RESUMEN
Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.
Asunto(s)
Proteínas Bacterianas , Pared Celular , Clostridioides difficile , Peptidoglicano , Clostridioides difficile/enzimología , Clostridioides difficile/metabolismo , Peptidoglicano/metabolismo , Pared Celular/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Peptidoglicano Glicosiltransferasa/metabolismo , Peptidoglicano Glicosiltransferasa/química , Peptidoglicano Glicosiltransferasa/genéticaRESUMEN
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.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Proteómica , Femenino , Masculino , Ratas , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Amígdala del Cerebelo/metabolismo , Ubiquitina/metabolismo , Trastornos de la Memoria/metabolismo , Miedo/fisiología , Adenosina Trifosfato/metabolismoRESUMEN
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 â¼ 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.
Asunto(s)
Andrógenos , Glucocorticoides , Conducta Paterna , Urodelos , Animales , Masculino , Andrógenos/metabolismo , Andrógenos/sangre , Glucocorticoides/metabolismo , Urodelos/metabolismo , Urodelos/fisiología , Conducta Paterna/fisiología , Testosterona/metabolismo , Testosterona/sangre , Comportamiento de Nidificación/fisiología , Reproducción/fisiología , Estaciones del AñoRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Diabetes Mellitus Tipo 2 , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Proteínas Amiloidogénicas/metabolismo , Animales , Antraquinonas , Catecoles/farmacología , Catecoles/uso terapéutico , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/uso terapéutico , Oxidación-Reducción , Agregado de Proteínas , Quinonas , RatasRESUMEN
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.
Asunto(s)
Miedo , Hipocampo , Animales , Femenino , Masculino , ProteolisisRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Factores Quimiotácticos/metabolismo , Sinorhizobium meliloti/metabolismo , Proteínas Bacterianas/genética , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Factores Quimiotácticos/genética , Quimiotaxis , Eliminación de Gen , Glioxilatos/metabolismo , Ligandos , Sinorhizobium meliloti/genéticaRESUMEN
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.
Asunto(s)
Glucocorticoides , Urodelos , Andrógenos , Animales , Corticosterona , Femenino , Hidrocortisona , Masculino , Testosterona , Urodelos/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sinorhizobium meliloti/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Quimiotaxis , Transducción de Señal , Sinorhizobium meliloti/química , Sinorhizobium meliloti/genéticaRESUMEN
Corticosterone is widely regarded to be the predominant glucocorticoid produced in amphibians. However, we recently described unusually low baseline and stress-induced corticosterone profiles in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis), a giant, fully aquatic salamander. Here, we hypothesized that hellbenders might also produce cortisol, the predominant glucocorticoid used by fishes and non-rodent mammals. To test our hypothesis, we collected plasma samples in two field experiments and analyzed them using multiple analytical techniques to determine how plasma concentrations of cortisol and corticosterone co-varied after 1) physical restraint and 2) injection with adrenocorticotropic hormone (ACTH), the pituitary hormone responsible for triggering the release of glucocorticoids from amphibian interrenal glands. Using liquid chromatography-mass spectrometry, we found that baseline and restraint-induced plasma concentrations of cortisol were more than five times those of corticosterone. We then demonstrated that plasma concentrations of both glucocorticoids increased in response to ACTH in a dose-dependent manner, but cortisol concentrations were consistently higher (up to 10-fold) than corticosterone. Cortisol and corticosterone concentrations were not correlated with one another at basal or induced conditions. The extremely low plasma concentrations of corticosterone in hellbenders suggests that corticosterone could simply be a byproduct of cortisol production, and raises questions as to whether corticosterone has any distinct physiological function in hellbenders. Our results indicate that hellbenders produce cortisol as their predominant glucocorticoid, supporting a small and inconclusive body of literature indicating that some other amphibians may produce appreciable quantities of cortisol. We hypothesize that the use of cortisol by hellbenders could be an adaptation to their fully aquatic life history due to cortisol's ability to fulfill both mineralocorticoid and glucocorticoid functions, similar to its functions in fishes. Given the large number of amphibian species that are fully aquatic or have aquatic life stages, we suggest that the broadly held assumption that corticosterone is the predominant glucocorticoid in all amphibians requires further scrutiny. Ultimately, multi-species tests of this assumption will reveal the ecological factors that influenced the evolution of endocrine adaptations among amphibian lineages, and may provide insight into convergent evolution of endocrine traits in paedomorphic species.
Asunto(s)
Glucocorticoides/metabolismo , Hidrocortisona/metabolismo , Urodelos/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Animales , Corticosterona/sangre , Hidrocortisona/sangre , Masculino , Urodelos/sangreRESUMEN
Bacterial endospores produced by Bacillus and Clostridium species can remain dormant and highly resistant to environmental insults for long periods, but they can also rapidly germinate in response to a nutrient-rich environment. Multiple proteins involved in sensing and responding to nutrient germinants, initiating solute and water transport, and accomplishing spore wall degradation are associated with the membrane surrounding the spore core. In order to more fully catalog proteins that may be involved in spore germination, as well as to identify protein changes taking place during germination, unbiased proteomic analyses of membrane preparations isolated from dormant and germinated spores of Bacillus anthracis and Bacillus subtilis were undertaken. Membrane-associated proteins were fractionated by SDS-PAGE, gel slices were trypsin digested, and extracted peptides were fractionated by liquid chromatography and analyzed by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. More than 500 proteins were identified from each preparation. Bioinformatic methods were used to characterize proteins with regard to membrane association, cellular function, and conservation across species. Numerous proteins not previously known to be spore associated, 6 in B. subtilis and 68 in B. anthracis, were identified. Relative quantitation based on spectral counting indicated that the majority of spore membrane proteins decrease in abundance during the first 20 min of germination. The spore membranes contained several proteins thought to be involved in the transport of metal ions, a process that plays a major role in spore formation and germination. Analyses of mutant strains lacking these transport proteins implicated YloB in the accumulation of calcium within the developing forespore.IMPORTANCE Bacterial endospores can remain dormant and highly resistant to environmental insults for long periods but can also rapidly germinate in response to a nutrient-rich environment. The persistence and subsequent germination of spores contribute to their colonization of new environments and to the spread of certain diseases. Proteins of Bacillus subtilis and Bacillus anthracis were identified that are associated with the spore membrane, a position that can allow them to contribute to germination. A set of identified proteins that are predicted to carry out ion transport were examined for their contributions to spore formation, stability, and germination. Greater knowledge of spore formation and germination can contribute to the development of better decontamination strategies.
Asunto(s)
Bacillus anthracis/química , Bacillus subtilis/química , Proteínas de Transporte de Membrana/análisis , Proteoma/análisis , Esporas Bacterianas/química , Cromatografía Liquida , Biología Computacional , Electroforesis en Gel de Poliacrilamida , Proteolisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismoRESUMEN
Bacteriophage 7-7-1, a member of the family Myoviridae, infects the soil bacterium Agrobacterium sp. strain H13-3. Infection requires attachment to actively rotating bacterial flagellar filaments, with flagellar number, length, and rotation speed being important determinants for infection efficiency. To identify the secondary receptor(s) on the cell surface, we isolated motile, phage-resistant Agrobacterium sp. H13-3 transposon mutants. Transposon insertion sites were pinpointed using arbitrary primed PCR and bioinformatics analyses. Three genes were recognized, whose corresponding proteins had the following computationally predicted functions: AGROH133_07337, a glycosyltransferase; AGROH133_13050, a UDP-glucose 4-epimerase; and AGROH133_08824, an integral cytoplasmic membrane protein. The first two gene products are part of the lipopolysaccharide (LPS) synthesis pathway, while the last is predicted to be a relatively small (13.4-kDa) cytosolic membrane protein with up to four transmembrane helices. The phenotypes of the transposon mutants were verified by complementation and site-directed mutagenesis. Additional characterization of motile, phage-resistant mutants is also described. Given these findings, we propose a model for Agrobacterium sp. H13-3 infection by bacteriophage 7-7-1 where the phage initially attaches to the flagellar filament and is propelled down toward the cell surface by clockwise flagellar rotation. The phage then attaches to and degrades the LPS to reach the outer membrane and ejects its DNA into the host using its syringe-like contractile tail. We hypothesize that the integral membrane protein plays an important role in events following viral DNA ejection or in LPS processing and/or deployment. The proposed two-step attachment mechanism may be conserved among other flagellotropic phages infecting Gram-negative bacteria.IMPORTANCE Flagellotropic bacteriophages belong to the tailed-phage order Caudovirales, the most abundant phages in the virome. While it is known that these viruses adhere to the bacterial flagellum and use flagellar rotation to reach the cell surface, their infection mechanisms are poorly understood. Characterizing flagellotropic-phage-host interactions is crucial to understanding how microbial communities are shaped. Using a transposon mutagenesis approach combined with a screen for motile, phage-resistant mutants, we identified lipopolysaccharides as the secondary cell surface receptor for phage 7-7-1. This is the first cell surface receptor identified for flagellotropic phages. One hypothetical membrane protein was also recognized as essential for infection. These new findings, together with previous results, culminated in an infection model for phage 7-7-1.
Asunto(s)
Agrobacterium/virología , Proteínas Bacterianas/metabolismo , Caudovirales/fisiología , Flagelos/metabolismo , Lipopolisacáridos/metabolismo , Receptores Virales/metabolismo , Agrobacterium/genética , Proteínas Bacterianas/genética , Elementos Transponibles de ADN , ADN Viral/genética , Flagelos/genética , Prueba de Complementación Genética , Mutagénesis , Receptores Virales/genética , Acoplamiento ViralRESUMEN
Sinorhizobium meliloti is a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors have been characterized, with McpU and McpX serving as general amino acid and quaternary ammonium compound sensors, respectively. Both receptors use a dual Cache (calcium channels and chemotaxis receptors) domain for ligand binding. We identified that the ligand-binding periplasmic region (PR) of McpV contains a single Cache domain. Homology modeling revealed that McpVPR is structurally similar to a sensor domain of a chemoreceptor with unknown function from Anaeromyxobacter dehalogenans, which crystallized with acetate in its binding pocket. We therefore assayed McpV for carboxylate binding and S. meliloti for carboxylate sensing. Differential scanning fluorimetry identified 10 potential ligands for McpVPR Nine of these are monocarboxylates with chain lengths between two and four carbons. We selected seven compounds for capillary assay analysis, which established positive chemotaxis of the S. meliloti wild type, with concentrations of peak attraction at 1 mM for acetate, propionate, pyruvate, and glycolate, and at 100 mM for formate and acetoacetate. Deletion of mcpV or mutation of residues essential for ligand coordination abolished positive chemotaxis to carboxylates. Using microcalorimetry, we determined that dissociation constants of the seven ligands with McpVPR were in the micromolar range. An McpVPR variant with a mutation in the ligand coordination site displayed no binding to isobutyrate or propionate. Of all the carboxylates tested as attractants, only glycolate was detected in alfalfa seed exudates. This work examines the relevance of carboxylates and their sensor to the rhizobium-legume interaction.IMPORTANCE Legumes share a unique association with certain soil-dwelling bacteria known broadly as rhizobia. Through concerted interorganismal communication, a legume allows intracellular infection by its cognate rhizobial species. The plant then forms an organ, the root nodule, dedicated to housing and supplying fixed carbon and nutrients to the bacteria. In return, the engulfed rhizobia, differentiated into bacteroids, fix atmospheric N2 into ammonium for the plant host. This interplay is of great benefit to the cultivation of legumes, such as alfalfa and soybeans, and is initiated by chemotaxis to the host plant. This study on carboxylate chemotaxis contributes to the understanding of rhizobial survival and competition in the rhizosphere and aids the development of commercial inoculants.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Carboxílicos/metabolismo , Factores Quimiotácticos/metabolismo , Quimiotaxis , Medicago sativa/microbiología , Sinorhizobium meliloti/fisiología , Aminoácidos/metabolismo , Proteínas Bacterianas/genética , Canales de Calcio , Calorimetría , Fluorometría , Ligandos , Modelos Moleculares , Periplasma/metabolismo , Exudados de Plantas , Dominios Proteicos , Sinorhizobium meliloti/genética , SimbiosisRESUMEN
The bacterium Sinorhizobium meliloti is attracted to seed exudates of its host plant alfalfa (Medicago sativa). Since quaternary ammonium compounds (QACs) are exuded by germinating seeds, we assayed chemotaxis of S. meliloti towards betonicine, choline, glycine betaine, stachydrine and trigonelline. The wild type displayed a positive response to all QACs. Using LC-MS, we determined that each germinating alfalfa seed exuded QACs in the nanogram range. Compared to the closely related nonhost species, spotted medic (Medicago arabica), unique profiles were released. Further assessments of single chemoreceptor deletion strains revealed that an mcpX deletion strain displayed little to no response to these compounds. Differential scanning fluorimetry showed interaction of the isolated periplasmic region of McpX (McpXPR and McpX34-306 ) with QACs. Isothermal titration calorimetry experiments revealed tight binding to McpXPR with dissociation constants (Kd ) in the nanomolar range for choline and glycine betaine, micromolar Kd for stachydrine and trigonelline and a Kd in the millimolar range for betonicine. Our discovery of S. meliloti chemotaxis to plant-derived QACs adds another role to this group of compounds, which are known to serve as nutrient sources, osmoprotectants and cell-to-cell signalling molecules. This is the first report of a chemoreceptor that mediates QACs taxis through direct binding.
Asunto(s)
Compuestos de Amonio Cuaternario/metabolismo , Sinorhizobium meliloti/metabolismo , Proteínas Bacterianas/metabolismo , Betaína/metabolismo , Células Quimiorreceptoras/metabolismo , Quimiotaxis , Colina/metabolismo , Medicago sativa/microbiología , Simbiosis/fisiologíaRESUMEN
UNLABELLED: Heat-resistant endospore formation plays an important role in Clostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores of C. perfringens strain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed of l-arginine-poly(l-aspartic acid), to ß-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species of Clostridium, but their role has not been defined. To determine the function of cyanophycin in C. perfringens, a mutation was introduced into the cphA gene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulating C. perfringens cells, an Escherichia coli strain expressing the cphA gene made copious amounts of cyanophycin, confirming that cphA encodes a cyanophycin synthetase. IMPORTANCE: Clostridium perfringens is a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. How C. perfringens controls the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role in C. perfringens than it does in cyanobacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridium perfringens/crecimiento & desarrollo , Proteoma/metabolismo , Esporas Bacterianas/enzimología , Proteínas Bacterianas/genética , Clostridium perfringens/química , Clostridium perfringens/enzimología , Clostridium perfringens/genética , Espectrometría de Masas , Proteoma/química , Proteoma/genética , Esporas Bacterianas/química , Esporas Bacterianas/genética , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
Amyloid formation of the 37-residue amylin is involved in the pathogenesis of type 2 diabetes and, potentially, diabetes-induced neurological deficits. Numerous flavonoids exhibit inhibitory effects against amylin amyloidosis, but the mechanisms of inhibition remain unclear. Screening a library of natural compounds uncovered a potent lead compound, the flavone baicalein. Baicalein inhibits amylin amyloid formation and reduces amylin-induced cytotoxicity. Analogue analyses demonstrated, for the first time, key roles of the vicinal hydroxyl groups on the A-ring. We provided mass spectrometric evidence that incubating baicalein and amylin leads to their conjugation, consistent with a Schiff base mechanism.
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Flavanonas/farmacología , Polipéptido Amiloide de los Islotes Pancreáticos/antagonistas & inhibidores , Polipéptido Amiloide de los Islotes Pancreáticos/química , Diabetes Mellitus Tipo 2/etiología , Evaluación Preclínica de Medicamentos , Flavanonas/química , Antagonistas de Hormonas/química , Antagonistas de Hormonas/farmacología , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/ultraestructura , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Relación Estructura-ActividadRESUMEN
Plant seeds and roots exude a spectrum of molecules into the soil that attract bacteria to the spermosphere and rhizosphere, respectively. The alfalfa symbiont Sinorhizobium meliloti utilizes eight chemoreceptors (McpT to McpZ and IcpA) to mediate chemotaxis. Using a modified hydrogel capillary chemotaxis assay that allows data quantification and larger throughput screening, we defined the role of S. meliloti chemoreceptors in sensing its host, Medicago sativa, and a closely related nonhost, Medicago arabica. S. meliloti wild type and most single-deletion strains displayed comparable chemotaxis responses to host or nonhost seed exudate. However, while the mcpZ mutant responded like wild type to M. sativa exudate, its reaction to M. arabica exudate was reduced by 80%. Even though the amino acid (AA) amounts released by both plant species were similar, synthetic AA mixtures that matched exudate profiles contributed differentially to the S. meliloti wild-type response to M. sativa (23%) and M. arabica (37%) exudates, with McpU identified as the most important chemoreceptor for AA. Our results show that S. meliloti is equally attracted to host and nonhost legumes; however, AA play a greater role in attraction to M. arabica than to M. sativa, with McpZ being specifically important in sensing M. arabica.
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Aminoácidos/química , Factores Quimiotácticos/metabolismo , Quimiotaxis/efectos de los fármacos , Plantas/microbiología , Semillas/metabolismo , Sinorhizobium meliloti/metabolismo , Aminoácidos/clasificación , Proteínas Bacterianas , Quimiotaxis/fisiología , Regulación Bacteriana de la Expresión Génica , Exudados de Plantas/química , Plantas/clasificación , Semillas/clasificación , Sinorhizobium meliloti/genética , Especificidad de la EspecieRESUMEN
BACKGROUND: Liver models that closely mimic the in vivo microenvironment are useful for understanding liver functions, capabilities, and intercellular communication processes. Three-dimensional (3D) liver models assembled using hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM) provide a functional system while also permitting isolation of individual cell types for proteomic analyses. METHODS: To better understand the mechanisms and processes that underlie liver model function, hepatocytes were maintained as monolayers and 3D PEM-based formats in the presence or absence of primary LSECs. The resulting hepatocyte proteomes, the proteins in the PEM, and extracellular levels of urea, albumin and glucose after three days of culture were compared. RESULTS: All systems were ketogenic and found to release glucose. The presence of the PEM led to increases in proteins associated with both mitochondrial and peroxisomal-based ß-oxidation. The PEMs also limited production of structural and migratory proteins associated with dedifferentiation. The presence of LSECs increased levels of Phase I and Phase II biotransformation enzymes as well as several proteins associated with the endoplasmic reticulum and extracellular matrix remodeling. The proteomic analysis of the PEMs indicated that there was no significant change after three days of culture. These results are discussed in relation to liver model function. CONCLUSIONS: Heterotypic cell-cell and cell-ECM interactions exert different effects on hepatocyte functions and phenotypes.
RESUMEN
BACKGROUND: Low phytic acid (lpa) crops are potentially eco-friendly alternative to conventional normal phytic acid (PA) crops, improving mineral bioavailability in monogastric animals as well as decreasing phosphate pollution. The lpa crops developed to date carry mutations that are directly or indirectly associated with PA biosynthesis and accumulation during seed development. These lpa crops typically exhibit altered carbohydrate profiles, increased free phosphate, and lower seedling emergence, the latter of which reduces overall crop yield, hence limiting their large-scale cultivation. Improving lpa crop yield requires an understanding of the downstream effects of the lpa genotype on seed development. Towards that end, we present a comprehensive comparison of gene-expression profiles between lpa and normal PA soybean lines (Glycine max) at five stages of seed development using RNA-Seq approaches. The lpa line used in this study carries single point mutations in a myo-inositol phosphate synthase gene along with two multidrug-resistance protein ABC transporter genes. RESULTS: RNA sequencing data of lpa and normal PA soybean lines from five seed-developmental stages (total of 30 libraries) were used for differential expression and functional enrichment analyses. A total of 4235 differentially expressed genes, including 512-transcription factor genes were identified. Eighteen biological processes such as apoptosis, glucan metabolism, cellular transport, photosynthesis and 9 transcription factor families including WRKY, CAMTA3 and SNF2 were enriched during seed development. Genes associated with apoptosis, glucan metabolism, and cellular transport showed enhanced expression in early stages of lpa seed development, while those associated with photosynthesis showed decreased expression in late developmental stages. The results suggest that lpa-causing mutations play a role in inducing and suppressing plant defense responses during early and late stages of seed development, respectively. CONCLUSIONS: This study provides a global perspective of transcriptomal changes during soybean seed development in an lpa mutant. The mutants are characterized by earlier expression of genes associated with cell wall biosynthesis and a decrease in photosynthetic genes in late stages. The biological processes and transcription factors identified in this study are signatures of lpa-causing mutations.
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Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Glycine max/efectos de los fármacos , Glycine max/genética , Ácido Fítico/farmacología , Semillas/efectos de los fármacos , Semillas/genética , Transcriptoma , Transporte Biológico , Análisis por Conglomerados , Biología Computacional/métodos , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Genes de Plantas , Glucanos/metabolismo , Anotación de Secuencia Molecular , Mutación , Fotosíntesis/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Clostridium difficile is a clinically important pathogen and the most common cause of hospital-acquired infectious diarrhea. Expression of the C. difficile gene csfV, which encodes σ(V), an extracytoplasmic function σ factor, is induced by lysozyme, which damages the peptidoglycan of bacteria. Here we show that σ(V) is required for lysozyme resistance in C. difficile. Using microarray analysis, we identified the C. difficile genes whose expression is dependent upon σ(V) and is induced by lysozyme. Although the peptidoglycan of wild-type C. difficile is intrinsically highly deacetylated, we have found that exposure to lysozyme leads to additional peptidoglycan deacetylation. This lysozyme-induced deacetylation is dependent upon σ(V). Expression of pdaV, which encodes a putative peptidoglycan deacetylase, was able to increase lysozyme resistance of a csfV mutant. The csfV mutant strain is severely attenuated compared to wild-type C. difficile in a hamster model of C. difficile-associated disease. We conclude that the σ(V) signal transduction system, which senses the host innate immune defense enzyme lysozyme, is required for lysozyme resistance and is necessary during C. difficile infection.
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Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/microbiología , Muramidasa/metabolismo , Factor sigma/fisiología , Animales , Antibacterianos/farmacología , Clostridioides difficile/efectos de los fármacos , Cricetinae , ADN Bacteriano/análisis , Modelos Animales de Enfermedad , Análisis por Micromatrices , Pruebas de Sensibilidad Microbiana , Factor sigma/metabolismo , Virulencia/fisiologíaRESUMEN
Bacterial chemotaxis is an important attribute that aids in establishing symbiosis between rhizobia and their legume hosts. Plant roots and seeds exude a spectrum of molecules into the soil to attract their bacterial symbionts. The alfalfa symbiont Sinorhizobium meliloti possesses eight chemoreceptors to sense its environment and mediate chemotaxis toward its host. The methyl accepting chemotaxis protein McpU is one of the more abundant S. meliloti chemoreceptors and an important sensor for the potent attractant proline. We established a dominant role of McpU in sensing molecules exuded by alfalfa seeds. Mass spectrometry analysis determined that a single germinating seed exudes 3.72 nmol of proline, producing a millimolar concentration near the seed surface which can be detected by the chemosensory system of S. meliloti. Complementation analysis of the mcpU deletion strain verified McpU as the key proline sensor. A structure-based homology search identified tandem Cache (calcium channels and chemotaxis receptors) domains in the periplasmic region of McpU. Conserved residues Asp-155 and Asp-182 of the N-terminal Cache domain were determined to be important for proline sensing by evaluating mutant strains in capillary and swim plate assays. Differential scanning fluorimetry revealed interaction of the isolated periplasmic region of McpU (McpU40-284) with proline and the importance of Asp-182 in this interaction. Using isothermal titration calorimetry, we determined that proline binds with a Kd (dissociation constant) of 104 µM to McpU40-284, while binding was abolished when Asp-182 was substituted by Glu. Our results show that McpU is mediating chemotaxis toward host plants by direct proline sensing.