity method, and 0.37 ± 0.10 (0.57 ± 0.08) phase bins for the center of mass method. Little dependence on ROI size was noted for the modified Amsterdam shroud and intensity methods while the Fourier transform and center of mass methods showed a noticeable dependence on the ROI size. Conclusions: The modified Amsterdam shroud, Fourier transform, and intensity respiratory signal methods are sufficiently accurate to be used for 4D imaging on the X-RAD system and show improvement over the existing center of mass method. The intensity and modified Amsterdam shroud methods are recommended due to their high accuracy and low dependence on ROI size.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Technical Note: Intrafractional changes in time lag relationship between anterior–posterior external and superior–inferior internal motion signals in abdominal tumor sites

Purpose: To investigate constancy, within a treatment session, of the time lag relationship between implanted markers in abdominal tumors and an external motion surrogate. Methods: Six gastroesophageal junction and three pancreatic cancer patients (IRB-approved protocol) received two cone-beam CTs (CBCT), one before and one after treatment. Time between scans was less than 30 min. Each patient had at least one implanted fiducial marker near the tumor. In all scans, abdominal displacement (Varian RPM) was recorded as the external motion signal. Purpose-built software tracked fiducials, representing internal signal, in CBCT projection images. Time lag between superior–inferior (SI) internal and anterior–posterior external signals was found by maximizing the correlation coefficient in each breathing cycle and averaging over all cycles. Time-lag-induced discrepancy between internal SI position and that predicted from the external signal (external prediction error) was also calculated. Results: Mean ± standard deviation time lag, over all scans and patients, was 0.10 ± 0.07 s (range 0.01–0.36 s). External signal lagged the internal in 17/18 scans. Change in time lag between pre- and post-treatment CBCT was 0.06 ± 0.07 s (range 0.01–0.22 s), corresponding to 3.1% ± 3.7% (range 0.6%–10.8%) of gate width (range 1.6–3.1 s). In only one patient, change in time lag exceeded 10% of the gate width. External prediction error over all scans of all patients varied from 0.1 ± 0.1 to 1.6 ± 0.4 mm. Conclusions: Time lag between internal motion along SI and external signals is small compared to the treatment gate width of abdominal patients examined in this study. Change in time lag within a treatment session, inferred from pre- to post-treatment measurements is also small, suggesting that a single measurement of time lag at the session start is adequate. These findings require confirmation in a larger number of patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Arm-in-Arm Response Regulator Dimers Promote Intermolecular Signal Transduction

ABSTRACT Bacteriophytochrome photoreceptors (BphPs) and their cognate response regulators make up two-component signal transduction systems which direct bacteria to mount phenotypic responses to changes in environmental light quality. Most of these systems utilize single-domain response regulators to transduce signals through unknown pathways and mechanisms. Here we describe the photocycle and autophosphorylation kinetics of RtBphP1, a red light-regulated histidine kinase from the desert bacterium Ramlibacter tataouinensis . RtBphP1 undergoes red to far-red photoconversion with rapid thermal reversion to the dark state. RtBphP1 is autophosphorylated in the dark; this activity is inhibited under red light. The RtBphP1 cognate response regulator, the R. tataouinensis bacteriophytochrome response regulator (RtBRR), and a homolog, AtBRR from Agrobacterium tumefaciens , crystallize unexpectedly as arm-in-arm dimers, reliant on a conserved hydrophobic motif, hFWAhL (where h is a hydrophobic M, V, L, or I residue). RtBRR and AtBRR dimerize distinctly from four structurally characterized phytochrome response regulators found in photosynthetic organisms and from all other receiver domain homodimers in the Protein Data Bank. A unique cacodylate-zinc-histidine tag metal organic framework yielded single-wavelength anomalous diffraction phases and may be of general interest. Examination of the effect of the BRR stoichiometry on signal transduction showed that phosphorylated RtBRR is accumulated more efficiently than the engineered monomeric RtBRR (RtBRR mon ) in phosphotransfer reactions. Thus, we conclude that arm-in-arm dimers are a relevant signaling intermediate in this class of two-component regulatory systems. IMPORTANCE BphP histidine kinases and their cognate response regulators comprise widespread red light-sensing two-component systems. Much work on BphPs has focused on structural understanding of light sensing and on enhancing the natural infrared fluorescence of these proteins, rather than on signal transduction or the resultant phenotypes. To begin to address this knowledge gap, we solved the crystal structures of two single-domain response regulators encoded by a region immediately downstream of that encoding BphPs. We observed a previously unknown arm-in-arm dimer linkage. Monomerization via deletion of the C-terminal dimerization motif had an inhibitory effect on net response regulator phosphorylation, underlining the importance of these unusual dimers for signal transduction.

Baker, Anna W.↗
DOE OSTI2023

A Mesorhizobium japonicum quorum sensing circuit that involves three linked genes and an unusual acyl-homoserine lactone signal

ABSTRACT Members of the genus Mesorhizobium, which are core components of the rhizosphere and specific symbionts of legume plants, possess genes for acyl-homoserine lactone (AHL) quorum sensing (QS). Here we show Mesorhizobium japonicum MAFF 303099 (formerly M. loti ) synthesizes and responds to N -[(2 E , 4 E )-2,4-dodecadienoyl] homoserine lactone (2 E, 4 E- C 12:2 -HSL). We show that the 2 E, 4 E- C 12:2 -HSL QS circuit involves one of four luxR-luxI -type genes found in the sequenced genome of MAFF 303099. We refer to this circuit, which appears to be conserved among Mesorhizobium species, as R1-I1. We show that two other Mesorhizobium strains also produce 2 E, 4 E- C 12:2 -HSL. The 2 E, 4 E- C 12:2 -HSL is unique among known AHLs in its arrangement of two trans double bonds. The R1 response to 2 E, 4 E- C 12:2 -HSL is extremely selective in comparison with other LuxR homologs, and the trans double bonds appear critical for R1 signal recognition. Most well-studied LuxI-like proteins use S -adenosylmethionine and an acyl-acyl carrier protein as substrates for synthesis of AHLs. Others that form a subgroup of LuxI-type proteins use acyl-coenzyme A substrates rather than acyl-acyl carrier proteins. I1 clusters with the acyl-coenzyme A-type AHL synthases. We show that a gene linked to the I1 AHL synthase is involved in the production of the QS signal. The discovery of the unique I1 product enforces the view that further study of acyl-coenzyme A-dependent LuxI homologs will expand our knowledge of AHL diversity. The involvement of an additional enzyme in AHL generation leads us to consider this system a three-component QS circuit. IMPORTANCE We report a Mesorhizobium japonicum quorum sensing (QS) system involving a novel acyl-homoserine lactone (AHL) signal. This system is known to be involved in root nodule symbiosis with host plants. The chemistry of the newly described QS signal indicated that there may be a dedicated cellular enzyme involved in its synthesis in addition to the types known for production of other AHLs. Indeed, we report that an additional gene is required for synthesis of the unique signal, and we propose that this is a three-component QS circuit as opposed to the canonical two-component AHL QS circuits. The signaling system is exquisitely selective. The selectivity may be important when this species resides in the complex microbial communities around host plants and may make this system useful in various synthetic biology applications of QS circuits.

59 BASIC BIOLOGICAL SCIENCES↗