Androgen and glucocorticoid profiles throughout extended uniparental paternal care in the eastern hellbender salamander (Cryptobranchus a. alleganiensis).

The behavioral endocrinology associated with reproduction and uniparental male care has been studied in teleosts, but little is known about hormonal correlates of uniparental male care in other ectotherms. To address this gap, we are the first to document the seasonal steroid endocrinology of uniparental male hellbender salamanders during the transition from pre-breeding to nest initiation, and through the subsequent eight months of paternal care. In doing so, we investigated the correlates of nest fate and clutch size, exploring hellbenders' alignment with several endocrinological patterns observed in uniparental male fish. Understanding the endocrinology of hellbender paternal care is also vital from a conservation perspective because high rates of nest failure were recently identified as a factor causing population declines in this imperiled species. We corroborated previous findings demonstrating testosterone and dihydrotestosterone (DHT) to be the primary androgens in hellbender reproduction, and that cortisol circulates as the most abundant glucocorticoid. However, we were unable to identify a prolactin or a "prolactin-like" peptide in circulation prior to or during parental care. We observed ∼ 80 % declines in both primary androgens during the transition from pre-breeding to nest initiation, and again as paternal care progressed past its first month. In the days immediately following nest initiation, testosterone and DHT trended higher in successful individuals, but did not differ with males' clutch size. We did not observe meaningful seasonality in baseline glucocorticoids associated with breeding or nesting. In contrast, stress-induced glucocorticoids were highest at pre-breeding and through the first two months of care, before declining during the latter-most periods of care as larvae approach emergence from the nest. Neither baseline nor stress-induced glucocorticoids varied significantly with either nest fate or clutch size. Both stress-induced cortisol and corticosterone were positively correlated with total length, a proxy for age in adult hellbenders. This is consistent with age-related patterns in some vertebrates, but the first such pattern observed in a wild amphibian population. Generally, we found that nesting hellbenders adhere to some but not all of the endocrinological patterns observed in uniparental male teleosts prior to and during parental care.

Identification of a new family of peptidoglycan transpeptidases reveals atypical crosslinking is essential for viability in Clostridioides difficile.

Clostridioides difficile, the leading cause of antibiotic-associated diarrhea, relies primarily on 3-3 crosslinks created by L,D-transpeptidases (LDTs) to fortify its peptidoglycan (PG) cell wall. This is unusual, as in most bacteria the vast majority of PG crosslinks are 4-3 crosslinks, which are created by penicillin-binding proteins (PBPs). Here we report the unprecedented observation that 3-3 crosslinking is essential for viability in C. difficile. We also report the discovery of a new family of LDTs that use a VanW domain to catalyze 3-3 crosslinking rather than a YkuD domain as in all previously known LDTs. Bioinformatic analyses indicate VanW domain LDTs are less common than YkuD domain LDTs and are largely restricted to Gram-positive bacteria. Our findings suggest that LDTs might be exploited as targets for antibiotics that kill C. difficile without disrupting the intestinal microbiota that is important for keeping C. difficile in check.

FurC (PerR) contributes to the regulation of peptidoglycan remodeling and intercellular molecular transfer in the cyanobacterium Anabaena sp. strain PCC 7120.

Microbial extracellular proteins and metabolites provide valuable information concerning how microbes adapt to changing environments. In cyanobacteria, dynamic acclimation strategies involve a variety of regulatory mechanisms, being ferric uptake regulator proteins as key players in this process. In the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120, FurC (PerR) is a global regulator that modulates the peroxide response and several genes involved in photosynthesis and nitrogen metabolism. To investigate the possible role of FurC in shaping the extracellular environment of Anabaena, the analysis of the extracellular metabolites and proteins of a furC-overexpressing variant was compared to that of the wild-type strain. There were 96 differentially abundant proteins, 78 of which were found for the first time in the extracellular fraction of Anabaena. While these proteins belong to different functional categories, most of them are predicted to be secreted or have a peripheral location. Several stress-related proteins, including PrxA, flavodoxin, and the Dps homolog All1173, accumulated in the exoproteome of furC-overexpressing cells, while decreased levels of FurA and a subset of membrane proteins, including several export proteins and amiC gene products, responsible for nanopore formation, were detected. Direct repression by FurC of some of those genes, including amiC1 and amiC2, could account for odd septal nanopore formation and impaired intercellular molecular transfer observed in the furC-overexpressing variant. Assessment of the exometabolome from both strains revealed the release of two peptidoglycan fragments in furC-overexpressing cells, namely 1,6-anhydro-N-acetyl-ß-D-muramic acid (anhydroMurNAc) and its associated disaccharide (ß-D-GlcNAc-(1-4)-anhydroMurNAc), suggesting alterations in peptidoglycan breakdown and recycling.IMPORTANCECyanobacteria are ubiquitous photosynthetic prokaryotes that can adapt to environmental stresses by modulating their extracellular contents. Measurements of the organization and composition of the extracellular milieu provide useful information about cyanobacterial adaptive processes, which can potentially lead to biomimetic approaches to stabilizing biological systems to adverse conditions. Anabaena sp. strain PCC 7120 is a multicellular, nitrogen-fixing cyanobacterium whose intercellular molecular exchange is mediated by septal junctions that traverse the septal peptidoglycan through nanopores. FurC (PerR) is an essential transcriptional regulator in Anabaena, which modulates the response to several stresses. Here, we show that furC-overexpressing cells result in a modified exoproteome and the release of peptidoglycan fragments. Phenotypically, important alterations in nanopore formation and cell-to-cell communication were observed. Our results expand the roles of FurC to the modulation of cell-wall biogenesis and recycling, as well as in intercellular molecular transfer.

Sex-differences in proteasome-dependent K48-polyubiquitin signaling in the amygdala are developmentally regulated in rats.

BACKGROUND: Sex differences have been observed in several brain regions for the molecular mechanisms involved in baseline (resting) and memory-related processes. The ubiquitin proteasome system (UPS) is a major protein degradation pathway in cells. Sex differences have been observed in lysine-48 (K48)-polyubiquitination, the canonical degradation mark of the UPS, both at baseline and during fear memory formation within the amygdala. Here, we investigated when, how, and why these baseline sex differences arise and whether both sexes require the K48-polyubiquitin mark for memory formation in the amygdala. METHODS: We used a combination of molecular, biochemical and proteomic approaches to examine global and protein-specific K48-polyubiquitination and DNA methylation levels at a major ubiquitin coding gene (Uba52) at baseline in the amygdala of male and female rats before and after puberty to determine if sex differences were developmentally regulated. We then used behavioral and genetic approaches to test the necessity of K48-polyubiquitination in the amygdala for fear memory formation. RESULTS: We observed developmentally regulated baseline differences in Uba52 methylation and total K48-polyubiquitination, with sexual maturity altering levels specifically in female rats. K48-polyubiquitination at specific proteins changed across development in both male and female rats, but sex differences were present regardless of age. Lastly, we found that genetic inhibition of K48-polyubiquitination in the amygdala of female, but not male, rats impaired fear memory formation. CONCLUSIONS: These results suggest that K48-polyubiquitination differentially targets proteins in the amygdala in a sex-specific manner regardless of age. However, sexual maturity is important in the developmental regulation of K48-polyubiquitination levels in female rats. Consistent with these data, K48-polyubiquitin signaling in the amygdala is selectively required to form fear memories in female rats. Together, these data indicate that sex-differences in baseline K48-polyubiquitination within the amygdala are developmentally regulated, which could have important implications for better understanding sex-differences in molecular mechanisms involved in processes relevant to anxiety-related disorders such as post-traumatic stress disorder (PTSD).

Male and female brains have differences in size, development, and cellular processes. Further, males and females have differences in likelihood of developing certain anxiety-related disorders, such as post-traumatic stress disorder (PTSD). We previously observed sex differences in a cellular mechanism that controls the destruction of proteins via tagging by the protein modifier ubiquitin in resting and behaviorally trained animals. We found that adult female rats "ubiquitinated" different proteins during learning and had more ubiquitin than male rats at rest in the amygdala, the brain region that controls emotional regulation. This study investigated if the sex difference in ubiquitin at rest changed as animals age, including the proteins being ubiquitinated and how the amount of ubiquitin was controlled. We also investigated if male and female rats need ubiquitin for memory formation. We found that males and females ubiquitinate different proteins, but that aging also contributes to changes in this, suggesting that sexual maturity may be important for controlling the amount of ubiquitin in females. Lastly, we found that only female rats needed ubiquitin in the amygdala for forming a fear memory. These results are important for understanding the role of ubiquitin activity at different developmental stages and for forming fear-based memories in both sexes. Since females are more likely to develop PTSD than males, these data could help understand how different cellular processes work together in PTSD development to create better treatment options.

Toward Automatic Inference of Glycan Linkages Using MSn and Machine Learning─Proof of Concept Using Sialic Acid Linkages.

Glycosidic linkages in oligosaccharides play essential roles in determining their chemical properties and biological activities. MSn has been widely used to infer glycosidic linkages but requires a substantial amount of starting material, which limits its application. In addition, there is a lack of rigorous research on what MSn protocols are proper for characterizing glycosidic linkages. In this work, to deliver high-quality experimental data and analysis results, we propose a machine learning-based framework to establish appropriate MSn protocols and build effective data analysis methods. We demonstrate the proof-of-principle by applying our approach to elucidate sialic acid linkages (α2'-3' and α2'-6') in a set of sialyllactose standards and NIST sialic acid-containing N-glycans as well as identify several protocol configurations for producing high-quality experimental data. Our companion data analysis method achieves nearly 100% accuracy in classifying α2'-3' vs α2'-6' using MS5, MS4, MS3, or even MS2 spectra alone. The ability to determine glycosidic linkages using MS2 or MS3 is significant as it requires substantially less sample, enabling linkage analysis for quantity-limited natural glycans and synthesized materials, as well as shortens the overall experimental time. MS2 is also more amenable than MS3/4/5 to automation when coupled to direct infusion or LC-MS. Additionally, our method can predict the ratio of α2'-3' and α2'-6' in a mixture with 8.6% RMSE (root-mean-square error) across data sets using MS5 spectra. We anticipate that our framework will be generally applicable to analysis of other glycosidic linkages.

Proteasome-independent K63 polyubiquitination selectively regulates ATP levels and proteasome activity during fear memory formation in the female amygdala.

Females are more likely than males to develop post-traumatic stress disorder (PTSD). However, the neurobiological mechanisms responsible for these sex differences remain elusive. The ubiquitin proteasome system (UPS) is involved in fear memory formation and implicated in PTSD development. Despite this, proteasome-independent functions of the UPS have rarely been studied in the brain. Here, using a combination of molecular, biochemical, proteomic, behavioral, and novel genetic approaches, we investigated the role of proteasome-independent lysine-63 (K63)-polyubiquitination, the second most abundant ubiquitin modification in cells, in the amygdala during fear memory formation in male and female rats. Only females had increased levels of K63-polyubiquitination targeting in the amygdala following fear conditioning, which targeted proteins involved in ATP synthesis and proteasome function. CRISPR-dCas13b-mediated knockdown of K63-polyubiquitination in the amygdala via editing of the K63 codon in the major ubiquitin gene, Ubc, impaired fear memory in females, but not males, and caused a reduction in learning-related increases in ATP levels and proteasome activity in the female amygdala. These results suggest that proteasome-independent K63-polyubiquitination is selectively involved in fear memory formation in the female amygdala, where it is involved in the regulation of ATP synthesis and proteasome activity following learning. This indicates the first link between proteasome-independent and proteasome-dependent UPS functions in the brain during fear memory formation. Importantly, these data are congruent with reported sex differences in PTSD development and may contribute to our understanding of why females are more likely to develop PTSD than males.

The Chlamydia trachomatis p-aminobenzoate synthase CADD is a manganese-dependent oxygenase that uses its own amino acid residues as substrates.

CADD (chlamydia protein associating with death domains) is a p-aminobenzoate (pAB) synthase involved in a noncanonical route for tetrahydrofolate biosynthesis in Chlamydia trachomatis. Although previously implicated to employ a diiron cofactor, here, we show that pAB synthesis by CADD requires manganese and the physiological cofactor is most likely a heterodinuclear Mn/Fe cluster. Isotope-labeling experiments revealed that the two oxygen atoms in the carboxylic acid portion of pAB are derived from molecular oxygen. Further, mass spectrometry-based proteomic analyses of CADD-derived peptides demonstrated a glycine substitution at Tyr27, providing strong evidence that this residue is sacrificed for pAB synthesis. Additionally, Lys152 was deaminated and oxidized to aminoadipic acid, supporting its proposed role as a sacrificial amino group donor.

Selective Detection of Misfolded Tau From Postmortem Alzheimer's Disease Brains.

Tau aggregates are present in multiple neurodegenerative diseases known as "tauopathies," including Alzheimer's disease, Pick's disease, progressive supranuclear palsy, and corticobasal degeneration. Such misfolded tau aggregates are therefore potential sources for selective detection and biomarker discovery. Six human tau isoforms present in brain tissues and both 3R and 4R isoforms have been observed in the neuronal inclusions. To develop selective markers for AD and related rare tauopathies, we first used an engineered tau protein fragment 4RCF as the substrate for ultrasensitive real-time quaking-induced conversion analyses (RT-QuIC). We showed that misfolded tau from diseased AD and other tauopathy brains were able to seed recombinant 4RCF substrate. We further expanded to use six individual recombinant tau isoforms as substrates to amplify misfolded tau seeds from AD brains. We demonstrated, for the first time to our knowledge, that misfolded tau from the postmortem AD brain tissues was able to specifically seed all six full-length human tau isoforms. Our results demonstrated that RT-QuIC analysis can discriminate AD and other tauopathies from non-AD normal controls. We further uncovered that 3R-tau isoforms displayed significantly faster aggregation kinetics than their 4R-tau counterparts under conditions of both no seeding and seeding with AD brain homogenates. In summary, our work offers potential new avenues of misfolded tau detection as potential biomarkers for diagnosis of AD and related tauopathies and provides new insights into isoform-specific human tau aggregation.

Catechol-containing compounds are a broad class of protein aggregation inhibitors: Redox state is a key determinant of the inhibitory activities.

A range of neurodegenerative and related aging diseases, such as Alzheimer's disease and type 2 diabetes, are linked to toxic protein aggregation. Yet the mechanisms of protein aggregation inhibition by small molecule inhibitors remain poorly understood, in part because most protein targets of aggregation assembly are partially unfolded or intrinsically disordered, which hinders detailed structural characterization of protein-inhibitor complexes and structural-based inhibitor design. Herein we employed a parallel small molecule library-screening approach to identify inhibitors against three prototype amyloidogenic proteins in neurodegeneration and related proteinopathies: amylin, Aß and tau. One remarkable class of inhibitors identified from these screens against different amyloidogenic proteins was catechol-containing compounds and redox-related quinones/anthraquinones. Secondary assays validated most of the identified inhibitors. In vivo efficacy evaluation of a selected catechol-containing compound, rosmarinic acid, demonstrated its strong mitigating effects of amylin amyloid deposition and related diabetic pathology in transgenic HIP rats. Further systematic investigation of selected class of inhibitors under aerobic and anaerobic conditions revealed that the redox state of the broad class of catechol-containing compounds is a key determinant of the amyloid inhibitor activities. The molecular insights we gained not only explain why a large number of catechol-containing polyphenolic natural compounds, often enriched in healthy diet, have anti-neurodegeneration and anti-aging activities, but also could guide the rational design of therapeutic or nutraceutical strategies to target a broad range of neurodegenerative and related aging diseases.

Protein SUMOylation is a sex-specific regulator of fear memory formation in the amygdala.

Strong evidence has implicated ubiquitin signaling in the process of fear memory formation. While less abundant than ubiquitination, evidence suggests that protein SUMOylation may also be involved in fear memory formation in neurons. However, the importance of amygdala protein SUMOylation in fear memory formation has never been directly examined. Furthermore, while recent evidence indicates that males and females differ significantly in the requirement for ubiquitin signaling during fear memory formation, whether sex differences also exist in the importance of protein SUMOylation to this process remains unknown. Here we found that males and females differ in the requirement for protein SUMOylation in the amygdala during fear memory formation. Western blot analysis revealed that while females had higher resting levels of SUMOylation, both sexes showed global increases following fear conditioning. However, SUMOylation-specific proteomic analysis revealed that only females have increased targeting of individual proteins by SUMOylation following fear conditioning, some of which were heat shock proteins. This suggests that protein SUMOylation is more robustly engaged in the amygdala of females following fear conditioning. In vivo siRNA mediated knockdown of Ube2i, the coding gene for the essential E2 ligase for SUMOylation conjugation, in the amygdala impaired fear memory in males without any effect in females. Importantly, higher siRNA concentrations than what was needed to impair memory in males reduced Ube2i levels in the amygdala of females but resulted in an increase in SUMOylation levels, suggesting a compensatory effect in females that was not observed in males. Collectively, these data reveal a novel, sex-specific role for protein SUMOylation in the amygdala during fear memory formation and expand our understanding of how ubiquitin-like signaling regulates memory formation.

The unusual cell wall of the Lyme disease spirochaete Borrelia burgdorferi is shaped by a tick sugar.

Peptidoglycan-a mesh sac of glycans that are linked by peptides-is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N-acetylglucosamine (GlcNAc) ß-(1-4)-linked to N-acetylmuramic acid (MurNAc). Borrelia burgdorferi, the tick-borne Lyme disease pathogen, produces glycan chains in which MurNAc is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography-mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain GlcNAc-GlcNAc. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility.

Proteomic Analysis Reveals Sex-Specific Protein Degradation Targets in the Amygdala During Fear Memory Formation.

Ubiquitin-proteasome mediated protein degradation has been widely implicated in fear memory formation in the amygdala. However, to date, the protein targets of the proteasome remain largely unknown, limiting our understanding of the functional significance for protein degradation in fear memory formation. Additionally, whether similar proteins are targeted by the proteasome between sexes has yet to be explored. Here, we combined a degradation-specific K48 Tandem Ubiquitin Binding Entity (TUBE) with liquid chromatography mass spectrometry (LC/MS) to identify the target substrates of the protein degradation process in the amygdala of male and female rats following contextual fear conditioning. We found that males (43) and females (77) differed in the total number of proteins that had significant changes in K48 polyubiquitin targeting in the amygdala following fear conditioning. Many of the identified proteins (106) had significantly reduced levels in the K48-purified samples 1 h after fear conditioning, suggesting active degradation of the substrate due to learning. Interestingly, only 3 proteins overlapped between sexes, suggesting that targets of the protein degradation process may be sex-specific. In females, many proteins with altered abundance in the K48-purified samples were involved in vesicle transport or are associated with microtubules. Conversely, in males, proteins involved in the cytoskeleton, ATP synthesis and cell signaling were found to have significantly altered abundance. Only 1 protein had an opposite directional change in abundance between sexes, LENG1, which was significantly enhanced in males while lower in females. This suggests a more rapid degradation of this protein in females during fear memory formation. Interestingly, GFAP, a critical component of astrocyte structure, was a target of K48 polyubiquitination in both males and females, indicating that protein degradation is likely occurring in astrocytes following fear conditioning. Western blot assays revealed reduced levels of these target substrates following fear conditioning in both sexes, confirming that the K48 polyubiquitin was targeting these proteins for degradation. Collectively, this study provides strong evidence that sex differences exist in the protein targets of the degradation process in the amygdala following fear conditioning and critical information regarding how ubiquitin-proteasome mediated protein degradation may contribute to fear memory formation in the brain.

Pre-breeding androgen and glucocorticoid profiles in the eastern hellbender salamander (Cryptobranchus alleganiensis alleganiensis).

Seasonally breeding species exhibit cyclical changes in circulating steroid hormone profiles that correspond with changes to their reproductive behavior and ecology. Such information is critical to the conservation of imperiled and data-deficient species, such as the eastern hellbender salamander (Cryptobranchus alleganiensis alleganiensis). We determined changes in plasma testosterone (T), dihydrotestosterone (DHT), 11-ketotestosterone (11-KT), 11-ketoandrostenedione (11-KA), dehydroepiandrosterone (DHEA), cortisol, corticosterone, and progesterone (P4) during a four-month period preceding breeding in adult male and female eastern hellbenders. This pre-breeding period is characterized by increased diel movement and aggression by both sexes, follicular development and yolk production in females, and sperm production, territoriality, and nest site establishment in males. In both males and females, we observed a progressive increase in circulating T and DHT during the pre-reproductive season, both peaking in August (17 days before breeding), but concentrations of both hormones were higher in males. Conversely, 11-KT was higher in females, but did not vary significantly by date. These results suggest that T and DHT are the predominant androgens in eastern hellbenders and are likely important regulators of reproductive processes in both males and females. The detection of significant quantities of DHT and 11-KT in females is particularly interesting, considering that unlike T, neither of these androgens can be converted to estrogens. Therefore, it seems possible that aggression or some aspect of reproduction in the female eastern hellbender may be directly mediated by androgen signaling. Baseline cortisol did not vary throughout the pre-breeding period but was higher in females than males, and also became highly variable in females leading up to breeding. Progesterone, 11-KA, DHEA, and corticosterone were rarely or never detected, and thus, do not appear to be important during the pre-reproductive season. This study provides a physiological framework for future studies of hellbender reproductive biology, which could ultimately be important for their conservation.

Comparative Metabolomics of Fruits and Leaves in a Hyperdiverse Lineage Suggests Fruits Are a Key Incubator of Phytochemical Diversification.

Interactions between plants and leaf herbivores have long been implicated as the major driver of plant secondary metabolite diversity. However, other plant-animal interactions, such as those between fruits and frugivores, may also be involved in phytochemical diversification. Using 12 species of Piper, we conducted untargeted metabolomics and molecular networking with extracts of fruits and leaves. We evaluated organ-specific secondary metabolite composition and compared multiple dimensions of phytochemical diversity across organs, including richness, structural complexity, and variability across samples at multiple scales within and across species. Plant organ identity, species identity, and the interaction between the two all significantly influenced secondary metabolite composition. Leaves and fruit shared a majority of compounds, but fruits contained more unique compounds and had higher total estimated chemical richness. While the relative levels of chemical richness and structural complexity across organs varied substantially across species, fruit diversity exceeded leaf diversity in more species than the reverse. Furthermore, the variance in chemical composition across samples was higher for fruits than leaves. By documenting a broad pattern of high phytochemical diversity in fruits relative to leaves, this study lays groundwork for incorporating fruit into a comprehensive and integrative understanding of the ecological and evolutionary factors shaping secondary metabolite composition at the whole-plant level.

Females, but not males, require protein degradation in the hippocampus for contextual fear memory formation.

Strong evidence supports a role for protein degradation in fear memory formation. However, these data have been largely done in only male animals. Here, we found that following contextual fear conditioning, females, but not males, had increased levels of proteasome activity and K48 polyubiquitin protein targeting in the dorsal hippocampus, the latter of which occurred at chaperones or RNA processing proteins. In vivo CRISPR-dCas9-mediated repression of protein degradation in the dorsal hippocampus impaired contextual fear memory in females, but not males. These results suggest a sex-specific role for protein degradation in the hippocampus during the consolidation of a contextual fear memory.

Sex-Specific Linear Polyubiquitination Is a Critical Regulator of Contextual Fear Memory Formation.

Strong evidence supports that protein ubiquitination is a critical regulator of fear memory formation. However, as this work has focused on protein degradation, it is currently unknown whether polyubiquitin modifications that are independent of the proteasome are involved in learning-dependent synaptic plasticity. Here, we present the first evidence that atypical linear (M1) polyubiquitination, the only ubiquitin chain that does not occur at a lysine site and is largely independent of the proteasome, is critically involved in contextual fear memory formation in the amygdala in a sex-specific manner. Using immunoblot and unbiased proteomic analyses, we found that male (49) and female (14) rats both had increased levels of linear polyubiquitinated substrates following fear conditioning, though none of these protein targets overlapped between sexes. In males, target protein functions involved cell junction and axonal guidance signaling, while in females the primary target was Adiponectin A, a critical regulator of neuroinflammation, synaptic plasticity, and memory, suggesting sex-dependent functional roles for linear polyubiquitination during fear memory formation. Consistent with these increases, in vivo siRNA-mediated knockdown of Rnf31, an essential component of the linear polyubiquitin E3 complex LUBAC, in the amygdala impaired contextual fear memory in both sexes without affecting memory retrieval. Collectively, these results provide the first evidence that proteasome-independent linear polyubiquitination is a critical regulator of fear memory formation, expanding the potential roles of ubiquitin-signaling in learning-dependent synaptic plasticity. Importantly, our data identify a novel sex difference in the functional role of, but not a requirement for, linear polyubiquitination in fear memory formation.

McpT, a Broad-Range Carboxylate Chemoreceptor in Sinorhizobium meliloti.

Chemoreceptors enable the legume symbiont Sinorhizobium meliloti to detect and respond to specific chemicals released from their host plant alfalfa, which allows the establishment of a nitrogen-fixing symbiosis. The periplasmic region (PR) of transmembrane chemoreceptors act as the sensory input module for chemotaxis systems via binding of specific ligands, either directly or indirectly. S. meliloti has six transmembrane and two cytosolic chemoreceptors. However, the function of only three of the transmembrane receptors have been characterized so far, with McpU, McpV, and McpX serving as general amino acid, short-chain carboxylate, and quaternary ammonium compound sensors, respectively. In the present study, we analyzed the S. meliloti chemoreceptor McpT. High-throughput differential scanning fluorimetry assays, using Biolog phenotype microarray plates, identified 15 potential ligands for McpTPR, with the majority classified as mono-, di-, and tricarboxylates. S. meliloti exhibited positive chemotaxis toward seven selected carboxylates, namely, α-ketobutyrate, citrate, glyoxylate, malate, malonate, oxalate, and succinate. These carboxylates were detected in seed exudates of the alfalfa host. Deletion of mcpT resulted in a significant decrease of chemotaxis to all carboxylates except for citrate. Isothermal titration calorimetry revealed that McpTPR bound preferentially to the monocarboxylate glyoxylate and with lower affinity to the dicarboxylates malate, malonate, and oxalate. However, no direct binding was detected for the remaining three carboxylates that elicited an McpT-dependent chemotaxis response. Taken together, these results demonstrate that McpT is a broad-range carboxylate chemoreceptor that mediates chemotactic response via direct ligand binding and an indirect mechanism that needs to be identified. IMPORTANCE Nitrate pollution is one of the most widespread and challenging environmental problems that is mainly caused by the agricultural overapplication of nitrogen fertilizers. Biological nitrogen fixation by the endosymbiont Sinorhizobium meliloti enhances the growth of its host Medicago sativa (alfalfa), which also efficiently supplies the soil with nitrogen. Establishment of the S. meliloti-alfalfa symbiosis relies on the early exchange and recognition of chemical signals. The present study contributes to the disclosure of this complex molecular dialogue by investigating the underlying mechanisms of carboxylate sensing in S. meliloti. Understanding individual steps that govern the S. meliloti-alfalfa molecular cross talk helps in the development of efficient, commercial bacterial inoculants that promote the growth of alfalfa, which is the most cultivated forage legume in the world, and improves soil fertility.

An Updated Perspective on Sinorhizobium meliloti Chemotaxis to Alfalfa Flavonoids.

The symbiotic interaction between leguminous plants and their cognate rhizobia allows for the fixation of gaseous dinitrogen into bioavailable ammonia. The perception of host-derived flavonoids is a key initial step for the signaling events that must occur preceding the formation of the nitrogen-fixing organ. Past work investigating chemotaxis - the directed movement of bacteria through chemical gradients - of Bradyrhizobium japonicum, Rhizobium leguminosarum, and Rhizobium meliloti discovered chemotaxis to various organic compounds, but focused on chemotaxis to flavonoids because of their relevance to the symbiosis biochemistry. The current work sought to replicate and further examine Sinorhizobium (Ensifer) meliloti chemotaxis to the flavonoids previously thought to act as the principal attractant molecules prior to the initial signaling stage. Exudate from germinating alfalfa seedlings was analyzed for composition and quantities of different flavonoid compounds using mass spectrometry. The abundance of four prevalent flavonoids in germinating alfalfa seed exudates (SEs) was at a ratio of 200:5:5:1 for hyperoside, luteolin, luteolin-7-glucoside, and chrysoeriol. Using quantitative chemotaxis capillary assays, we did not detect chemotaxis of motile S. meliloti cells to these, and two other flavonoids identified in seed exudates. In support of these findings, the flavonoid fraction of seed exudates was found to be an insignificant attractant relative to the more hydrophilic fraction. Additionally, we observed that cosolvents commonly used to dissolve flavonoids confound the results. We propose that the role flavonoids play in S. meliloti chemotaxis is insignificant relative to other components released by alfalfa seeds.

Cellular Stoichiometry of Chemotaxis Proteins in Sinorhizobium meliloti.

Chemotaxis systems enable microbes to sense their immediate environment, moving toward beneficial stimuli and away from those that are harmful. In an effort to better understand the chemotaxis system of Sinorhizobium meliloti, a symbiont of the legume alfalfa, the cellular stoichiometries of all ten chemotaxis proteins in S. meliloti were determined. A combination of quantitative immunoblot and mass spectrometry revealed that the protein stoichiometries in S. meliloti varied greatly from those in Escherichia coli and Bacillus subtilis To compare protein ratios to other systems, values were normalized to the central kinase CheA. All S. meliloti chemotaxis proteins exhibited increased ratios to various degrees. The 10-fold higher molar ratio of adaptor proteins CheW1 and CheW2 to CheA might result in the formation of rings in the chemotaxis array that consist of only CheW instead of CheA and CheW in a 1:1 ratio. We hypothesize that the higher ratio of CheA to the main response regulator CheY2 is a consequence of the speed-variable motor in S. meliloti, instead of a switch-type motor. Similarly, proteins involved in signal termination are far more abundant in S. meliloti, which utilizes a phosphate sink mechanism based on CheA retrophosphorylation to inactivate the motor response regulator versus CheZ-catalyzed dephosphorylation as in E. coli and B. subtilis Finally, the abundance of CheB and CheR, which regulate chemoreceptor methylation, was increased compared to CheA, indicative of variations in the adaptation system of S. meliloti Collectively, these results mark significant differences in the composition of bacterial chemotaxis systems.IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti contributes greatly to host-plant growth by fixing atmospheric nitrogen. The provision of nitrogen as ammonium by S. meliloti leads to increased biomass production of its legume host alfalfa and diminishes the use of environmentally harmful chemical fertilizers. To better understand the role of chemotaxis in host-microbe interaction, a comprehensive catalogue of the bacterial chemotaxis system is vital, including its composition, function, and regulation. The stoichiometry of chemotaxis proteins in S. meliloti has very few similarities to the systems in Escherichia coli and Bacillus subtilis In addition, total amounts of proteins are significantly lower. S. meliloti exhibits a chemotaxis system distinct from known models by incorporating new proteins as exemplified by the phosphate sink mechanism.

The dynamic response of the Arabidopsis root metabolome to auxin and ethylene is not predicted by changes in the transcriptome.

While the effects of phytohormones on plant gene expression have been well characterized, comparatively little is known about how hormones influence metabolite profiles. This study examined the effects of elevated auxin and ethylene on the metabolome of Arabidopsis roots using a high-resolution 24 h time course, conducted in parallel to time-matched transcriptomic analyses. Mass spectrometry using orthogonal UPLC separation strategies (reversed phase and HILIC) in both positive and negative ionization modes was used to maximize identification of metabolites with altered levels. The findings show that the root metabolome responds rapidly to hormone stimulus and that compounds belonging to the same class of metabolites exhibit similar changes. The responses were dominated by changes in phenylpropanoid, glucosinolate, and fatty acid metabolism, although the nature and timing of the response was unique for each hormone. These alterations in the metabolome were not directly predicted by the corresponding transcriptome data, suggesting that post-transcriptional events such as changes in enzyme activity and/or transport processes drove the observed changes in the metabolome. These findings underscore the need to better understand the biochemical mechanisms underlying the temporal reconfiguration of plant metabolism, especially in relation to the hormone-metabolome interface and its subsequent physiological and morphological effects.