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DOE OSTI2021

Sensor Domain of Histidine Kinase VxrA of Vibrio cholerae : Hairpin-Swapped Dimer and Its Conformational Change

VxrA and VxrB are cognate histidine kinase (HK)-response regulator (RR) pairs of a two-component signaling system (TCS) found in Vibrio cholerae, a bacterial pathogen that causes cholera. The VxrAB TCS positively regulates virulence, the type VI secretion system, biofilm formation, and cell wall homeostasis in V. cholerae, providing protection from environmental stresses and contributing to the transmission and virulence of the pathogen. The VxrA HK has a unique periplasmic sensor domain (SD) and, remarkably, lacks a cytoplasmic linker domain between the second transmembrane helix and the dimerization and histidine phosphotransfer (DHp) domain, indicating that this system may utilize a potentially unique signal sensing and transmission TCS mechanism. Here, we have determined several crystal structures of VxrA-SD and its mutants. These structures reveal a novel structural fold forming an unusual β hairpin-swapped dimer. A conformational change caused by relative rotation of the two monomers in a VxrA-SD dimer could potentially change the association of transmembrane helices and, subsequently, the pairing of cytoplasmic DHp domains. Based on the structural observation, we propose a putative scissor-like closing regulation mechanism for the VxrA HK. IMPORTANCE V. cholerae has a dynamic life cycle, which requires rapid adaptation to changing external conditions. Two-component signal transduction (TCS) systems allow V. cholerae to sense and respond to these environmental changes. The VxrAB TCS positively regulates a number of important V. cholerae phenotypes, including virulence, the type six secretion system, biofilm formation, and cell wall homeostasis. Here, we provide the crystal structures of the VxrA sensor histidine kinase sensing domain and propose a mechanism for signal transduction. The cognate signal for VxrAB remains unknown; however, in this work we couple our structural analysis with functional assessments of key residues to further our understanding of this important TCS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Selective Detection of Intermediate-Amplitude Motion by Solid-State NMR

The coexistence of rigid and mobile molecules or molecular segments abounds in biomolecular assemblies. Examples include the carbohydrate-rich cell walls of plants and intrinsically disordered proteins that contain rigid β-sheet cores. In solid-state nuclear magnetic resonance (NMR) spectroscopy, dipolar polarization transfer experiments are well suited for detecting rigid components, whereas scalar-coupling experiments are well suited for detecting highly mobile components. However, few NMR methods are available to detect the segments that undergo intermediate-amplitude fast motion. Here, we introduce two NMR experiments, a two-dimensional T 2H -filtered CP-hCH correlation and a three-dimensional J-INADEQUATE CCH correlation, to observe this intermediate-amplitude motion. Both experiments involve 1 H detection under fast magic-angle spinning (MAS). By combining 1 H transverse relaxation (T 2H ) filters with dipolar polarization transfer, we suppress the signals of both highly rigid and highly mobile species, thus revealing the signals of intermediate mobile species. 1 H detection under fast MAS is crucial for distinguishing the different motional amplitudes. Here, we demonstrate these techniques on several plant cell wall samples and show that they allow the selective detection and resolution of certain hemicellulose and pectin signals, which are usually masked by the signals of the rigid cellulose and the highly dynamic pectins in purely dipolar and scalar NMR spectra.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Engineering microalgal cell wall-anchored proteins using GP1 PPSPX motifs and releasing with intein-mediated fusion

AbstractHarnessing and controlling the localization of recombinant proteins is critical for advancing applications in synthetic biology, industrial biotechnology, and drug delivery. This study explores protein anchoring and controlled release inChlamydomonas reinhardtii, providing innovative tools for these fields. Using truncated variants of the GP1 glycoprotein fused to the plastic-degrading enzyme PHL7, we identified the PPSPX motif as essential for anchoring proteins to the cell wall. Constructs with increased PPSPX content exhibited reduced secretion but improved anchoring, pinpointing the potential anchor-signal sites of GP1 and highlighting the distinct roles of these motifs in protein localization. Building on the anchoring capabilities established with these glycomodules, we also demonstrated a controlled release system using a pH-sensitive intein derived from RecA fromMycobacterium tuberculosis. This intein efficiently cleaved and released PHL7 and mCherry that was fused to GP1 under acidic conditions, enabling precise temporal and environmental control. At pH 5.5, fluorescence kinetics demonstrated significant mCherry release from the pJPW4mCherry construct within 4 hours. In contrast, release was minimal under pH 8.0 conditions and negligible for the pJPW2mCherry (W2) control, irrespective of the pH. Additionally, bands on the Western blot at the expected size of mCherry also showed its efficient release from the mCherry::intein::GP1 fusion protein at pH 5.5. Conversely, at pH 8.0, no bands were detected. This anchor-release approach offers significant potential for drug delivery, biocatalysis, and environmental monitoring applications. By integrating glycomodules and pH-sensitive inteins, this study establishes a versatile framework for optimizing protein localization and release inC. reinhardtii, with broad implications for proteomics, biofilm engineering, and scalable therapeutic delivery systems.Graphical Abstract

Kang, Kalisa (ORCID:0009000619398129)↗
DOE OSTI2023

Deciphering the molecular components of the quorum sensing system in the fungus Ophiostoma piceae

Quorum sensing (QS) is a complex cell-cell communication mechanism that coordinates population-level behaviors in microbes. In eukaryotes, this phenomenon has been extensively described in the dimorphic yeast Candida albicans as its main QS molecule, the sesquiterpene alcohol farnesol, is responsible for various phenotypic (i.e., inhibition of yeast-to-hyphae transition, biofilm formation, and, hence, pathogenesis) and metabolic (i.e., induction of oxidative stress and apoptosis) changes. Ophiostoma piceae CECT 20416 is a dimorphic saprotrophic ascomycete with biotechnological interest that also produces farnesol as QS molecule, but in this case, the alcohol promotes the morphological transition to the mycelial form, biofilm formation, enzyme secretion, and melanin production. Here, we characterized the physiological response of Ophiostoma piceae to farnesol, the molecular components of the QS system of this fungus have been investigated using a “multiomics” approach that involved genomic, transcriptomic, and proteomic analyses. Some genes identified in this work are proposed as key factors in farnesol transport and signaling. We have also cataloged the genes undergoing major transcriptional changes triggered by the presence of the autoinducer, such as cell-wall remodeling, reactive oxygen species (ROS) protection, and melanin biosynthesis, using self-organizing maps. This analysis could be useful for applications in the forestry industry, for enzymes production, and for the valorization of residues. Furthermore, it might as well help to investigate the QS mechanisms of clinically relevant fungi phylogenetically related to Ophiostoma.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Formin nanoclustering-mediated actin assembly during plant flagellin and DSF signaling

Plants respond to bacterial infection acutely with actin remodeling during plant immune responses. The mechanisms by which bacterial virulence factors (VFs) modulate plant actin polymerization remain enigmatic. Here, we show that plant-type-I formin serves as the molecular sensor for actin remodeling in response to two bacterial VFs: Xanthomonas campestris pv. campestris (Xcc) diffusible signal factor (DSF), and pathogen-associated molecular pattern (PAMP) flagellin in pattern-triggered immunity (PTI). Both VFs regulate actin assembly by tuning the clustering and nucleation activity of formin on the plasma membrane (PM) at the nano-sized scale. By being integrated within the cell-wall-PM-actin cytoskeleton (CW-PM-AC) continuum, the dynamic behavior and function of formins are highly dependent on each scaffold layer’s composition within the CW-PM-AC continuum during both DSF and PTI signaling. Our results reveal a central mechanism for rapid actin remodeling during plant-bacteria interactions, in which bacterial signaling molecules fine tune plant formin nanoclustering in a host mechanical-structure-dependent manner.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Evolutionary history of arbuscular mycorrhizal fungi and genomic signatures of obligate symbiosis

The colonization of land and the diversification of terrestrial plants is intimately linked to the evolutionary history of their symbiotic fungal partners. Extant representatives of these fungal lineages include mutualistic plant symbionts, the arbuscular mycorrhizal (AM) fungi in Glomeromycota and fine root endophytes in Endogonales (Mucoromycota), as well as fungi with saprotrophic, pathogenic and endophytic lifestyles. These fungal groups separate into three monophyletic lineages but their evolutionary relationships remain enigmatic confounding ancestral reconstructions. Their taxonomic ranks are currently fluid. In this study, we recognize these three monophyletic linages as phyla, and use a balanced taxon sampling and broad taxonomic representation for phylogenomic analysis that rejects a hard polytomy and resolves Glomeromycota as sister to a clade composed of Mucoromycota and Mortierellomycota. Low copy numbers of genes associated with plant cell wall degradation could not be assigned to the transition to a plant symbiotic lifestyle but appears to be an ancestral phylogenetic signal. Both plant symbiotic lineages, Glomeromycota and Endogonales, lack numerous thiamine metabolism genes but the lack of fatty acid synthesis genes is specific to AM fungi. Many genes previously thought to be missing specifically in Glomeromycota are either missing in all analyzed phyla, or in some cases, are actually present in some of the analyzed AM fungal lineages, e.g. the high affinity phosphorus transporter Pho89. Based on a broad taxon sampling of fungal genomes we present a well-supported phylogeny for AM fungi and their sister lineages. We show that among these lineages, two independent evolutionary transitions to mutualistic plant symbiosis happened in a genomic background profoundly different from that known from the emergence of ectomycorrhizal fungi in Dikarya. These results call for further reevaluation of genomic signatures associated with plant symbiosis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis of broad-spectrum β-lactam resistance in Staphylococcus aureus

Abstract Broad-spectrum β-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden 1,2 . In clinical strains, resistance is largely controlled by BlaR1 3 , a receptor that senses β-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain 4 . The metalloprotease domain has a role in BlaI derepression, inducing blaZ (β-lactamase PC1) and mecA (β-lactam-resistant cell-wall transpeptidase PBP2a) expression 3–7 . Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously 8 . Cryo-electron microscopy structures of BlaR1—the wild type and an autocleavage-deficient F284A mutant, with or without β-lactam—reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from β-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action—in this case, antibiotic resistance—through the direct cleavage of a repressor.

Science & Technology - Other Topics↗
DOE OSTI2025

Label-free structural imaging of plant roots and microbes using third-harmonic generation microscopy

Root biology is pivotal in addressing global challenges including sustainable agriculture and climate change. However, roots have been relatively understudied among plant organs, partly due to the difficulties in imaging root structures in their natural environment. Here we used microfabricated ecosystems (EcoFABs) to establish growing environments with optical access and employed nonlinear multimodal microscopy of third-harmonic generation (THG) and three-photon fluorescence (3PF) to achieve label-free, in situ imaging of live roots and microbes at high spatiotemporal resolution. THG enabled us to observe key plant root structures including the vasculature, Casparian strips, dividing meristematic cells, and root cap cells, as well as subcellular features including nuclear envelopes, nucleoli, starch granules, and putative stress granules. THG from the cell walls of bacteria and fungi also provides label-free contrast for visualizing these microbes in the root rhizosphere. With simultaneously recorded 3PF signal, we demonstrated our ability to investigate root-microbe interactions by achieving single-bacterium tracking and subcellular imaging of fungal spores and hyphae in the rhizosphere.

Pan, Daisong [University of California, Berkeley, ↗
DOE OSTI2022

Editorial: Wood Development and Physiology in a Changing Climate

Wood of forest trees is a renewable, sustainable and easily workable material and has been widely used in construction, paper making, furniture, and as a feedstock for biofuels. Wood formation is the result of a highly ordered and finely regulated process. Secondary xylem (wood) and phloem are the inner and outer derivative products of the vascular cambium, respectively. Xylem is mainly composed of dead cells with thickened cell walls rich in cellulose, hemicelluloses, and lignin, which are responsible for providing mechanical support and conducting water and minerals for the trees. Phloem transports photoassimilates and signaling molecules, including phytohormones and peptides. Wood structure varies greatly between, and within, tree species. A thorough understanding of developmental processes and metabolic pathways during wood formation would provide a basis for improving wood biomass and modifying properties in forest trees.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis

Abstract Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2. In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Pectic-AGP is a major form of Arabidopsis AGPs

As a key component in cell walls of numerous organisms ranging from green algae to higher plants, AGPs play principal roles in many biological processes such as cell-cell adhesion and regulating Ca 2+ signaling pathway as a Ca 2+ -capacitor. Consistently, AGP structures vary from species to species and from tissue to tissue. To understand the functions of AGPs, it is vital to know their structural differences relative to their location in the plant. Thus, AGPs were purified from different Arabidopsis tissues. Analyses of these AGPs demonstrated that the AGPs comprised covalently linked pectin and AGP, referred to as pectic-AGPs. Importantly, these pectic-AGPs were glycosylated with a remarkable variety of polysaccharides including homogalacturonan, rhamnogalacturonan-I, and type II arabinogalactan at different ratios and lengths. Furthermore, this result not only suggests that pectic-AGP is a major form of Arabidopsis AGPs, but also supports AGPs serve as crosslinkers covalently connecting pectins with structures tailored for tissue-specific functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Pith–specific lignification in Nicotiana attenuata as a defense against a stem–boring herbivore

Plants have developed tissue-specific defense strategies in response to various herbivores with different feeding habits. Although defense responses to leaf-chewing insects have been well studied, little is known about stem-specific responses, particularly in the pith, to stem-boring herbivores. To understand the stem-specific defense, we first conducted a comparative transcriptomic analysis of the wild tobacco Nicotiana attenuata before and after attack by the leaf-chewing herbivore Manduca sexta and the stem borer Trichobaris mucorea. When the stem-boring herbivore attacked, lignin-associated genes were upregulated specifically in the inner parenchymal cells of the stem, the pith; lignin also accumulated highly in the attacked pith. Silencing the lignin biosynthetic gene cinnamyl alcohol dehydrogenase enhanced the performance of the stem-boring herbivore but had no effect on the growth of the leaf-chewing herbivore. Two-dimensional nuclear magnetic resonance results revealed that lignified pith contains feruloyltyramine as an unusual lignin component in the cell wall, as a response against stem-boring herbivore attack. Pith-specific lignification induced by the stem-boring herbivore was modulated by both jasmonate and ethylene signaling. Furthermore, these results suggest that lignin provides a stem-specific inducible barrier, protecting plants against stem-boring insects.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A systems genetic analysis identifies putative mechanisms and candidate genes regulating vessel traits in poplar wood

Wood is the water conducting tissue of tree stems. Like most angiosperm trees, poplar wood contains water-conducting vessel elements whose functional properties affect water transport and growth rates, as well as susceptibility to embolism and hydraulic failure during water stress and drought. Here we used a unique hybrid poplar pedigree carrying genomically characterized chromosomal insertions and deletions to undertake a systems genomics analysis of vessel traits. We assayed gene expression in wood forming tissues from clonal replicates of genotypes covering dosage quantitative trait loci with insertions and deletions, genotypes with extreme vessel trait phenotypes, and control genotypes. A gene co-expression analysis was used to assign genes to modules, which were then used in integrative analyses to identify modules associated with traits, to identify putative molecular and cellular processes associated with each module, and finally to identify candidate genes using multiple criteria including dosage responsiveness. These analyses identified known processes associated with vessel traits including stress response, abscisic acid and cell wall biosynthesis, and in addition identified previously unexplored processes including cell cycle and protein ubiquitination. We discuss our findings relative to component processes contributing to vessel trait variation including signaling, cell cycle, cell expansion, and cell differentiation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Lipopolysaccharide and lipoteichoic acid influence milk production ability via different early responses in bovine mammary epithelial cells

Highlights: • LPS and LTA affect on the milk production of lactating BMECs in different manners. • LPS/TLR4 and LTA/TLR2 signaling induce different innate immune responses in BMECs. • Inhibition of p38 and NF-κB may help recover decrease in milk production in mastitis. Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are cell wall components of Escherichia coli and Staphylococcus aureus, which cause clinical and subclinical mastitis, respectively. However, the reason of the difference in symptoms by pathogen type remains unclear. In this study, the influence of LPS and LTA on early response and milk production in lactating bovine mammary epithelial cells (BMECs) was comparatively investigated. The results showed that LPS decreased the secretion of β-casein, lactose, and triglycerides, whereas LTA decreased the secretion of lactose and triglycerides but increased lactoferrin production without any influence on β-casein secretion. In addition, the influence of milk lipid droplet size in BMECs and gene expression related to milk fat synthesis was different between LPS and LTA. LPS increased the gene expression of interleukin (IL)-1β, tumor necrosis factor-α, and IL-8 through the activation of the nuclear factor-κB (NF-κB), p38, and c-Jun N-terminal kinase pathways, whereas LTA increased IL-1β and CC chemokine ligand 5 expression through the activation of the NF-κB pathway. Moreover, these cytokines and chemokines differently affected the milk production ability of BMECs. These results suggested that the pathogen-specific symptoms may be related to the differences in the early response of BMECs to bacterial toxins.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Leakless end-to-end transport of small molecules through micron-length DNA nanochannels

Designed and engineered protein and DNA nanopores can be used to sense and characterize single molecules and control transmembrane transport of molecular species. However, designed biomolecular pores are less than 100 nm in length and are used primarily for transport across lipid membranes. Nanochannels that span longer distances could be used as conduits for molecules between nonadjacent compartments or cells. We design micrometer-long, 7-nm-diameter DNA nanochannels that small molecules can traverse according to the laws of continuum diffusion. Binding DNA origami caps to channel ends eliminates transport and demonstrates that molecules diffuse from one channel end to the other rather than permeating through channel walls. These micrometer-length nanochannels can also grow, form interconnects, and interface with living cells. This work thus shows how to construct multifunctional, dynamic agents that control molecular transport, opening ways of studying intercellular signaling and modulating molecular transport between synthetic and living cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Selective One-Dimensional 13 C– 13 C Spin-Diffusion Solid-State Nuclear Magnetic Resonance Methods to Probe Spatial Arrangements in Biopolymers Including Plant Cell Walls, Peptides, and Spider Silk

Two-dimensional (2D) and 3D through-space 13 C– 13 C homonuclear spin-diffusion techniques are powerful solid-state nuclear magnetic resonance (NMR) tools for extracting structural information from 13 C-enriched biomolecules, but necessarily long acquisition times restrict their applications. In this work, we explore the broad utility and underutilized power of a chemical shift-selective one-dimensional (1D) version of a 2D 13 C– 13 C spin-diffusion solid-state NMR technique. The method, which is called 1D dipolar-assisted rotational resonance (DARR) difference, is applied to a variety of biomaterials including lignocellulosic plant cell walls, microcrystalline peptide fMLF, and black widow dragline spider silk. 1D 13 C– 13 C spin-diffusion methods described here apply in select cases in which the 1D 13 C solid-state NMR spectrum displays chemical shift-resolved moieties. Furthermore, this is analogous to the selective 1D nuclear Overhauser effect spectroscopy (NOESY) experiment utilized in liquid-state NMR as a faster (1D instead of 2D) and often less ambiguous (direct sampling of the time domain data, coupled with increased signal averaging) alternative to 2D NOESY. Selective 1D 13 C– 13 C spin-diffusion methods are more time-efficient than their 2D counterparts such as proton-driven spin diffusion (PDSD) and dipolar-assisted rotational resonance. The additional time gained enables measurements of 13 C– 13 C spin-diffusion buildup curves and extraction of spin-diffusion time constants TSD, yielding detailed structural information. Specifically, selective 1D DARR difference buildup curves applied to 13 C-enriched hybrid poplar woody stems confirm strong spatial interaction between lignin and acetylated xylan polymers within poplar plant secondary cell walls, and an interpolymer distance of ~0.45–0.5 nm was estimated. Additionally, Tyr/Gly long-range correlations were observed on isotopically enriched black widow spider dragline silks.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗