RESUMO
Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.
Assuntos
Chromobacterium , Ferro , Sideróforos , Humanos , Sideróforos/genética , Bactérias/metabolismo , Homeostase/genética , Antibacterianos/química , Peptídeo HidrolasesRESUMO
Thyroid hormones (THs) are crucial in bodily functions, while iron is essential for processes like oxygen transport. Specialized proteins maintain iron balance, including ferritin, transferrin, ferroportin, and hepcidin. Research suggests that THs can influence iron homeostasis by affecting mRNA and protein expression, such as ferritin and transferrin. Our study focused on male rats to assess mRNA expression of iron homeostasis-related proteins and metabolomics in thyroid dysfunction. We found altered gene expression across various tissues (liver, duodenum, spleen, and kidney) and identified disrupted metabolite patterns in thyroid dysfunction. These findings highlight tissue-specific effects of thyroid dysfunction on essential iron homeostasis proteins and provide insights into associated metabolic changes. Our research contributes to understanding the intricate interplay between thyroid hormones and iron balance. By unveiling tissue-specific gene expression alterations and metabolic disruptions caused by thyroid dysfunction, our work lays a foundation for future investigations to explore underlying mechanisms and develop targeted strategies for managing iron-related complications in thyroid disorders.
Assuntos
Ferro , Doenças da Glândula Tireoide , Ratos , Masculino , Animais , Ferritinas/genética , Ferritinas/metabolismo , Transferrina/metabolismo , Homeostase , Doenças da Glândula Tireoide/genética , Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Hormônios TireóideosRESUMO
Serious concerns have recently been raised regarding the association of Fe excess with neurodegenerative diseases in mammals and nutritional and oxidative disorders in plants. Therefore, the current study aimed to understand the physiological changes induced by Fe excess in Pistia stratiotes, a species often employed in phytoremediation studies. P. stratiotes were subjected to five concentrations of Fe: 0.038 (control), 1.0, 3.0, 5.0 and 7.0 mM. Visual symptoms of Fe-toxicity such as bronzing of leaf edges in 5.0 and 7.0 mM-grown plants were observed after 5 days. Nevertheless, no major changes were observed in photosynthesis-related parameters at this time-point. In contrast, plants growing for 10 days in high Fe concentrations showed decreased chlorophyll concentrations and lower net CO2 assimilation rate. Notwithstanding, P. stratiotes accumulated high amounts of Fe, especially in roots (maximum of 10,000 µg g-1 DW) and displayed a robust induction of the enzymatic antioxidant system. In conclusion, we demonstrated that P. stratiotes can be applied to clean up Fe-contaminated water, as the species displays high Fe bioaccumulation, mostly in root apoplasts, and can maintain physiological processes under Fe excess. Our results further revealed that by monitoring visual symptoms, P. stratiotes could be applied for bioindication purposes.
Assuntos
Araceae , Hydrocharitaceae , Poluentes Químicos da Água , Animais , Ferro , Biodegradação Ambiental , Bioacumulação , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/análise , Água , MamíferosRESUMO
Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicusfur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicusfur protein was able to complement an E. colifur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus, as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus.
Assuntos
Gluconacetobacter , Ferro , Proteínas de Bactérias/metabolismo , Meios de Cultura/farmacologia , Ferro/metabolismo , TranscriptomaRESUMO
Chromobacterium violaceum is an environmental Gram-negative beta-proteobacterium that causes systemic infections in humans. C. violaceum uses siderophore-based iron acquisition systems to overcome the host-imposed iron limitation, but its capacity to use other iron sources is unknown. In this work, we characterized ChuPRSTUV as a heme utilization system employed by C. violaceum to explore an important iron reservoir in mammalian hosts, free heme and hemoproteins. We demonstrate that the chuPRSTUV genes comprise a Fur-repressed operon that is expressed under iron limitation. The chu operon potentially encodes a small regulatory protein (ChuP), an outer membrane TonB-dependent receptor (ChuR), a heme degradation enzyme (ChuS), and an inner membrane ABC transporter (ChuTUV). Our nutrition growth experiments using C. violaceum chu deletion mutants revealed that, with the exception of chuS, all genes of the chu operon are required for heme and hemoglobin utilization in C. violaceum. The mutant strains without chuP displayed increased siderophore halos on CAS plate assays. Significantly, we demonstrate that ChuP connects heme and siderophore utilization by acting as a positive regulator of chuR and vbuA, which encode the TonB-dependent receptors for the uptake of heme (ChuR) and the siderophore viobactin (VbuA). Our data favor a model of ChuP as a heme-binding post-transcriptional regulator. Moreover, our virulence data in a mice model of acute infection demonstrate that C. violaceum uses both heme and siderophore for iron acquisition during infection, with a preference for siderophores over the Chu heme utilization system.
Assuntos
Heme , Sideróforos , Animais , Chromobacterium , Heme/metabolismo , Ferro/metabolismo , Mamíferos/metabolismo , Camundongos , Sideróforos/metabolismo , Fatores de Transcrição , VirulênciaRESUMO
It is known that iron transporter proteins and their regulation can modulate the fish's immune system, suggesting these proteins as a potential candidate for fish vaccines. Previous studies have evidenced the effects of Atlantic salmon immunized with the chimeric iron-related protein named IPath® against bacterial and ectoparasitic infections. The present study aimed to explore the transcriptome modulation and the morphology of the sea louse Caligus rogercresseyi in response to Atlantic salmon injected with IPath®. Herein, Atlantic salmon were injected with IPath® and challenged to sea lice in controlled laboratory conditions. Then, female adults were collected after 25 days post-infection for molecular and morphological evaluation. Transcriptome analysis conducted in lice collected from immunized fish revealed high modulation of transcripts compared with the control groups. Notably, the low number of up/downregulated transcripts was mainly found in lice exposed to the IPath® fish group. Among the top-25 differentially expressed genes, Vitellogenin, Cytochrome oxidases, and proteases genes were strongly downregulated, suggesting that IPath® can alter lipid transport, hydrogen ion transmembrane transport, and proteolysis. The morphological analysis in lice collected from IPath® fish revealed abnormal embryogenesis and inflammatory processes of the genital segment. Furthermore, head kidney, spleen, and skin were also analyzed in immunized fish to evaluate the transcription expression of immune and iron homeostasis-related genes. The results showed downregulation of TLR22, MCHII, IL-1ß, ALAs, HO, BLVr, GSHPx, and Ferritin genes in head kidney and skin tissues; meanwhile, those genes did not show significant differences in spleen tissue. Overall, our findings suggest that IPath® can be used to enhance the fish immune response, showing a promissory commercial application against lice infections.
Assuntos
Copépodes/genética , Ectoparasitoses/prevenção & controle , Doenças dos Peixes/prevenção & controle , Proteínas Recombinantes/administração & dosagem , Salmo salar/parasitologia , Transcriptoma , Vacinas/administração & dosagem , Animais , Ectoparasitoses/veterinária , Feminino , Ferritinas/genética , Salmo salar/imunologia , Transferrina/genética , VacinaçãoRESUMO
Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions. The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target. This review aims to give an overview on Grxs, starting by a phylogenetic analysis of vertebrate Grxs, followed by an analysis of the mechanisms of action, the specific characteristics of the different human isoforms and a discussion on aspects related to human physiology and diseases.
Assuntos
Glutarredoxinas , Glutationa , Catálise , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Humanos , Oxirredução , FilogeniaRESUMO
Iron is a highly reactive metal that participates in several processes in prokaryotic and eukaryotic cells. Hosts and pathogens compete for iron in the context of infection. Chromobacterium violaceum, an environmental Gram-negative bacterial pathogen, relies on siderophores to overcome iron limitation in the host. In this work, we studied the role of the ferric uptake regulator Fur in the physiology and virulence of C. violaceum A Δfur mutant strain showed decreased growth and fitness under regular in vitro growth conditions and presented high sensitivity to iron and oxidative stresses. Furthermore, the absence of fur caused derepression of siderophore production and reduction in swimming motility and biofilm formation. Consistent with these results, the C. violaceum Δfur mutant was highly attenuated for virulence and liver colonization in mice. In contrast, a manganese-selected spontaneous fur mutant showed only siderophore overproduction and sensitivity to oxidative stress, indicating that Fur remained partially functional in this strain. We found that mutations in genes related to siderophore biosynthesis and a putative CRISPR-Cas locus rescued the Δfur mutant growth defects, indicating that multiple Fur-regulated processes contribute to maintaining bacterial cell fitness. Overall, our data indicated that Fur is conditionally essential in C. violaceum mainly by protecting cells from iron overload and oxidative damage. The requirement of Fur for virulence highlights the importance of iron in the pathogenesis of C. violaceumIMPORTANCE Maintenance of iron homeostasis, i.e., avoiding both deficiency and toxicity of this metal, is vital to bacteria and their hosts. Iron sequestration by host proteins is a crucial strategy to combat bacterial infections. In bacteria, the ferric uptake regulator Fur coordinates the expression of several iron-related genes. Sometimes, Fur can also regulate several other processes. In this work, we performed an in-depth phenotypic characterization of fur mutants in the human opportunistic pathogen Chromobacterium violaceum We determined that fur is a conditionally essential gene necessary for proper growth under regular conditions and is fully required for survival under iron and oxidative stresses. Fur also controlled several virulence-associated traits, such as swimming motility, biofilm formation, and siderophore production. Consistent with these results, a C. violaceumfur null mutant showed attenuation of virulence. Therefore, our data established Fur as a major player required for C. violaceum to manage iron, including during infection in the host.
Assuntos
Proteínas de Bactérias/genética , Chromobacterium/fisiologia , Chromobacterium/patogenicidade , Ferro/toxicidade , Estresse Oxidativo , Proteínas Repressoras/genética , Sideróforos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Repressoras/metabolismo , VirulênciaRESUMO
AIMS: The importance of bacterioferritin in the virulence and pathogenicity of the genus Mycobacterium is still unclear. The aim of this study was to analyse if the expression of a recombinant bacterioferritin from M. tuberculosis (Mtb) by Mycma could improve the capacity of this bacillus to resist the host defence mechanisms. METHODS AND RESULTS: Recombinant Mycma, expressing bacterioferritin (Rv1876) from Mtb, was developed by transformation with pMIP12_Rv1876. To determine bacterioferritin influence on Mycma physiology and virulence, the mycobacteria growth was analysed in vitro and in vivo. It was observed that the expression of bacterioferritin improved the growth rate of recombinant Mycma_BfrA under iron excess and oxidative stress, as compared to the wild type. Furthermore, in the murine model of infection, it was observed that Mycma_BfrA-infected mice had higher bacillary load and a more pronounced lesion in the lungs when compared with the wild type. CONCLUSION: This study showed that bacterioferritin confers additional resistance to stress conditions, resulting in increased pathogenicity of Mycma during mice infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new insights about the importance of bacterioferritin in the virulence and pathogenicity of the Mycobacterium genus.
Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/metabolismo , Ferritinas/metabolismo , Mycobacterium abscessus/fisiologia , Mycobacterium abscessus/patogenicidade , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Grupo dos Citocromos b/genética , Ferritinas/genética , Camundongos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium abscessus/genética , Mycobacterium abscessus/crescimento & desenvolvimento , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , VirulênciaRESUMO
KEY MESSAGE: There is a link between PAP/SAL retrograde pathway, ethylene signaling and Fe metabolism in Arabidopsis. Nuclear gene expression is regulated by a diversity of retrograde signals that travel from organelles to the nucleus in a lineal or classical model. One such signal molecule is 3'-phosphoadenisine-5'-phosphate (PAP) and it's in vivo levels are regulated by SAL1/FRY1, a phosphatase enzyme located in chloroplast and mitochondria. This metabolite inhibits the action of a group of exorribonucleases which participate in post-transcriptional gene expression regulation. Transcriptome analysis of Arabidopsis thaliana mutant plants in PAP-SAL1 pathway revealed that the ferritin genes AtFER1, AtFER3, and AtFER4 are up-regulated. In this work we studied Fe metabolism in three different mutants of the PAP/SAL1 retrograde pathway. Mutant plants showed increased Fe accumulation in roots, shoots and seeds when grown in Fe-sufficient condition, and a constitutive activation of the Strategy I Fe uptake genes. As a consequence, they grew more vigorously than wild type plants in Fe-deficient medium. However, when mutant plants grown in Fe-deficient conditions were sprayed with Fe in their leaves, they were unable to deactivate root Fe uptake. Ethylene synthesis inhibition revert the constitutive Fe uptake phenotype. We propose that there is a link between PAP/SAL pathway, ethylene signaling and Fe metabolism.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ferro/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Difosfato de Adenosina/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Clorofila , Cloroplastos/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Homeostase , Mitocôndrias/metabolismo , Mutação , Monoéster Fosfórico Hidrolases/genética , Folhas de Planta/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismoRESUMO
The ability of Rhodococcus equi to survive in macrophages and cause pneumonia in foals depends on vapA and rhbC genes, which produce the virulence-associated protein A (VapA) and the rhequichelin siderophore, respectively. Virulent R. equi acquires Fe from transferrin by unknown mechanisms. Our objectives were to determine the role of GAPDH in Fe homeostasis, to further characterize GAPDH, rhbC, and vapA expression under iron homeostasis, and to document the occurrence of rhbC gene in R. equi isolates. Therefore, vapA + R. equi was cultured under excessive, physiologic, and restricted iron concentrations, and quantitative culture and gene expression were performed. The relative expression of GAPDH, rhbC, and vapA after 48 h of culture were analyzed by qPCR. To determine the rhbC occurrence, total DNA was extracted from R. equi isolated from foals with clinical rhodococcosis (n = 22), healthy horses (feces, n = 16; nasal swab, n = 9), soil (n = 6), and 2 ATCC reference strains. Conventional PCR was performed to identify genus/species, vapA, and rhbC genes. Iron restriction proportionally decreased R. equi growth rates, and induced high expression of both GAPDH and vapA. The putative role of GAPDH in R. equi iron homeostasis should be further investigated. rhbC was significantly up-regulated under both Fe excess and critical starvation. The rhbC gene was identified in all clinical isolates and soil, but it was absent in 2 isolates from healthy horses, suggesting that rhequichelin is not required for R. equi nasal and intestinal colonization.
Assuntos
Infecções por Actinomycetales/microbiologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Ferro/metabolismo , Rhodococcus equi/genética , Rhodococcus equi/metabolismo , Homeostase , Rhodococcus equi/crescimento & desenvolvimento , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Prenatal ethanol exposure (PAE) induces behavioral maladptations in offspring, including a deficit in memory formation which is part of the umbrella sign of fetal alcohol spectrum disorder. Clinical and preclinical studies have shown that iron depletion exacerbates cognitive problems in offspring exposed to ethanol in utero and that PAE promotes dysregulation in brain iron homeostasis. However, the mechanisms underlying brain iron dysregulation and neuronal activity defects in adolescent offspring of PAE are unclear and poorly understand. Here, we used a PAE rat model to analyze messenger RNA (mRNA) and protein expression of iron homeostasis genes such as transferrin receptor (TfR), divalent metal transporter (DMT1), ferroportin (FPN1), and ferritin (FT) in brain areas associated with memory formation such as the prefrontal cortex (PFC), ventral tegmental area, and hippocampus. Interestingly, we found that 21 day old PAE rats have higher mRNA expression of DMT1 in the PFC, and TfR in the hippocampus, compared to control animals. In contrast FPN has lower mRNA expression in the PFC, and FT and FPN1 have lower expression in the hippocampus. In agreement with these results, we found a 1.5-2 fold increase of TfR and DMT1 protein levels both in the hippocampus and the PFC. Additionally, using an electrophysiological approach, we found that in hippocampal slices from PAE rats, iron treatment decreased long-term potentiation (LTP), but not AMPAR basal transmission (AMPAR fEPSP). In contrast, in control slices Fe-NTA did not affect LTP but decreased significantly the AMPAR fEPSP. Meanwhile, iron chelation with deferiprone decreased AMPAR transmission in PAE and control slices and decreased LTP only in controls slices. These results suggest that PAE affects iron homeostasis of specific brain areas-PFC and hippocampus-which could be involved in maladaptive cognition observed in this animal model.
RESUMO
This review provides a synopsis of the published literature over the past two years on the heme-binding protein hemopexin (HPX), with some background information on the biochemistry of the HPX system. One focus is on the mechanisms of heme-driven pathology in the context of heme and iron homeostasis in human health and disease. The heme-binding protein hemopexin is a multi-functional protectant against hemoglobin (Hb)-derived heme toxicity as well as mitigating heme-mediated effects on immune cells, endothelial cells, and stem cells that collectively contribute to driving inflammation, perturbing vascular hemostasis and blood-brain barrier function. Heme toxicity, which may lead to iron toxicity, is recognized increasingly in a wide range of conditions involving hemolysis and immune system activation and, in this review, we highlight some newly identified actions of heme and hemopexin especially in situations where normal processes fail to maintain heme and iron homeostasis. Finally, we present preliminary data showing that the cytokine IL-6 cross talks with activation of the c-Jun N-terminal kinase pathway in response to heme-hemopexin in models of hepatocytes. This indicates another level of complexity in the cell responses to elevated heme via the HPX system when the immune system is activated and/or in the presence of inflammation.
RESUMO
BACKGROUND: Iron is an essential micronutrient for the growth and development of virtually all living organisms, playing a pivotal role in the proliferative capability of many bacterial pathogens. The impact that the bioavailability of iron has on the transcriptional response of bacterial species in the CMNR group has been widely reported for some members of the group, but it hasn't yet been as deeply explored in Corynebacterium pseudotuberculosis. Here we describe for the first time a comprehensive RNA-seq whole transcriptome analysis of the T1 wild-type and the Cp13 mutant strains of C. pseudotuberculosis under iron restriction. The Cp13 mutant strain was generated by transposition mutagenesis of the ciuA gene, which encodes a surface siderophore-binding protein involved in the acquisition of iron. Iron-regulated acquisition systems are crucial for the pathogenesis of bacteria and are relevant targets to the design of new effective therapeutic approaches. RESULTS: Transcriptome analyses showed differential expression in 77 genes within the wild-type parental T1 strain and 59 genes in Cp13 mutant under iron restriction. Twenty-five of these genes had similar expression patterns in both strains, including up-regulated genes homologous to the hemin uptake hmu locus and two distinct operons encoding proteins structurally like hemin and Hb-binding surface proteins of C. diphtheriae, which were remarkably expressed at higher levels in the Cp13 mutant than in the T1 wild-type strain. These hemin transport protein genes were found to be located within genomic islands associated with known virulent factors. Down-regulated genes encoding iron and heme-containing components of the respiratory chain (including ctaCEF and qcrCAB genes) and up-regulated known iron/DtxR-regulated transcription factors, namely ripA and hrrA, were also identified differentially expressed in both strains under iron restriction. CONCLUSION: Based on our results, it can be deduced that the transcriptional response of C. pseudotuberculosis under iron restriction involves the control of intracellular utilization of iron and the up-regulation of hemin acquisition systems. These findings provide a comprehensive analysis of the transcriptional response of C. pseudotuberculosis, adding important understanding of the gene regulatory adaptation of this pathogen and revealing target genes that can aid the development of effective therapeutic strategies against this important pathogen.
Assuntos
Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/metabolismo , Perfilação da Expressão Gênica , Deficiências de Ferro , Corynebacterium pseudotuberculosis/crescimento & desenvolvimento , Corynebacterium pseudotuberculosis/fisiologia , Redes Reguladoras de Genes , Ilhas Genômicas/genética , Viabilidade Microbiana/genética , Mutação , Transcrição GênicaRESUMO
The ethanol stress response in ethanologenic yeast during fermentation involves the swishing of several adaptation mechanisms. In Saccharomyces cerevisiae, the Jac1p and Isu1p proteins constitute the scaffold system for the Fe-S cluster assembly. This study was performed using the over-expression of the Jac1p and Isu1p in the industrially utilized S. cerevisiae UMArn3 strain, with the objective of improving the Fe-S assembly/recycling, and thus counteracting the toxic effects of ethanol stress during fermentation. The UMArn3 yeast was transformed with both the JAC1-His and ISU1-His genes-plasmid contained. The Jac1p and Isu1p His-tagged proteins over-expression in the engineered yeasts was confirmed by immunodetection, rendering increases in ethanol tolerance level from a DL50 = ~ 4.5% ethanol (v/v) to DL50 = ~ 8.2% ethanol (v/v), and survival up 90% at 15% ethanol (v/v) comparing to ~ 50% survival in the control strain. Fermentation by the engineered yeasts showed that the ethanol production was increased, producing 15-20% more ethanol than the control yeast. The decrease of ROS and free-iron accumulation was observed in the engineered yeasts under ethanol stress condition. The results indicate that Jac1p and Isu1p over-expression in the S. cerevisiae UMArn3.3 yeast increased its ethanol tolerance level and ethanol production by a mechanism that involves ROS and iron homeostasis related to the biogenesis/recycling of Fe-S clusters dependent proteins.
Assuntos
Etanol/metabolismo , Homeostase , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fermentação , Ferro/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Superóxidos/metabolismoRESUMO
Heme may represent a major iron-source for bacteria. In the symbiotic nitrogen-fixing bacterium Ensifer meliloti 1021, iron acquisition from heme depends on the outer-membrane heme-receptor ShmR. Expression of shmR gene is repressed by iron in a RirA dependent manner while under iron-limitation its expression requires the small protein HmuP. In this work, we identified highly conserved nucleotide motifs present upstream the shmR gene. These motifs are widely distributed among Alpha and Beta Proteobacteria, and correlate with the presence of HmuP coding sequences in bacterial genomes. According to data presented in this work, we named these new motifs as HmuP-responsive elements (HPREs). In the analyzed genomes, the HPREs were always present upstream of genes encoding putative heme-receptors. Moreover, in those Alpha and Beta Proteobacteria where transcriptional start sites for shmR homologs are known, HPREs were located in the 5'UTR region. In this work we show that in E. meliloti 1021, HPREs are involved in HmuP-dependent shmR expression. Moreover, we show that changes in sequence composition of the HPREs correlate with changes in a predicted RNA secondary structure element and affect shmR gene expression.
Assuntos
Regiões 5' não Traduzidas/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Sequência Conservada/genética , Motivos de Nucleotídeos/genética , Receptores de Superfície Celular/genética , Sinorhizobium meliloti/genética , Reação em Cadeia da Polimerase em Tempo Real , Sinorhizobium meliloti/crescimento & desenvolvimentoRESUMO
The [Fe-S] late-acting subsystem comprised of Isa1p/Isa2p, Grx5p, and Iba57p proteins (Fe-S-IBG subsystem) is involved in [4Fe-4S]-cluster protein assembly. The effect of deleting IBA57 in Saccharomyces cerevisiae on mitochondrial respiratory complex integration and functionality associated with Rieske protein maturation was evaluated. The iba57Δ mutant showed decreased expression and maturation of the Rieske protein. The loss of Rieske protein caused by IBA57 deletion affected the structure of supercomplexes III2IV2 and III2IV1 and their integration into the mitochondria, causing dysfunction in the electron transport chain. These effects were correlated with decreased cytochrome functionality and content in the iba57Δ mutant. These findings suggest that Iba57p participates in maturation of the [2Fe-2S]-cluster into the Rieske protein and that Rieske protein plays important roles in the conformation and functionality of mitochondrial supercomplex III/IV in the electron transport chain.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citocromos/deficiência , Deleção de Genes , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Phosphorus (Pi) deficiency induces root morphological remodeling in plants. The primary root length of rice increased under Pi deficiency stress; however, the underlying mechanism is not well understood. In this study, transcriptome analysis (RNA-seq) and Real-time quantitative PCR (qRT-PCR) techniques were combined with the determination of physiological and biochemical indexes to research the regulation mechanisms of iron (Fe) accumulation and callose deposition in rice roots, to illuminate the relationship between Fe accumulation and primary root growth under Pi deficient conditions. RESULTS: Induced expression of LPR1 genes was observed under low Pi, which also caused Fe accumulation, resulting in iron plaque formation on the root surface in rice; however, in contrast to Arabidopsis, low Pi promoted primary root lengthening in rice. This might be due to Fe accumulation and callose deposition being still appropriately regulated under low Pi. The down-regulated expression of Fe-uptake-related key genes (including IRT, NAS, NAAT, YSLs, OsNRAMP1, ZIPs, ARF, and Rabs) inhibited iron uptake pathways I, II, and III in rice roots under low Pi conditions. In contrast, due to the up-regulated expression of the VITs gene, Fe was increasingly stored in both root vacuoles and cell walls. Furthermore, due to induced expression and increased activity of ß-1-3 glucanase, callose deposition was more controlled in low Pi treated rice roots. In addition, low Pi and low Fe treatment still caused primary root lengthening. CONCLUSIONS: The obtained results indicate that Low phosphorus induces iron and callose homeostatic regulation in rice roots. Because of the Fe homeostatic regulation, Fe plays a small role in rice root morphological remodeling under low Pi.
Assuntos
Glucanos/metabolismo , Ferro/metabolismo , Oryza/metabolismo , Fósforo/deficiência , Raízes de Plantas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Homeostase , Oryza/crescimento & desenvolvimento , Fósforo/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The ferric uptake regulator (Fur) plays a major role in controlling the expression of iron homeostasis genes in bacterial organisms. In this work, we fully characterized the capacity of Fur to reconfigure the global transcriptional network and influence iron homeostasis in Enterococcus faecalis. The characterization of the Fur regulon from E. faecalis indicated that this protein (Fur) regulated the expression of genes involved in iron uptake systems, conferring to the system a high level of efficiency and specificity to respond under different iron exposure conditions. An RNAseq assay coupled with a systems biology approach allowed us to identify the first global transcriptional network activated by different iron treatments (excess and limited), with and without the presence of Fur. The results showed that changes in iron availability activated a complex network of transcriptional factors in E. faecalis, among them global regulators such as LysR, ArgR, GalRS, and local regulators, LexA and CopY, which were also stimulated by copper and zinc treatments. The deletion of Fur impacted the expression of genes encoding for ABC transporters, energy production and [Fe-S] proteins, which optimized detoxification and iron uptake under iron excess and limitation, respectively. Finally, considering the close relationship between iron homeostasis and pathogenesis, our data showed that the absence of Fur increased the internal concentration of iron in the bacterium and also affected its ability to produce biofilm. These results open new alternatives in the field of infection mechanisms of E. faecalis.