RESUMO
Management of the plant microbiome may help support food needs for the human population. Bacteria influence plants through enhancing nutrient uptake, metabolism, photosynthesis, biomass production and/or reinforcing immunity. However, information into how these microbes behave under different growth conditions is missing. In this work, we tested how carbon supplements modulate the interaction of Pseudomonas chlororaphis with Arabidopsis thaliana. P. chlororaphis streaks strongly repressed primary root growth, lateral root formation and ultimately, biomass production. Noteworthy, increasing sucrose availability into the media from 0 to 2.4% restored plant growth and promoted lateral root formation in bacterized seedlings. This effect could not be observed by supplementing sucrose to leaves only, indicating that the interaction was strongly modulated by bacterial access to sugar. Total phenazine content decreased in the bacteria grown in high (2.4%) sucrose medium, and conversely, the expression of phzH and pslA genes were diminished by sugar supply. Pyocyanin antagonized the promoting effects of sucrose in lateral root formation and biomass production in inoculated seedlings, indicating that this virulence factor accounts for growth repression during the plant-bacterial interaction. Defence reporter transgenes PR-1::GUS and LOX2::GUS were induced in leaves, while the expression of the auxin-inducible, synthetic reporter gene DR5::GUS was enhanced in the roots of bacterized seedlings at low and high sucrose treatments, which suggests that growth/defence trade-offs in plants are critically modulated by P. chlororaphis. Collectively, our data suggest that bacterial carbon nutrition controls the outcome of the relation with plants.
Assuntos
Arabidopsis , Ácidos Indolacéticos , Fenazinas , Raízes de Plantas , Pseudomonas chlororaphis , Sacarose , Sacarose/metabolismo , Arabidopsis/microbiologia , Arabidopsis/metabolismo , Arabidopsis/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/metabolismo , Pseudomonas chlororaphis/metabolismo , Fenazinas/metabolismo , Ácidos Indolacéticos/metabolismoRESUMO
The capacity of Pseudomonas aeruginosa to assimilate nutrients is essential for niche colonization and contributes to its pathogenicity. Isocitrate lyase (ICL), the first enzyme of the glyoxylate cycle, redirects isocitrate from the tricarboxylic acid cycle to render glyoxylate and succinate. P. aeruginosa ICL (PaICL) is regarded as a virulence factor due to its role in carbon assimilation during infection. The AceA/ICL protein family shares the catalytic domain I, triosephosphate isomerase barrel (TIM-barrel). The carboxyl terminus of domain I is essential for Escherichia coli ICL (EcICL) of subfamily 1. PaICL, which belongs to subfamily 3, has domain II inserted at the periphery of domain I, which is believed to participate in enzyme oligomerization. In addition, PaICL has the α13-loop-α14 (extended motif), which protrudes from the enzyme core, being of unknown function. This study investigates the role of domain II, the extended motif, and the carboxyl-terminus (C-ICL) and amino-terminus (N-ICL) regions in the function of the PaICL enzyme, also as their involvement in the virulence of P. aeruginosa PAO1. Deletion of domain II and the extended motif results in enzyme inactivation and structural instability of the enzyme. The His6-tag fusion at the C-ICL protein produced a less efficient enzyme than fusion at the N-ICL, but without affecting the acetate assimilation or virulence. The PaICL homotetrameric structure of the enzyme was more stable in the N-His6-ICL than in the C-His6-ICL, suggesting that the C-terminus is critical for the ICL quaternary conformation. The ICL-mutant A39 complemented with the recombinant proteins N-His6-ICL or C-His6-ICL were more virulent than the WT PAO1 strain. The findings indicate that the domain II and the extended motif are essential for the ICL structure/function, and the C-terminus is involved in its quaternary structure conformation, confirming that in P. aeruginosa, the ICL is essential for acetate assimilation and virulence.
Assuntos
Isocitrato Liase , Pseudomonas aeruginosa , Isocitrato Liase/genética , Isocitrato Liase/química , Isocitrato Liase/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ciclo do Ácido Cítrico , Glioxilatos/metabolismo , Acetatos/metabolismoRESUMO
In the current research, our work measured the effect of silver nanoparticles (AgNP) synthesized from Larrea tridentata (Sessé and Moc. ex DC.) on the mycelial growth and morphological changes in mycelia from different phytopathogenic and beneficial fungi. The assessment was conducted in Petri dishes, with Potato-Dextrose-Agar (PDA) as the culture medium; the AgNP concentrations used were 0, 60, 90, and 120 ppm. Alternaria solani and Botrytis cinerea showed the maximum growth inhibition at 60 ppm (70.76% and 51.75%). Likewise, Macrophomina spp. required 120 ppm of AgNP to achieve 65.43%, while Fusarium oxisporum was less susceptible, reaching an inhibition of 39.04% at the same concentration. The effect of silver nanoparticles was inconspicuous in Pestalotia spp., Colletotrichum gloesporoides, Phytophthora cinnamomi, Beauveria bassiana, Metarhizium anisopliae, and Trichoderma viridae fungi. The changes observed in the morphology of the fungi treated with nanoparticles were loss of definition, turgidity, and constriction sites that cause aggregations of mycelium, dispersion of spores, and reduced mycelium growth. AgNP could be a sustainable alternative to managing diseases caused by Alternaria solani and Macrophomina spp.
Assuntos
Ascomicetos , Fusarium , Nanopartículas Metálicas , Prata/farmacologia , Fungos , Alternaria , Meios de Cultura/farmacologiaRESUMO
Plants being sessile organisms are exposed to various biotic and abiotic factors, thus causing stress. The Pseudomonas aeruginosa bacterium is an opportunistic pathogen for animals, insects, and plants. Direct exposure of Arabidopsis thaliana to the P. aeruginosa PAO1 strain induces plant death by producing a wide variety of virulence factors, which are regulated mainly by quorum sensing systems. Besides virulence factors, P. aeruginosa PAO1 also produces cyclodipeptides (CDPs), which possess auxin-like activity and promote plant growth through activation of the target of the rapamycin (AtTOR) pathway. On the other hand, plant defense mechanisms are regulated through the production of phytohormones, such as salicylic acid (SA) and jasmonic acid (JA), which are induced in response to pathogen-associated molecular patterns (PAMPs), activating defense genes associated with SA and JA such as PATHOGENESIS-RELATED-1 (PR-1) and LIPOXYGENASE2 (LOX2), respectively. PR proteins are suggested to play critical roles in coordinating the Systemic Acquired Resistance (SAR). In contrast, LOX proteins (LOX2, LOX3, and LOX4) have been associated with the production of JA by producing its precursors, oxylipins. The activation of defense mechanisms involves signaling cascades such as Mitogen-Activated Protein Kinases (MAPKs) or the TOR pathway as a switch for re-directing energy towards defense or growth. In this work, we challenged A. thaliana (wild type, mpk6 or mpk3 mutants, and overexpressing TOR) seedlings with P. aeruginosa PAO1 strains to identify the role of bacterial CDPs in the plant immune response. Results showed that the pre-exposure of these Arabidopsis seedlings to CDPs significantly reduced plant infection of the pathogenic P. aeruginosa PAO1 strains, indicating that plants that over-express AtTOR or lack MPK3/MPK6 protein-kinases are more susceptible to the pathogenic effects. In addition, CDPs induced the GUS activity only in the LOX2::GUS plants, indicative of JA-signaling activation. Our findings indicate that the CDPs are molecules that trigger SA-independent and JA-dependent defense responses in A. thaliana; hence, bacterial CDPs may be considered elicitors of the Arabidopsis immune response to pathogens.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Imunidade , Oxilipinas/metabolismo , Desenvolvimento Vegetal , Doenças das Plantas/microbiologia , Pseudomonas aeruginosa , Ácido Salicílico/metabolismo , Fatores de Virulência/metabolismo , Fatores de Virulência/farmacologiaRESUMO
The pathogenic bacterium Pseudomonas aeruginosa possesses high metabolic versatility, with its effectiveness to cause infections likely due to its well-regulated genetic content. P. aeruginosa PAO1 has at least six fadD paralogous genes, which have been implicated in fatty acid (FA) degradation and pathogenicity. In this study, we used mutagenesis and a functional approach in P. aeruginosa PAO1 to determine the roles of the fadD4 gene in acyclic terpene (AT) and FA assimilation and on pathogenicity. The results indicate that fadD4 encodes a terpenoyl-CoA synthetase utilized for AT and FA assimilation. Additionally, mutations in fadD paralogs led to the modification of the quorum-sensing las/rhl systems, as well as the content of virulence factors pyocyanin, biofilm, rhamnolipids, lipopolysaccharides (LPS), and polyhydroxyalkanoates. In a Caenorhabditis elegans in vivo pathogenicity model, culture supernatants from the 24-h-grown fadD4 single mutant increased lethality compared to the PAO1 wild-type (WT) strain; however, the double mutants fadD1/fadD2, fadD1/fadD4, and fadD2/fadD4 and single mutant fadD2 increased worm survival. A correlation analysis indicated an interaction between worm death by the PAO1 strain, the fadD4 mutation, and the virulence factor LPS. Fatty acid methyl ester (FAME) analysis of LPS revealed that a proportion of the LPS and FA on lipid A were modified by the fadD4 mutation, suggesting that FadD4 is also involved in the synthesis/degradation and modification of the lipid A component of LPS. LPS isolated from the fadD4 mutant and double mutants fadD1/fadD4 and fadD2/fadD4 showed a differential behavior to induce an increase in body temperature in rats injected with LPS compared to the WT strain or from the fadD1 and fadD2 mutants. In agreement, LPS isolated from the fadD4 mutant and double mutants fadD1/fadD2 and fadD2/fadD4 increased the induction of IL-8 in rat sera, but IL1-ß cytokine levels decreased in the double mutants fadD1/fadD2 and fadD1/fadD4. The results indicate that the fadD genes are implicated in the degree of pathogenicity of P. aeruginosa PAO1 induced by LPS-lipid A, suggesting that FadD4 contributes to the removal of acyl-linked FA from LPS, rendering modification in its immunogenic response associated to Toll-like receptor TLR4. The genetic redundancy of fadD is important for bacterial adaptability and pathogenicity over the host.
RESUMO
Multi-walled carbon nanotubes (MWCNTs) are of multidisciplinary scientific interest due to their exceptional physicochemical properties and a broad range of applications. However, they are considered potentially toxic nanoparticles when they accumulate in the environment. Given their ability to oxidize resistant polymers, mycorremediation with lignocellulolytic fungi are suggested as biological alternatives to the mineralization of MWCNTs. Hence, this study involves the ability of two fungi specie to MWCNTs biotransformation by laccase and peroxidases induction and evaluation in vivo of its toxicity using Caenorhabditis elegans worms as a model. Results showed that the fungi Penicillium chrysogenum and Pleurotus ostreatus were capable to grow on media with MWCNTs supplemented with glucose or lignin. Activities of lignin-peroxidase, manganese-peroxidase, and laccase in cultures of both fungi were induced by MWCNTs. Raman, FTIR spectroscopy, HR-TEM, and TGA analyses of the residue from the cultures of both fungi revealed structural modifications on the surface of MWCNTs and its amount diminished, correlating the MWCNTs structural modifications with the laccase-peroxidase activities in the fungal cultures. Results indicate that the degree of toxicity of MWCNTs on the C. elegans model was enhanced by the structure modification associated with the fungal ligninolytic activity. The toxic effect of MWCNTs on the in vivo model of worms reveals the increment of reactive oxygen species as a mechanism of toxicity. Findings indicate that the MWCNTs can be subject in nature to biotransformation processes such as the fungal metabolism, which contribute to modify their toxicity properties on susceptible organisms and contributing to environmental elimination.
RESUMO
Cyclodipeptides (CDPs) are the smallest peptidic molecules that can be produced by diverse organisms such as bacteria, fungi, and animals. They have multiple biological effects. In this paper, we examined the CDPs produced by the bacteria Pseudomonas aeruginosa PAO1, which are known as opportunistic pathogens of humans and plants on TARGET OF RAPAMYCIN (TOR) signaling pathways, and regulation of root system architecture. This bacterium produces the bioactive CDPs: cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-L-Val). In a previous report, these molecules were found to modulate basic cellular programs not only via auxin mechanisms but also by promoting the phosphorylation of the S6 ribosomal protein kinase (S6K), a downstream substrate of the TOR kinase. In the present work, we found that the inoculation of Arabidopsis plants with P. aeruginosa PAO1, the non-pathogenic P. aeruginosa ΔlasI/Δrhll strain (JM2), or by direct exposure of plants to CDPs influenced growth and promoted root branching depending upon the treatment imposed, while genetic evidence using Arabidopsis lines with enhanced or decreased TOR levels indicated a critical role of this pathway in the bacterial phytostimulation.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Proteínas de Bactérias/fisiologia , Proteínas de Plantas/genética , Pseudomonas aeruginosa/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Dipeptídeos/fisiologia , Peptídeos Cíclicos/fisiologia , Proteínas de Plantas/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Melanoma is an aggressive cancer that utilizes multiple signaling pathways, including those that involve oncogenes, proto-oncogenes, and tumor suppressors. It has been suggested that melanoma formation requires cross-talk of the PI3K/Akt/mTOR and Ras-ERK pathways. This pathway cross-talk has been associated with aggressiveness, drug resistance, and metastasis; thus, simultaneous targeting of components of the different pathways involved in melanoma may aid in therapy. We have previously reported that bacterial cyclodipeptides (CDPs) are cytotoxic to HeLa cells and inhibit Akt phosphorylation. Here, we show that CDPs decreased melanoma size and tumor formation in a subcutaneous xenografted mouse melanoma model. In fact, CDPs accelerated death of B16-F0 murine melanoma cells. In mice, antitumor effect was improved by treatment with CDPs using cyclodextrins as drug vehicle. In tumors, CDPs caused nuclear fragmentation and changed the expression of the Bcl-2 and Ki67 apoptotic markers and promoted restoration of hyperactivation of the PI3K/Akt/mTOR pathway. Additionally, elements of several signaling pathways such as the Ras-ERK, PI3K/JNK/PKA, p27Kip1/CDK1/survivin, MAPK, HIF-1, epithelial-mesenchymal transition, and cancer stem cell pathways were also modified by treatment of xenografted melanoma mice with CDPs. The findings indicate that the multiple signaling pathways implicated in aggressiveness of the murine B16-F0 melanoma line are targeted by the bacterial CDPs. Molecular modeling of CDPs with protein kinases involved in neoplastic processes suggested that these compounds could indeed interact with the active site of the enzymes. The results suggest that CDPs may be considered as potential antineoplastic drugs, interfering with multiple pathways involved in tumor formation and progression.
RESUMO
Plants adapt to soil injury and biotic stress via cell regeneration. In Arabidopsis, root tip damage by genotoxic agents, antibiotics, UV light and cutting induces a program that recovers the missing tissues through activation of stem cells and involves ethylene response factor 115 (ERF115), which triggers cell replenishment. Here, we show that mutation of the gene encoding an MED18 subunit of the transcriptional MEDIATOR complex and chromate [Cr(VI)], an environmental pollutant, synergistically trigger a developmental program that enables the splitting of the meristem in vivo to produce twin roots. Expression of the quiescent centre gene marker WOX5, auxin-inducible DR5:GFP reporter and the ERF115 factor traced the changes in cell identity during the conversion of single primary root meristems into twin roots and were induced in an MED18 and chromate-dependent manner during the root twinning events, which also required auxin redistribution and signalling mediated by IAA14/SOLITARY ROOT (SLR1). Splitting of the root meristem allowed dichotomous root branching in Arabidopsis, a poorly understood process in which stem cells may act to enable whole organ regeneration.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Complexo Mediador/genética , Meristema/genética , Raízes de Plantas/genética , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Cromo/farmacologia , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Ácidos Indolacéticos/metabolismo , Complexo Mediador/metabolismo , Meristema/efeitos dos fármacos , Mutação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Fatores de Transcrição/genéticaRESUMO
Azospirillum brasilense colonizes plant roots and improves productivity, but the molecular mechanisms behind its phytostimulation properties remain mostly unknown. Here, we uncover an important role of TARGET OF RAPAMYCIN (TOR) signaling on the response of Arabidopsis thaliana to A. brasilense Sp245. The effect of the bacterium on TOR expression was analyzed in the transgenic line TOR/tor-1, which carries a translational fusion with the GUS reporter protein, and the activity of TOR was assayed thought the phosphorylation of its downstream signaling target S6K protein. Besides, the role of TOR on plant growth in inoculated plants was assessed using the ATP-competitive inhibitor AZD-8055. A decrease in growth of the primary root correlates with an improved branching and absorptive capacity via lateral root and root hair proliferation 6 days after transplant to different concentrations of the bacterium (103 or 105 CFU/mL). Bacterization increased the expression of TOR in shoot and root apexes and promoted phosphorylation of S6K 3 days after transplant. The TOR inhibitor AZD-8055 (1 µM) inhibited plant growth and cell division in root meristems and in lateral root primordia, interfering with the phytostimulation by A. brasilense. In addition, the role of auxin produced by the bacterium to stimulate TOR expression was explored. Noteworthy, the A. brasilense mutant FAJ009, impaired in auxin production, was unable to elicit TOR signaling to the level observed for the wild-type strain, showing the importance of this phyhormone to stimulate TOR signaling. Together, our findings establish an important role of TOR signaling for the probiotic traits elicited by A. brasilense in A. thaliana.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Azospirillum brasilense/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Arabidopsis/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Meristema/metabolismo , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosforilação , Desenvolvimento Vegetal , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Quinolinas/antagonistas & inibidores , Rhizobiaceae , Triazóis/antagonistas & inibidoresRESUMO
In fungi, heterotrimeric G proteins are key regulators of biological processes such as mating, virulence, morphology, among others. Mucor circinelloides is a model organism for many biological processes, and its genome contains the largest known repertoire of genes that encode putative heterotrimeric G protein subunits in the fungal kingdom: twelve Gα (McGpa1-12), three Gß (McGpb1-3), and three Gγ (McGpg1-3). Phylogenetic analysis of fungal Gα showed that they are divided into four distinct groups as reported previously. Fungal Gß and Gγ are also divided into four phylogenetic groups, and to our understanding this is the first report of a phylogenetic classification for fungal Gß and Gγ subunits. Almost all genes that encode putative heterotrimeric G subunits in M. circinelloides are differentially expressed during dimorphic growth, except for McGpg1 (Gγ) that showed very low mRNA levels at all developmental stages. Moreover, several of the subunits are expressed in a similar pattern and at the same level, suggesting that they constitute discrete complexes. For example, McGpb3 (Gß), and McGpg2 (Gγ), are co-expressed during mycelium growth, and McGpa1, McGpb2, and McGpg2, are co-expressed during yeast development. These findings provide the conceptual framework to study the biological role of these genes during M. circinelloides morphogenesis.
Assuntos
Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Mucor/crescimento & desenvolvimento , Mucor/metabolismo , Filogenia , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Dados de Sequência Molecular , Morfogênese , Mucor/química , Mucor/genética , Alinhamento de SequênciaRESUMO
Maize (Zea mays) root system architecture has a complex organization, with adventitious and lateral roots determining its overall absorptive capacity. To generate basic information about the earlier stages of root development, we compared the post-embryonic growth of maize seedlings germinated in water-embedded cotton beds with that of plants obtained from embryonic axes cultivated in liquid medium. In addition, the effect of four different auxins, namely indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on root architecture and levels of the heat shock protein HSP101 and the cell cycle proteins CKS1, CYCA1 and CDKA1 were analyzed. Our data show that during the first days after germination, maize seedlings develop several root types with a simultaneous and/or continuous growth. The post-embryonic root development started with the formation of the primary root (PR) and seminal scutellar roots (SSR) and then continued with the formation of adventitious crown roots (CR), brace roots (BR) and lateral roots (LR). Auxins affected root architecture in a dose-response fashion; whereas NAA and IBA mostly stimulated crown root formation, 2,4-D showed a strong repressing effect on growth. The levels of HSP101, CKS1, CYCA1 and CDKA in root and leaf tissues were differentially affected by auxins and interestingly, HSP101 registered an auxin-inducible and root specific expression pattern. Taken together, our results show the timing of early branching patterns of maize and indicate that auxins regulate root development likely through modulation of the HSP101 and cell cycle proteins.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ácidos Indolacéticos/farmacologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Plântula/metabolismo , Zea mays/metabolismo , Desenvolvimento Vegetal/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Plântula/efeitos dos fármacos , Zea mays/efeitos dos fármacos , Zea mays/crescimento & desenvolvimentoRESUMO
Superoxide dismutase (SOD) activities of the oomycete Phytophthora cinnamomi were examined. Five polypeptides with manganese superoxide dismutase (MnSOD) activity were found in mycelium growing in liquid culture with relative molecular weights ranging from approximately 25 to 100 kDa. Comparison with characterized avocado SODs showed no evidence for the presence of either iron or copper/zinc SODs in P. cinnamomi. The level of activity of the MnSOD polypeptides decreased in the presence of avocado root or cell wall components. Growth of P. cinnamomi, measured as dry weight, increased when the mycelium was grown in the presence of superoxide anion (O(2) (-)), which was added exogenously. Our results suggest that the metabolism of O(2) (-) has an important role in the development of P. cinnamomi.
Assuntos
Proteínas Fúngicas/química , Micélio/enzimologia , Phytophthora/enzimologia , Superóxido Dismutase/química , Parede Celular/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/química , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Oxidantes/farmacologia , Persea/microbiologia , Phytophthora/efeitos dos fármacos , Phytophthora/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Cianeto de Potássio/química , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo , Superóxidos/farmacologiaRESUMO
The enzymes involved in the catabolism of leucine are encoded by the liu gene cluster in Pseudomonas aeruginosa PAO1. A mutant in the liuE gene (ORF PA2011) of P. aeruginosa was unable to utilize both leucine/isovalerate and acyclic terpenes as the carbon source. The liuE mutant grown in culture medium with citronellol accumulated metabolites of the acyclic terpene pathway, suggesting an involvement of liuE in both leucine/isovalerate and acyclic terpene catabolic pathways. The LiuE protein was expressed as a His-tagged recombinant polypeptide purified by affinity chromatography in Escherichia coli. LiuE showed a mass of 33 kDa under denaturing and 79 kDa under nondenaturing conditions. Protein sequence alignment and fingerprint sequencing suggested that liuE encodes 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HMG-CoA lyase), which catalyzes the cleavage of HMG-CoA to acetyl-CoA and acetoacetate. LiuE showed HMG-CoA lyase optimal activity at a pH of 7.0 and 37 degrees C, an apparent K(m) of 100 microM for HMG-CoA and a V(max) of 21 micromol min(-1) mg(-1). These results demonstrate that the liuE gene of P. aeruginosa encodes for the HMG-CoA lyase, an essential enzyme for growth in both leucine and acyclic terpenes.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Leucina/metabolismo , Oxo-Ácido-Liases/genética , Oxo-Ácido-Liases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Terpenos/metabolismo , Acetoacetatos/metabolismo , Acil Coenzima A/metabolismo , Coenzima A/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Deleção de Genes , Expressão Gênica , Genes Bacterianos , Concentração de Íons de Hidrogênio , Cinética , Redes e Vias Metabólicas , Peso Molecular , Família Multigênica , Oxo-Ácido-Liases/química , Oxo-Ácido-Liases/isolamento & purificação , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , TemperaturaRESUMO
Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.