RESUMO
KEY MESSAGE: The ISC Fe-S cluster biosynthetic pathway would play a key role in the regulation of iron and sulfur homeostasis in plants. The Arabidopsis thaliana mitochondrial cysteine desulfurase AtNFS1 has an essential role in cellular ISC Fe-S cluster assembly, and this pathway is one of the main sinks for iron (Fe) and sulfur (S) in the plant. In different plant species it has been reported a close relationship between Fe and S metabolisms; however, the regulation of both nutrient homeostasis is not fully understood. In this study, we have characterized AtNFS1 overexpressing and knockdown mutant Arabidopsis plants. Plants showed alterations in the ISC Fe-S biosynthetic pathway genes and in the activity of Fe-S enzymes. Genes involved in Fe and S uptakes, assimilation, and regulation were up-regulated in overexpressing plants and down-regulated in knockdown plants. Furthermore, the plant nutritional status in different tissues was in accordance with those gene activities: overexpressing lines accumulated increased amounts of Fe and S and mutant plant had lower contents of S. In summary, our results suggest that the ISC Fe-S cluster biosynthetic pathway plays a crucial role in the homeostasis of Fe and S in plants, and that it may be important in their regulation.
Assuntos
Ferro/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Enxofre/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/genéticaRESUMO
OBJECTIVE: To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2. MATERIALS AND METHODS: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions. RESULTS: LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05). CONCLUSIONS: Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre/fisiologia , Enterococcus faecalis/crescimento & desenvolvimento , Percepção de Quorum/fisiologia , Análise de Variância , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Contagem de Colônia Microbiana , Enterococcus faecalis/genética , Técnicas de Inativação de Genes , Microscopia Confocal , Microscopia Eletrônica de Varredura , Plasmídeos , Percepção de Quorum/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de TempoRESUMO
Abstract Objective: To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2. Materials and Methods: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions. Results: LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05). Conclusions: Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.
Assuntos
Liases de Carbono-Enxofre/fisiologia , Proteínas de Bactérias/fisiologia , Enterococcus faecalis/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Percepção de Quorum/fisiologia , Plasmídeos , Liases de Carbono-Enxofre/genética , Fatores de Tempo , Proteínas de Bactérias/genética , Microscopia Eletrônica de Varredura , Contagem de Colônia Microbiana , Análise de Variância , Enterococcus faecalis/genética , Microscopia Confocal , Percepção de Quorum/genética , Técnicas de Inativação de Genes , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Escherichia coli is a highly adaptive microorganism, and its ability to form biofilms under certain conditions can be critical for antimicrobial resistance. The adhesion of four E. coli isolates from bovine mastitis to bovine mammary alveolar (MAC-T) cells, biofilm production on a polystyrene surface, and the expression profiles of the genes fliC, csgA, fimA, and luxS in the presence of enrofloxacin, gentamicin, co-trimoxazole, and ampicillin at half of the MIC were investigated. Increased adhesion of E. coli isolates in the presence of antimicrobials was not observed; however, increased internalization of some isolates was observed by confocal microscopy. All of the antimicrobials induced the formation of biofilms by at least one isolate, whereas enrofloxacin and co-trimoxazole decreased biofilm formation by at least one isolate. Quantitative PCR analysis revealed that all four genes were differentially expressed when bacteria were exposed to subinhibitory concentrations of antimicrobials, with expression altered on the order of 1.5- to 22-fold. However, it was not possible to associate gene expression with induction or reduction of biofilm formation in the presence of the antimicrobials. Taken together, the results demonstrate that antimicrobials could induce biofilm formation by some isolates, in addition to inducing MAC-T cell invasion, a situation that might occur in vivo, potentially resulting in a bacterial reservoir in the udder, which might explain some cases of persistent mastitis in herds.
Assuntos
Anti-Infecciosos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Escherichia coli/efeitos dos fármacos , Mastite Bovina/microbiologia , Leite/microbiologia , Ampicilina/farmacologia , Animais , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Liases de Carbono-Enxofre/genética , Bovinos , Enrofloxacina , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Feminino , Proteínas de Fímbrias/genética , Flagelina/genética , Fluoroquinolonas/farmacologia , Gentamicinas/farmacologia , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/tratamento farmacológico , Poliestirenos , Combinação Trimetoprima e Sulfametoxazol/farmacologiaRESUMO
Quorum sensing is a cell-cell signaling mechanism based on cell density and that involves the production of hormone-like molecules called autoinducers (AI). One of the most studied AIs has been termed AI-2, and its biosynthesis requires the enzyme encoded by luxS. We have previously described for the first time that Bacteroides species can produce molecules with AI-2 activity. In this study, we focus on the detection of luxS and its activity as the AI-2 synthase in Bacteroides species. The strains Bacteroides fragilis B3b and Bacteroides vulgatus ATCC 8482 were selected based on a positive phenotype for AI-2 production and the presence of a putative luxS in the genome, respectively. In order to identify the luxS gene, cloning and heterologous expression strategies were utilized. We demonstrate that both strains contain functional luxS orthologs that can complement AI-2 production in Escherichia coli.
Assuntos
Proteínas de Bactérias/genética , Bacteroides fragilis/genética , Bacteroides/genética , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre/genética , Regulação Bacteriana da Expressão Gênica , Homosserina/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Bacteroides/metabolismo , Bacteroides fragilis/metabolismo , Liases de Carbono-Enxofre/metabolismo , Sequência Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Homosserina/biossíntese , Lactonas , Dados de Sequência Molecular , Percepção de Quorum , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de SinaisRESUMO
In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk(-)ppx(-) or ppk(-)ppx(-)/ppk(+) strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.
Assuntos
Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Polifosfatos/metabolismo , Percepção de Quorum/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre/metabolismo , Meios de Cultura/metabolismo , Meios de Cultivo Condicionados/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Homosserina/análogos & derivados , Homosserina/biossíntese , Lactonas , Polifosfatos/farmacologia , Percepção de Quorum/efeitos dos fármacos , Fatores de Virulência/biossíntese , Fatores de Virulência/genéticaRESUMO
Genus Deinococcus is characterized by an increased resistance toward reactive oxygen species (ROS). The chromosome of five strains belonging to this genus has been sequenced and the presence of a luxS-like gene was deduced from their genome sequences. The aim of this study was to assess if a complete QS circuit is present in Deinococcus sp. and if this QS is associated with ROS. Primers for searching luxS-like gene and the putative receptor gene, namely ai2R, were designed. AI-2 signal production was evaluated by luminescence analysis using Vibrio harveyi BB170 as reporter strain. AI-2 signal was also evaluated by competitive assays using cinnamaldehyde, ascorbic acid, and 3-mercaptopropionic acid as interfering molecules. Potassium tellurite and metronidazole were used as oxidative stressors. A luxS-like gene as well as an ai2R gene was detected in strain UDEC-P1 by PCR. Cell-free supernatant of strain UDEC-P1 culture induced luminescence in V. harveyi BB170, and this property was inhibited with the three interfering molecules. The oxidative stressors metronidazole and potassium tellurite decreased Deinococcus sp. viability, but increased luminescence of the reporter strain. The results demonstrate that both a functional luxS-like gene and a putative receptor for AI-2 signal are present in Deinococcus sp. strain UDEC-P1. This finding also suggests that a complete QS circuit is present in this genus, which could be related to oxidative stress.
Assuntos
Deinococcus/efeitos dos fármacos , Farmacorresistência Bacteriana , Percepção de Quorum , Espécies Reativas de Oxigênio/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Deinococcus/classificação , Deinococcus/genética , Deinococcus/fisiologia , Regulação Bacteriana da Expressão Gênica , Homosserina/análogos & derivados , Homosserina/genética , Homosserina/metabolismo , Lactonas/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
AtNfs1 is the Arabidopsis thaliana mitochondrial homolog of the bacterial cysteine desulfurases NifS and IscS, having an essential role in cellular Fe-S cluster assembly. Homology modeling of AtNfs1m predicts a high global similarity with E. coli IscS showing a full conservation of residues involved in the catalytic site, whereas the chloroplastic AtNfs2 is more similar to the Synechocystis sp. SufS. Pull-down assays showed that the recombinant mature form, AtNfs1m, specifically binds to Arabidopsis frataxin (AtFH). A hysteretic behavior, with a lag phase of several minutes, was observed and hysteretic parameters were affected by pre-incubation with AtFH. Moreover, AtFH modulates AtNfs1m kinetics, increasing V(max) and decreasing the S(0.5) value for cysteine. Results suggest that AtFH plays an important role in the early steps of Fe-S cluster formation by regulating AtNfs1 activity in plant mitochondria.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , Mitocôndrias/enzimologia , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Liases de Carbono-Enxofre/genética , Cinética , Mitocôndrias/química , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de SequênciaRESUMO
BACKGROUND: Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. RESULTS: Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. CONCLUSIONS: Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression.
Assuntos
Liases de Carbono-Enxofre/metabolismo , Regulação da Expressão Gênica de Plantas , Glycine max/enzimologia , Proteínas de Soja/metabolismo , Liases de Carbono-Enxofre/genética , DNA de Plantas/genética , Genes Mitocondriais , Proteínas Ferro-Enxofre/biossíntese , Filogenia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Regiões Promotoras Genéticas , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas de Soja/genética , Glycine max/genética , Estresse FisiológicoRESUMO
Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe-S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe-S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe-S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe-S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe-4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.