RESUMO
Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.
Assuntos
Blastocladiella/crescimento & desenvolvimento , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Transporte de Elétrons , Oxirredução , Oxigênio/metabolismoRESUMO
Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K(+) channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K(+) selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K(+) channel and the rhodopsin-guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K(+) channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus.
Assuntos
Blastocladiomycota/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas Fúngicas/metabolismo , Canais de Potássio/metabolismo , Blastocladiomycota/genética , Blastocladiomycota/fisiologia , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Proteínas Fúngicas/genética , Potenciais da Membrana , Potássio/metabolismo , Canais de Potássio/genética , Esporos Fúngicos/metabolismoRESUMO
BACKGROUND: With the aim of remaining viable, bacteria must deal with changes in environmental conditions, including increases in external osmolarity. While studies concerning bacterial response to this stress condition have focused on soil, marine and enteric species, this report is about Caulobacter crescentus, a species inhabiting freshwater oligotrophic habitats. RESULTS: A genomic analysis reported in this study shows that most of the classical genes known to be involved in intracellular solute accumulation under osmotic adaptation are missing in C. crescentus. Consistent with this observation, growth assays revealed a restricted capability of the bacterium to propagate under hyperosmotic stress, and addition of the compatible solute glycine betaine did not improve bacterial resistance. A combination of transcriptomic and proteomic analyses indicated quite similar changes triggered by the presence of either salt or sucrose, including down-regulation of many housekeeping processes and up-regulation of functions related to environmental adaptation. Furthermore, a GC-MS analysis revealed some metabolites at slightly increased levels in stressed cells, but none of them corresponding to well-established compatible solutes. CONCLUSION: Despite a clear response to hyperosmotic stress, it seems that the restricted capability of C. crescentus to tolerate this unfavorable condition is probably a consequence of the inability to accumulate intracellular solutes. This finding is consistent with the ecology of the bacterium, which inhabits aquatic environments with low nutrient concentration.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/metabolismo , Betaína/farmacologia , Transporte Biológico , Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/metabolismo , Água Doce/microbiologia , Perfilação da Expressão Gênica , Anotação de Sequência Molecular , Concentração Osmolar , Pressão Osmótica , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Sacarose/farmacologiaRESUMO
Sensing light is the fundamental property of visual systems, with vision in animals being based almost exclusively on opsin photopigments [1]. Rhodopsin also acts as a photoreceptor linked to phototaxis in green algae [2, 3] and has been implicated by chemical means as a light sensor in the flagellated swimming zoospores of the fungus Allomyces reticulatus [4]; however, the signaling mechanism in these fungi remains unknown. Here we use a combination of genome sequencing and molecular inhibition experiments with light-sensing phenotype studies to examine the signaling pathway involved in visual perception in the closely related fungus Blastocladiella emersonii. Our data show that in these fungi, light perception is accomplished by the function of a novel gene fusion (BeGC1) of a type I (microbial) rhodopsin domain and guanylyl cyclase catalytic domain. Photobleaching of rhodopsin function prevents accumulation of cGMP levels and phototaxis of fungal zoospores exposed to green light, whereas inhibition of guanylyl cyclase activity negatively affects fungal phototaxis. Immunofluorescence microscopy localizes the BeGC1 protein to the external surface of the zoospore eyespot positioned close to the base of the swimming flagellum [4, 5], demonstrating this is a photoreceptive organelle composed of lipid droplets. Taken together, these data indicate that Blastocladiomycota fungi have a cGMP signaling pathway involved in phototaxis similar to the vertebrate vision-signaling cascade but composed of protein domain components arranged as a novel gene fusion architecture and of distant evolutionary ancestry to type II rhodopsins of animals.
Assuntos
Blastocladiella/fisiologia , Proteínas Fúngicas/genética , Guanilato Ciclase/genética , Luz , Rodopsina/genética , Transdução de Sinais , Sequência de Aminoácidos , Sequência de Bases , Blastocladiella/genética , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas Fúngicas/metabolismo , Fusão Gênica , Guanilato Ciclase/metabolismo , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Rodopsina/metabolismo , Alinhamento de Sequência , Percepção VisualRESUMO
BACKGROUND: The α-proteobacterium Caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metals in these sites. It has been reported that C. crescentus responds to exposure to various heavy metals by altering the expression of a large number of genes. RESULTS: In this work, we show that the ECF sigma factor σF is one of the regulatory proteins involved in the control of the transcriptional response to chromium and cadmium. Microarray experiments indicate that σF controls eight genes during chromium stress, most of which were previously described as induced by heavy metals. Surprisingly, σF itself is not strongly auto-regulated under metal stress conditions. Interestingly, σF-dependent genes are not induced in the presence of agents that generate reactive oxygen species. Promoter analyses revealed that a conserved σF-dependent sequence is located upstream of all genes of the σF regulon. In addition, we show that the second gene in the sigF operon acts as a negative regulator of σF function, and the encoded protein has been named NrsF (Negative regulator of sigma F). Substitution of two conserved cysteine residues (C131 and C181) in NrsF affects its ability to maintain the expression of σF-dependent genes at basal levels. Furthermore, we show that σF is released into the cytoplasm during chromium stress and in cells carrying point mutations in both conserved cysteines of the protein NrsF. CONCLUSION: A possible mechanism for induction of the σF-dependent genes by chromium and cadmium is the inactivation of the putative anti-sigma factor NrsF, leading to the release of σF to bind RNA polymerase core and drive transcription of its regulon.
Assuntos
Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/fisiologia , Regulação Bacteriana da Expressão Gênica , Metais Pesados/toxicidade , Fator sigma/metabolismo , Estresse Fisiológico , Cádmio/toxicidade , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Cromo/toxicidade , Perfilação da Expressão Gênica , Análise em Microsséries , ÓperonRESUMO
The extracytoplasmic function sigma factor σ(T) is the master regulator of general stress response in Caulobacter crescentus and controls the expression of its paralogue σ(U). In this work we showed that PhyR and NepR act, respectively, as positive and negative regulators of σ(T) expression and function. Biochemical data also demonstrated that NepR directly binds σ(T) and the phosphorylated form of PhyR. We also described the essential role of the histidine kinase gene CC3474, here denominated phyK, for expression of σ(T)-dependent genes and for resistance to stress conditions. Additionally, in vivo evidence of PhyK-dependent phosphorylation of PhyR is presented. This study also identified a conserved cysteine residue (C95) located in the periplasmic portion of PhyK that is crucial for the function of the protein. Furthermore, we showed that PhyK, PhyR and σ(T) regulate the same set of genes and that σ(T) apparently directly controls most of its regulon. In contrast, σ(U) seems to have a very modest contribution to the expression of a subset of σ(T)-dependent genes. In conclusion, this report describes the molecular mechanism involved in the control of general stress response in C. crescentus.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Caulobacter crescentus/enzimologia , Caulobacter crescentus/genética , Histidina Quinase , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Fator sigma/genética , Estresse FisiológicoRESUMO
Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAT1 genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1Δ) of this gene. The gat1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gat1 is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans.
Assuntos
Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica , Nitrogênio/metabolismo , Transativadores/metabolismo , Animais , Aspergillus nidulans/genética , Candida albicans/genética , Criptococose/microbiologia , Cryptococcus neoformans/genética , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/genética , Fatores de Transcrição GATA/genética , Deleção de Genes , Perfilação da Expressão Gênica , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Análise em Microsséries , Regulon , Homologia de Sequência de Aminoácidos , Análise de Sobrevida , Transativadores/genética , Virulência , Dedos de ZincoRESUMO
BACKGROUND: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. RESULTS: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. CONCLUSIONS: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Nitrogênio/metabolismo , RNA Polimerase Sigma 54/metabolismo , Regulon , Xylella/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/genética , Xylella/química , Xylella/genéticaRESUMO
Global gene expression analysis was carried out with Blastocladiella emersonii cells subjected to oxygen deprivation (hypoxia) using cDNA microarrays. In experiments of gradual hypoxia (gradual decrease in dissolved oxygen) and direct hypoxia (direct decrease in dissolved oxygen), about 650 differentially expressed genes were observed. A total of 534 genes were affected directly or indirectly by oxygen availability, as they showed recovery to normal expression levels or a tendency to recover when cells were reoxygenated. In addition to modulating many genes with no putative assigned function, B. emersonii cells respond to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favor anaerobic metabolism through the upregulation of genes encoding glycolytic enzymes and lactate dehydrogenase and the downregulation of most genes coding for tricarboxylic acid (TCA) cycle enzymes. Furthermore, genes involved in energy-costly processes, like protein synthesis, amino acid biosynthesis, protein folding, and transport, had their expression profiles predominantly downregulated during oxygen deprivation, indicating an energy-saving effort. Data also revealed similarities between the transcriptional profiles of cells under hypoxia and under iron(II) deprivation, suggesting that Fe(2+) ion could have a role in oxygen sensing and/or response to hypoxia in B. emersonii. Additionally, treatment of fungal cells prior to hypoxia with the antibiotic geldanamycin, which negatively affects the stability of mammalian hypoxia transcription factor HIF-1alpha, caused a significant decrease in the levels of certain upregulated hypoxic genes.
Assuntos
Blastocladiella/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Blastocladiella/metabolismo , Hipóxia Celular , Proteínas Fúngicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oxigênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca(2+), and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources.
Assuntos
Blastocladiella/fisiologia , Perfilação da Expressão Gênica , Esporos Fúngicos/fisiologia , Blastocladiella/citologia , Blastocladiella/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Análise por Conglomerados , Proteínas do Citoesqueleto/genética , Regulação para Baixo/genética , Proteínas Fúngicas/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Glucose/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/genética , Biossíntese de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esporos Fúngicos/efeitos dos fármacos , Fatores de Tempo , Triptofano/farmacologia , Regulação para Cima/genéticaRESUMO
The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor Nomega-Nitro-L-arginine methyl ester (L-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when L-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca(2+)-(*)NO-cGMP signaling pathway playing a role in zoospore biogenesis.
Assuntos
Blastocladiella/fisiologia , Cálcio/farmacologia , Ativadores de Enzimas/farmacologia , Óxido Nítrico/metabolismo , Transdução de Sinais , Esporos Fúngicos/crescimento & desenvolvimento , Sequência de Aminoácidos , GMP Cíclico/análise , Citosol/química , Inibidores Enzimáticos/farmacologia , Etiquetas de Sequências Expressas , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/genética , Dados de Sequência Molecular , Óxido Nítrico Sintase/metabolismo , Oxidiazóis/farmacologia , Quinoxalinas/farmacologia , Alinhamento de SequênciaRESUMO
Caulobacter crescentussigma(E) belongs to the ECF (extracytoplasmic function) subfamily of RNA polymerase sigma factors, whose members regulate gene expression in response to distinct environmental stresses. During physiological growth conditions, data indicate that sigma(E) is maintained in reduced levels due to the action of ChrR, a negative regulator of rpoE gene expression and function. However, once bacterial cells are exposed to cadmium, organic hydroperoxide, singlet oxygen or UV-A irradiation, transcription of rpoE is induced in a sigma(E)-dependent manner. Site-directed mutagenesis indicated that residue C188 in ChrR is critical for the cadmium response while residues H140 and H142 are required for the bacterial response to organic hydroperoxide, singlet oxygen and UV-A. Global transcriptional analysis showed that sigma(E) regulates genes involved in protecting cells against oxidative damages. A combination of transcriptional start site identification and promoter prediction revealed that some of these genes contain a putative sigma(E)-dependent motif in their upstream regions. Furthermore, deletion of rpoE and two sigma(E)-dependent genes (cfaS and hsp20) impairs Caulobacter survival when singlet oxygen is constantly generated in the cells.
Assuntos
Cádmio/metabolismo , Caulobacter crescentus/genética , Peróxido de Hidrogênio/metabolismo , Fator sigma/metabolismo , Oxigênio Singlete/metabolismo , Raios Ultravioleta , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Caulobacter crescentus/efeitos da radiação , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Mutagênese Sítio-Dirigida , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , RNA Bacteriano/genética , Transcrição GênicaRESUMO
Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class. During germination, the zoospore, a motile nongrowing cell, goes through a cascade of morphological changes that culminates with its differentiation into the germling cell, capable of coenocytic vegetative growth. Transcriptome analyses of B. emersonii cells were carried out during germination induced under various environmental conditions. Microarray data analyzing 3,563 distinct B. emersonii genes revealed that 26% of them are differentially expressed during germination in nutrient medium at at least one of the time points investigated. Over 500 genes are upregulated during the time course of germination under those conditions, most being related to cell growth, including genes involved in protein biosynthesis, DNA transcription, energetic metabolism, carbohydrate and oligopeptide transport, and cell cycle control. On the other hand, several transcripts stored in the zoospores are downregulated during germination in nutrient medium, such as genes involved in signal transduction, amino acid transport, and chromosome organization. In addition, germination induced in the presence of nutrients was compared with that triggered either by adenine or potassium ions in inorganic salt solution. Several genes involved in cell growth, induced during germination in nutrient medium, do not show increased expression when B. emersonii zoospores germinate in inorganic solution, suggesting that nutrients exert a positive effect on gene transcription. The transcriptome data also revealed that most genes involved in cell signaling show the same expression pattern irrespective of the initial germination stimulus.
Assuntos
Blastocladiella/fisiologia , Perfilação da Expressão Gênica , Esporos Fúngicos/fisiologia , Blastocladiella/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Esporos Fúngicos/genética , Transcrição GênicaRESUMO
The mitochondrial genome of the chytrid Blastocladiella emersonii was sequenced and annotated, revealing the complete set of oxidative phosphorylation genes and tRNAs/rRNAs necessary for the translation process. Phylogenetic reconstructions reinforce the proposal of the new phylum Blastocladiomycota. Evidences of gene duplication due to inserted elements suggest shared susceptibility to gene invasion/exchange between chytrids and zygomycetes. The gene content of B. emersonii is very similar to Allomyces macrogynus but the content of intronic and changeable elements is much lower suggesting a stronger resistance to this kind of exchange. In addition, a total of 401 potential nuclear transcripts encoding mitochondrial proteins were obtained after B. emersonii EST database scanning using Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana data as probes and TargetP tool to find N-terminal mitochondrial signal in translated sequences.
Assuntos
Blastocladiella/genética , DNA Fúngico/genética , DNA Mitocondrial/genética , Adenina , Allomyces/genética , Blastocladiella/classificação , Códon/genética , DNA Fúngico/isolamento & purificação , Biblioteca Gênica , Genoma Fúngico , Fases de Leitura Aberta , Filogenia , TiminaRESUMO
HSP90 proteins are important molecular chaperones involved in multiple cellular processes. This work reports the characterization of cDNAs encoding two distinct HSP90 proteins (named HSP90A and HSP90B) from the chytridiomycete Blastocladiella emersonii. Deduced amino acid sequences of HSP90A and HSP90B exhibit signatures of the cytosolic and endoplasmic reticulum (ER) HSP90 proteins, respectively. A genomic clone encoding HSP90A was also characterized indicating the presence of a single intron of 184 bp interrupting the coding region, located near the amino-terminus of the protein. Expression of both HSP90A and HSP90B genes increases significantly during heat shock at 38 degrees C, with highest induction ratios observed in cells stressed during germination of the fungus. Changes in the amount of HSP90A transcript were also evaluated during B. emersonii life cycle at physiological temperature (27 degrees C), and its levels were found to increase both during germination and sporulation of the fungus. HSP90A protein levels were analyzed during B. emersonii life cycle and significant changes were observed only during sporulation. Furthermore, during heat stress a large increase in the amount of HSP90A protein was observed. Induction of HSP90A and HSP90B genes during heat stress indicates the importance of both genes in the response to high temperature in B. emersonii.
Assuntos
Blastocladiella/genética , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP90/genética , Sequência de Aminoácidos , Blastocladiella/crescimento & desenvolvimento , Clonagem Molecular , DNA Complementar , Expressão Gênica , Genes Fúngicos , Temperatura Alta , Dados de Sequência Molecular , Elementos Reguladores de Transcrição , Sítio de Iniciação de TranscriçãoRESUMO
The phytopathogen Xylella fastidiosa produces long type IV pili and short type I pili involved in motility and adhesion. In this work, we have investigated the role of sigma factor sigma(54) (RpoN) in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from the non-pathogenic citrus strain J1a12, and microarray analyses of global gene expression comparing the wild type and rpoN mutant strains showed few genes exhibiting differential expression. In particular, gene pilA1 (XF2542), which encodes the structural pilin protein of type IV pili, showed decreased expression in the rpoN mutant, whereas two-fold higher expression of an operon encoding proteins of type I pili was detected, as confirmed by quantitative RT-PCR (qRT-PCR) analysis. The transcriptional start site of pilA1 was determined by primer extension, downstream of a sigma(54)-dependent promoter. Microarray and qRT-PCR data demonstrated that expression of only one of the five pilA paralogues, pilA1, was significantly reduced in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that sigma(54) differentially regulates genes involved in type IV and type I fimbrial biogenesis, and is involved in biofilm formation in X. fastidiosa.
Assuntos
Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , RNA Polimerase Sigma 54/genética , RNA Polimerase Sigma 54/metabolismo , Xylella/genética , Xylella/metabolismo , Sequência de Aminoácidos , Aderência Bacteriana/genética , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Citrus , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/ultraestrutura , Deleção de Genes , Perfilação da Expressão Gênica , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Sítio de Iniciação de Transcrição , Xylella/crescimento & desenvolvimento , Xylella/ultraestruturaRESUMO
Sigma factors of the ECF subfamily are important regulators of stress responses in bacteria. Analysis of Caulobacter crescentus genome sequence has indicated the presence of 13 members of the ECF (extracytoplasmic function) subfamily, suggesting that these regulators play an important role in C. crescentus physiology. This work describes the characterization of two highly similar C. crescentus ECF sigma factors, sigma(U) and sigma(T). The corresponding genes are not essential under normal growth conditions and absence of sigma(U) does not impair bacterial resistance to the environmental stresses tested. However, absence of sigma(T) significantly affects the ability of C. crescentus cells to survive osmotic and oxidative stress. Using transcription fusions to sigT and sigU upstream regions we demonstrate that both genes are induced by osmotic stress in a sigma(T)-dependent manner. Determination of sigU and sigT transcription start sites revealed an identical promoter motif, typical of ECF-dependent promoters. Transcriptome analysis revealed 40 putative members of the sigma(T) regulon, including sigU and sigR, encoding another ECF subfamily member, and genes involved in general stress responses and cell envelope functions. Twenty of those genes exhibit the sigT/sigU promoter motif in their upstream regions. Our data indicate a role of sigma(T) in distinct stress responses in C. crescentus.
Assuntos
Proteínas de Bactérias/fisiologia , Caulobacter crescentus/fisiologia , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , Fator sigma/fisiologia , Fusão Gênica Artificial , Sítios de Ligação/genética , Caulobacter crescentus/genética , Perfilação da Expressão Gênica , Genes Reporter , Viabilidade Microbiana/genética , Pressão Osmótica , Sítio de Iniciação de Transcrição , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
The global transcriptional response of the chytridiomycete Blastocladiella emersonii to environmental stress conditions was explored by sequencing a large number of expressed sequence tags (ESTs) from three distinct cDNA libraries, constructed with mRNA extracted from cells exposed to heat shock and different concentrations of cadmium chloride. A total of 6,350 high-quality EST sequences were obtained and assembled into 2,326 putative unigenes, 51% of them not previously described in B. emersonii. To approximately 59% of the unigenes it was possible to assign an orthologue in another organism, whereas 41% of them remained without a putative identification, with transcripts related to protein folding and antioxidant activity being highly enriched in the stress libraries. A microarray chip was constructed encompassing 3,773 distinct ESTs from the B. emersonii transcriptome presently available, which correspond to a wide range of biological processes. Global gene expression analysis of B. emersonii cells exposed to stress conditions revealed a large number of differentially expressed genes: 122 up- and 60 downregulated genes during heat shock and 189 up- and 110 downregulated genes during exposure to cadmium. The main functional categories represented among the upregulated genes were protein folding and proteolysis, proteins with antioxidant properties, and cellular transport. Interestingly, in response to cadmium stress, B. emersonii cells induced genes encoding six different glutathione S-transferases and six distinct metacaspases, as well as genes coding for several proteins of sulfur amino acid metabolism, indicating that cadmium causes oxidative stress and apoptosis in this fungus. All sequences described in this study have been submitted to the GenBank EST section with the accession numbers EE 730389 to EE 736848.
Assuntos
Blastocladiella , Cádmio/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Resposta ao Choque Térmico/fisiologia , Proteoma/análise , Blastocladiella/genética , Blastocladiella/metabolismo , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos TestesRESUMO
Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, named rpoE and rseA, predicted to encode an extracytoplasmic function (ECF) sigma factor and an anti-sigma factor, respectively. In this work, an rpoE null mutant was constructed in the citrus strain J1a12 and shown to be sensitive to exposure to heat shock and ethanol. To identify the X. fastidiosa sigma(E) regulon, global gene expression profiles were obtained by DNA microarray analysis of bacterial cells under heat shock, identifying 21 sigma(E)-dependent genes. These genes encode proteins belonging to different functional categories, such as enzymes involved in protein folding and degradation, signal transduction, and DNA restriction modification and hypothetical proteins. Several putative sigma(E)-dependent promoters were mapped by primer extension, and alignment of the mapped promoters revealed a consensus sequence similar to those of ECF sigma factor promoters of other bacteria. Like other ECF sigma factors, rpoE and rseA were shown to comprise an operon in X. fastidiosa, together with a third open reading frame (XF2241). However, upon heat shock, rpoE expression was not induced, while rseA and XF2241 were highly induced at a newly identified sigma(E)-dependent promoter internal to the operon. Therefore, unlike many other ECF sigma factors, rpoE is not autoregulated but instead positively regulates the gene encoding its putative anti-sigma factor.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Fator sigma/genética , Xylella/genética , Motivos de Aminoácidos , Proteínas de Bactérias/fisiologia , Sequência de Bases , Perfilação da Expressão Gênica , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regulon/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Fator sigma/fisiologia , Sítio de Iniciação de Transcrição , Transcrição Gênica , Xylella/metabolismoRESUMO
Misfolding and aggregation of protein molecules are major threats to all living organisms. Therefore, cells have evolved quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins. DnaK/DnaJ and GroES/GroEL are the best-characterized molecular chaperone systems in bacteria. In Caulobacter crescentus these chaperone machines are the products of essential genes, which are both induced by heat shock and cell cycle regulated. In this work, we characterized the viabilities of conditional dnaKJ and groESL mutants under different types of environmental stress, as well as under normal physiological conditions. We observed that C. crescentus cells with GroES/EL depleted are quite resistant to heat shock, ethanol, and freezing but are sensitive to oxidative, saline, and osmotic stresses. In contrast, cells with DnaK/J depleted are not affected by the presence of high concentrations of hydrogen peroxide, NaCl, and sucrose but have a lower survival rate after heat shock, exposure to ethanol, and freezing and are unable to acquire thermotolerance. Cells lacking these chaperones also have morphological defects under normal growth conditions. The absence of GroE proteins results in long, pinched filamentous cells with several Z-rings, whereas cells lacking DnaK/J are only somewhat more elongated than normal predivisional cells, and most of them do not have Z-rings. These findings indicate that there is cell division arrest, which occurs at different stages depending on the chaperone machine affected. Thus, the two chaperone systems have distinct roles in stress responses and during cell cycle progression in C. crescentus.