RESUMO
A rhizosphere strain, Achromobacter insolitus LCu2, was isolated from alfalfa (Medicago sativa L.) roots. It was able to degrade of 50% glyphosate as the sole phosphorus source, and was found resistant to 10 mM copper (II) chloride, and 5 mM glyphosate-copper complexes. Inoculation of alfalfa seedlings and potato microplants with strain LCu2 promoted plant growth by 30-50%. In inoculated plants, the toxicity of the glyphosate-copper complexes to alfalfa seedlings was decreased, as compared with the noninoculated controls. The genome of A. insolitus LCu2 consisted of one circular chromosome (6,428,890 bp) and encoded 5843 protein genes and 76 RNA genes. Polyphasic taxonomic analysis showed that A. insolitus LCu2 was closely related to A. insolitus DSM23807T on the basis of the average nucleotide identity of the genomes of 22 type strains and the multilocus sequence analysis. Genome analysis revealed genes putatively responsible for (1) plant growth promotion (osmolyte, siderophore, and 1-aminocyclopropane-1-carboxylate deaminase biosynthesis and auxin metabolism); (2) degradation of organophosphonates (glyphosate oxidoreductase and multiple phn clusters responsible for the transport, regulation and C-P lyase cleavage of phosphonates); and (3) tolerance to copper and other heavy metals, effected by the CopAB-CueO system, responsible for the oxidation of copper (I) in the periplasm, and by the efflux Cus system. The putative catabolic pathways involved in the breakdown of phosphonates are predicted. A. insolitus LCu2 is promising in the production of crops and the remediation of soils contaminated with organophosphonates and heavy metals.
Assuntos
Achromobacter , Cobre , Glicina , Glifosato , Medicago sativa , Rizosfera , Glicina/análogos & derivados , Glicina/metabolismo , Cobre/metabolismo , Achromobacter/genética , Achromobacter/metabolismo , Achromobacter/classificação , Achromobacter/efeitos dos fármacos , Medicago sativa/microbiologia , Filogenia , Genoma Bacteriano , Microbiologia do Solo , Raízes de Plantas/microbiologia , Genômica , Biodegradação AmbientalRESUMO
BACKGROUND AND AIMS: Plant diseases caused by Pectobacterium atrosepticum are often accompanied by extensive rot symptoms. In addition, these bacteria are able to interact with host plants without causing disease for long periods, even throughout several host plant generations. There is, to date, no information on the comparative physiology/biochemistry of symptomatic and asymptomatic plant-P. atrosepticum interactions. Typical (symptomatic) P. atrosepticum infections are associated with the induction of plant responses mediated by jasmonates, which are one of the products of the lipoxygenase cascade that gives origin to many other oxylipins with physiological activities. In this study, we compared the functioning of the lipoxygenase cascade following typical and latent (asymptomatic) infections to gain better insight into the physiological basis of the asymptomatic and antagonistic coexistence of plants and pectobacteria. METHODS: Tobacco plants were mock-inoculated (control) or infected with the wild type P. atrosepticum (typical infection) or its coronafacic acid-deficient mutant (latent infection). The expression levels of the target lipoxygenase cascade-related genes were assessed by Illumina RNA sequencing. Oxylipin profiles were analysed by GC-MS. With the aim of revising the incorrect annotation of one of the target genes, its open reading frame was cloned to obtain the recombinant protein, which was further purified and characterized using biochemical approaches. KEY RESULTS: The obtained data demonstrate that when compared to the typical infection, latent asymptomatic P. atrosepticum infection is associated with (and possibly maintained due to) decreased levels of 9-lipoxygenase branch products and jasmonic acid and increased level of cis-12-oxo-10,15-phytodienoic acid. The formation of 9-oxononanoic acid and epoxyalcohols in tobacco plants was based on the identification of the first tobacco hydroperoxide lyase (HPL) with additional epoxyalcohol synthase (EAS) activity. CONCLUSIONS: Our results contribute to the hypothesis of the oxylipin signature, indicating that different types of plant interactions with a particular pathogen are characterized by the different oxylipin profiles of the host plant. In addition, the tobacco LOC107825278 gene was demonstrated to encode an NtHPL (CYP74C43) enzyme yielding volatile aldehydes and aldoacids (HPL products) as well as oxiranyl carbinols (EAS products).
Assuntos
Lipoxigenase , Pectobacterium , Lipoxigenase/genética , Lipoxigenase/metabolismo , Pectobacterium/metabolismo , Doenças das Plantas/microbiologia , NicotianaRESUMO
The bacterial pathogen Salmonella enterica, which causes enteritis, has a broad host range and extensive environmental longevity. In water and soil, Salmonella interacts with protozoa and multiplies inside their phagosomes. Although this relationship resembles that between Salmonella and mammalian phagocytes, the interaction mechanisms and bacterial genes involved are unclear. Here, we characterized global gene expression patterns of S. enterica serovar Typhimurium within Acanthamoeba castellanii at the early stage of infection by Cappable-Seq. Gene expression features of S. Typhimurium within A. castellanii were presented with downregulation of glycolysis-related, and upregulation of glyoxylate cycle-related genes. Expression of Salmonella Pathogenicity Island-1 (SPI-1), chemotaxis system, and flagellar apparatus genes was upregulated. Furthermore, expression of genes mediating oxidative stress response and iron uptake was upregulated within A. castellanii as well as within mammalian phagocytes. Hence, global S. Typhimurium gene expression patterns within A. castellanii help better understand the molecular mechanisms of Salmonella adaptation to an amoeba cell and intracellular persistence in protozoa inhabiting water and soil ecosystems.
Assuntos
Acanthamoeba castellanii/microbiologia , Salmonella typhimurium/genética , Virulência/genética , Animais , Proteínas de Bactérias/genética , Ecossistema , Regulação Bacteriana da Expressão Gênica/genética , Ilhas Genômicas/genética , Mamíferos/microbiologiaRESUMO
A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta, O. anadyrensis, O. kamtschatica, and O. pumilio originating from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S ribosomal RNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium), Phyllobacteriaceae (genera Mesorhizobium, Phyllobacterium), and Bradyrhizobiaceae (genera Bosea, Tardiphaga). A plant nodulation assay showed that only strains belonging to genus Mesorhizobium could form nitrogen-fixing nodules on Oxytropis plants. The strains M. loti 582 and M. huakuii 583, in addition to symbiotic clusters, possessed genes of the type III and type VI secretion systems (T3SS and T6SS, respectively), which can influence the host specificity of strains. These strains formed nodules of two types (elongated and rounded) on O. kamtschatica roots. We suggest this phenomenon may result from Nod factor-dependent and -independent nodulation strategies. The obtained strains are of interest for further study of the T3SS and T6SS gene function and their role in the development of rhizobium-legume symbiosis. The prospects of using rhizobia having both gene systems related to symbiotic and nonsymbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.
Assuntos
Bradyrhizobiaceae , Mesorhizobium , Oxytropis/microbiologia , Rhizobium , Simbiose , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo VI/genética , Bradyrhizobiaceae/genética , Mesorhizobium/genética , Filogenia , RNA Ribossômico 16S/genética , Rhizobium/genética , Nódulos Radiculares de Plantas/microbiologiaRESUMO
Herein, for the first time the complexation ability of a homological series of triphenylphosphonium surfactants (TPPB-n) toward DNA decamers has been explored. Formation of lipoplexes was confirmed by alternative techniques, including dynamic light scattering, indicating the occurrence of nanosized complexes (ca. 100-150 nm), and monitoring the charge neutralization of nucleotide phosphate groups and the fluorescence quenching of dye-intercalator ethidium bromide. The complexation efficacy of TPPB-surfactants toward an oligonucleotide (ONu) is compared with that of reference cationic surfactants. Strong effects of the alkyl chain length and the structure of the head group on the surfactant/ONu interaction are revealed, which probably occur via different mechanisms, with electrostatic and hydrophobic forces or intercalation imbedding involved. Phosphonium surfactants are shown to be capable of disordering lipid bilayers, which is supported by a decrease in the temperature of the main phase transition, Tm. This effect enhances with an increase in the alkyl chain length, indicating the integration of TPPB-n with lipid membranes. This markedly differs from the behavior of typical cationic surfactant cetyltrimethylammonium bromide, which induces an increase in the Tm value. It was demonstrated that the cytotoxicity of TPPB-n in terms of the MTT-test on a human cell line 293T nonmonotonically changes within the homological series, with the highest cytotoxicity exhibited by the dodecyl and tetradecyl homologs.
Assuntos
DNA/química , Bicamadas Lipídicas/química , Ácidos Nucleicos/química , Tensoativos/química , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Tensoativos/toxicidadeRESUMO
In our study, the rare earth element ytterbium (Yb3+) was demonstrated to affect water exchange in roots of Zea mays seedlings. Herewith, the overall membrane permeability (Pd) increased. The Pd increase was determined by aquaporin activity but not the membrane lipid component since the closure of aquaporin channels due to low intracellular pH abolished the positive effect of Yb3+ on Pd. Additionally, the expression level of aquaporin genes ZmPIP2;2, ZmPIP2;6 and ZmTIP2;2 was increased when plants were grown in the presence of Yb3+. Our results indicate that previously described positive influence of rare earth metals on plant growth and productivity may be mediated (at least partially) by the modification of the plant hydraulic system.
Assuntos
Aquaporinas/metabolismo , Membrana Celular/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Água/metabolismo , Itérbio/farmacologia , Zea mays/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Raízes de Plantas/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Água/química , Itérbio/química , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismoRESUMO
Nonclassical P450s of CYP74 family control the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. At least ten genes attributed to four novel CYP74 subfamilies have been revealed by the recent sequencing of the spikemoss Selaginella moellendorffii Hieron genome. Two of these genes CYP74M1 and CYP74M3 have been cloned in the present study. Both recombinant proteins CYP74M1 and CYP74M3 were active towards the 13(S)-hydroperoxides of α-linolenic and linoleic acids (13-HPOT and 13-HPOD, respectively) and exhibited the activity of divinyl ether synthase (DES). Products were analyzed by gas chromatography-mass spectrometry. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the (1)H NMR, 2D-COSY, HSQC and HMBC. CYP74M1 (SmDES1) specifically converted 13-HPOT to (11Z)-etherolenic acid and 13-HPOD to (11Z)-etheroleic acid. CYP74M3 (SmDES2) turned 13-HPOT and 13-HPOD mainly to etherolenic and etheroleic acids, respectively. CYP74M1 and CYP74M3 are the first DESs detected in non-flowering plants. The obtained results demonstrate the existence of the sophisticated oxylipin biosynthetic machinery in the oldest taxa of vascular plants.
Assuntos
Clonagem Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Selaginellaceae/enzimologia , Compostos de Vinila/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Cinética , Ácidos Linoleicos/metabolismo , Ácidos Linolênicos/metabolismo , Peróxidos Lipídicos/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/genética , Proteínas Recombinantes/metabolismo , Selaginellaceae/classificação , Selaginellaceae/genética , Especificidade por SubstratoRESUMO
Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [18O2]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Compostos de Epóxi/metabolismo , Oxilipinas/metabolismo , Phaeophyceae/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Peróxido de Hidrogênio/metabolismo , Ácidos Linoleicos/metabolismo , Peróxidos Lipídicos/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Ácido alfa-LinolênicoRESUMO
Enzymes of the CYP74 family, including the divinyl ether synthase (DES), play important roles in plant cell signalling and defence. The potent DES activities have been detected before in the leaves of the meadow buttercup (Ranunculus acris L.) and few other Ranunculaceae species. The nature of these DESs and their genes remained unrevealed. The PCR with degenerate primers enabled to detect the transcript of unknown P450 gene assigned as CYP74Q1. Besides, two more CYP74Q1 isoforms with minimal sequence variations have been found. The full length recombinant CYP74Q1 protein was expressed in Escherichia coli. The preferred substrates of this enzyme are the 13-hydroperoxides of α-linolenic and linoleic acids, which are converted to the divinyl ether oxylipins (ω5Z)-etherolenic acid, (9Z,11E)-12-[(1'Z,3'Z)-hexadienyloxy]-9,11-dodecadienoic acid, and (ω5Z)-etheroleic acid, (9Z,11E)-12-[(1'Z)-hexenyloxy]-9,11-dodecadienoic acid, respectively, as revealed by the data of mass spectrometry, NMR and UV spectroscopy. Thus, CYP74Q1 protein was identified as the R. acris DES (RaDES), a novel DES type and the opening member of new CYP74Q subfamily.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Filogenia , Folhas de Planta/química , Proteínas de Plantas/metabolismo , Ranunculus/química , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/genética , Primers do DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ácidos Linoleicos/metabolismo , Dados de Sequência Molecular , Oxilipinas/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase , Ranunculus/enzimologia , Ranunculus/genética , Proteínas Recombinantes/classificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Ácido alfa-Linolênico/metabolismoRESUMO
Enzymes of the CYP74 family (P450 superfamily) play a key role in the plant lipoxygenase signalling cascade. Recently we detected a pathogen inducible divinyl ether synthase (DES) in flax leaves [Chechetkin, Blufard, Hamberg, Grechkin, 2008]. This prompted us to examine the CYP74 genes in the flax leaf transcriptome. Since the flax genome is not sequenced, we used the PCR approach with degenerate primers related to the conserved domains of selected CYP74 genes; this revealed several CYP74 transcripts in flax leaves. One transcript belongs to the previously described allene oxide synthase (LuAOS, CYP74A, GenBank ID: U00428.1). Another one contains the ORF (1473 bp) of an unknown CYP74B16 gene. Three more nearly identical sequences, including one expressed pseudogene, were also identified. The recombinant CYP74B16 protein expressed in Escherichia coli had 491 amino acid residues and MW of 56 kDa. The preferred substrate of this enzyme is the 13-hydroperoxide of α-linolenic acid, and the reaction product was identified by mass spectrometry, NMR and UV spectroscopy as the divinyl ether (9Z,11E)-12-[(1'Z,3'Z)-hexadienyloxy]-9,11-dodecadienoic acid, (ω5Z)-etherolenic acid. All previously known CYP74B subfamily enzymes are hydroperoxide lyases. The novel flax enzyme CYP74B16 (LuDES) is an unprecedented DES member of the CYP74B subfamily.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Linho/enzimologia , Linho/genética , Genes de Plantas/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Primers do DNA/metabolismo , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
We report the discovery of a new abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism by the rhizosphere bacterium Rhodococcus sp. P1Y. Analogue of (+)-ABA, enriched in tritium in the cyclohexene moiety, was fed in bacterial cells, and extracts containing radioactive metabolites were purified and analyzed to determine their structure. We obtained mass spectral fragmentation patterns and nuclear magnetic resonance spectra of a new metabolite of ABA identified as 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid, which we named rhodococcal acid (RA) and characterized using several other techniques. This metabolite is the second bacterial ABA degradation product in addition to dehydrovomifoliol that we described earlier. Taken together, these data reveal an unknown ABA catabolic pathway that begins with side chain disassembly, as opposed to the conversion of the cyclohexene moiety in plants. The role of ABA-utilizing bacteria in interactions with other microorganisms and plants is also discussed.
Assuntos
Ácido Abscísico , Ácido Acético , Ácido Abscísico/metabolismo , Trítio , Transformação Bacteriana , Extratos VegetaisRESUMO
The incredible success of crop breeding and agricultural innovation in the last century greatly contributed to the Green Revolution, which significantly increased yields and ensures food security, despite the population explosion. However, new challenges such as rapid climate change, deteriorating soil, and the accumulation of pollutants require much faster responses and more effective solutions that cannot be achieved through traditional breeding. Further prospects for increasing the efficiency of agriculture are undoubtedly associated with the inclusion in the breeding strategy of new knowledge obtained using high-throughput technologies and new tools in the future to ensure the design of new plant genomes and predict the desired phenotype. This article provides an overview of the current state of research in these areas, as well as the study of soil and plant microbiomes, and the prospective use of their potential in a new field of microbiome engineering. In terms of genomic and phenomic predictions, we also propose an integrated approach that combines high-density genotyping and high-throughput phenotyping techniques, which can improve the prediction accuracy of quantitative traits in crop species.
RESUMO
The phytohormone abscisic acid (ABA) plays an important role in plant growth and in response to abiotic stress factors. At the same time, its accumulation in soil can negatively affect seed germination, inhibit root growth and increase plant sensitivity to pathogens. ABA is an inert compound resistant to spontaneous hydrolysis and its biological transformation is scarcely understood. Recently, the strain Rhodococcus sp. P1Y was described as a rhizosphere bacterium assimilating ABA as a sole carbon source in batch culture and affecting ABA concentrations in plant roots. In this work, the intermediate product of ABA decomposition by this bacterium was isolated and purified by preparative HPLC techniques. Proof that this compound belongs to ABA derivatives was carried out by measuring the molar radioactivity of the conversion products of this phytohormone labeled with tritium. The chemical structure of this compound was determined by instrumental techniques including high-resolution mass spectrometry, NMR spectrometry, FTIR and UV spectroscopies. As a result, the metabolite was identified as (4RS)-4-hydroxy-3,5,5-trimethyl-4-[(E)-3-oxobut-1-enyl]cyclohex-2-en-1-one (dehydrovomifoliol). Based on the data obtained, it was concluded that the pathway of bacterial degradation and assimilation of ABA begins with a gradual shortening of the acyl part of the molecule.
Assuntos
Ácido Abscísico/metabolismo , Cicloexanonas/metabolismo , Rizosfera , Rhodococcus/metabolismo , Regulação da Expressão Gênica de Plantas , Espectroscopia de Ressonância Magnética , Reguladores de Crescimento de Plantas/metabolismoRESUMO
In enteric bacteria, DNA supercoiling is highly responsive to environmental conditions. Host specific features of environment serve as cues for the expression of genes required for colonization of host niches via changing supercoiling [1]. It has been shown that substitution at position 87 of GyrA of Salmonella enterica str. SL1344 influences global supercoiling and results in an altered transcriptome with increased expression of stress response pathways [2]. Aminocoumarin antibiotics, such as novobiocin, can be used to relax supercoiling and alter the expression of supercoiling-sensitive genes. Meanwhile, Salmonella enterica demonstrates a significant resistance to this antibiotic and relatively small variability of supercoiling in response to the growth phase, osmotic pressure, and novobiocin treatment. Here we present for the first time transcriptome data of Salmonella enterica subsp. Enterica serovar Typhimurium str. 14028S grown in the presence of novobiocin. These data will help identify genes involved in novobiocin resistance and adaptation processes associated with torsion perturbations in S. enterica. Cleaned FASTQ files for the RNA-seq libraries are deposited in the NCBI Sequence Read Archive (SRA, Identifier: SRP239815) and have been assigned BioProject accession PRJNA599397.
RESUMO
Plant growth-promoting rhizobacteria (PGPR) improve plant productivity and stress resistance. The mechanisms involved in plant-microbe interactions include the modulation of plant hormone status. The Novosphingobium sp. strain P6W was previously described as the bacterium capable of abscisic acid (ABA) degradation, and its inoculation decreased ABA concentrations in planta. The metabolic pathway for the ABA degradation in bacteria is still unknown. Here we present transcriptome data of Novosphingobium sp. P6W grown in the medium supplemented with ABA or fructose as the carbon source. Cleaned FASTQ files for the RNA-seq libraries are deposited in the NCBI Sequence Read Archive (SRA, Identifier: SRP189498) and have been assigned BioProject accession PRJNA529223.
RESUMO
Salmonella enterica is an ubiquitous pathogen throughout the world causing gastroenteritis in humans and animals. Survival of pathogenic bacteria in the external environment may be associated with the ability to overcome the stress caused by starvation. The bacterial response to starvation is well understood in laboratory cultures with a sufficiently high cell density. However, bacterial populations often have a small size when facing this challenge in natural biotopes. The aim of this work was to find out if there are differences in the transcriptomes of S. enterica depending on the factor of cell density during starvation. Here we present transcriptome data of Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S grown in carbon rich or carbon deficient medium with high or low cell density. These data will help identify genes involved in adaptation of low-density bacterial populations to starvation conditions.
RESUMO
Mechanisms of microbial catabolism of phytohormone abscisic acid (ABA) are still unknown. Here, we report the complete genome sequence of ABA-utilizing Rhodococcus sp. strain P1Y, isolated from the rice (Oryza sativa L.) rhizosphere. The sequence was obtained using an approach combining Oxford Nanopore Technologies MinION and Illumina MiSeq sequence data.
RESUMO
Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site-directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild-type LeAOS3. Thus, both sites 295 and 297 localized within the "I-helix central domain" ("oxygen binding domain") are the primary determinants of CYP74 type of catalysis.
Assuntos
Aldeído Liases/química , Sistema Enzimático do Citocromo P-450/química , Oxirredutases Intramoleculares/química , Proteínas de Plantas/química , Aldeído Liases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Catálise , Biologia Computacional , Sistema Enzimático do Citocromo P-450/genética , Oxirredutases Intramoleculares/genética , Solanum lycopersicum/enzimologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Análise de Sequência de ProteínaRESUMO
The conversion of linoleic acid 9-hydroperoxide (9-HPOD) by recombinant melon (Cucumis melo L.) hydroperoxide lyase (HPL, CYP74C subfamily) was studied. Short (5 s-1 min) incubations at 0 degrees C followed by rapid extraction and trimethylsilylation made it possible to trap a new unstable (t(1/2) <30 s) product, i.e. the hemiacetal (1'E,3'Z)-9-hydroxy-9-(1',3'-nonadienyloxy)-nonanoic acid. Identification was performed by GC-MS analysis and substantiated by the formation of trimethylsilyl 9-trimethylsilyloxy-9-nonyloxy-nonanoate upon catalytic hydrogenation and by (2)H-labelling experiments. Both (18)O atoms of [(18)O(2)-hydroperoxy]9-HPOD were incorporated into the hemiacetal. Along with the hemiacetal, three chain-cleavage products, i.e. the enol (1E,3Z)-nonadienol and the hydrates of 3(Z)-nonenal and 9-oxononanoic acid, were trapped as their trimethylsilyl derivatives. The kinetics of (18)O incorporation from [(18)O(2)]9-HPOD provided strong evidence that the cleavage products originated in the hemiacetal. Linolenic and linoleic acid 13-hydroperoxides served as substrates for recombinant HPLs of melon, alfalfa (Medicago sativa) and guava (Psidium guajava), and in each case hemiacetals and enols were detectable by the trapping technique. The data obtained demonstrated that CYP74C and CYP74B HPLs act as isomerases performing a homolytic rearrangement of fatty acid hydroperoxides into short-lived hemiacetals which upon decomposition produce 3(Z)-nonenal, 3(Z)-hexenal and other short chain aldehydes.
Assuntos
Aldeído Liases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Peróxidos Lipídicos/metabolismo , Acetais/química , Acetais/metabolismo , Aldeído Liases/genética , Cucumis melo/enzimologia , Cucumis melo/genética , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Isomerismo , Ácidos Linoleicos/química , Ácidos Linoleicos/metabolismo , Peróxidos Lipídicos/química , Medicago sativa/enzimologia , Medicago sativa/genética , Estrutura Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Psidium/enzimologia , Psidium/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Defensins are part of the innate immune system in plants with activity against a broad range of pathogens, including bacteria, fungi and viruses. Several defensins from conifers, including Scots pine defensin 1 (Pinus sylvestris defensin 1, (PsDef1)) have shown a strong antifungal activity, however structural and physico-chemical properties of the family, needed for establishing the structure-dynamics-function relationships, remain poorly characterized. We use several spectroscopic and computational methods to characterize the structure, dynamics, and oligomeric state of PsDef1. The three-dimensional structure was modeled by comparative modeling using several programs (Geno3D, SWISS-MODEL, I-TASSER, Phyre(2), and FUGUE) and verified by circular dichroism (CD) and infrared (FTIR) spectroscopy. Furthermore, FTIR data indicates that the structure of PsDef1 is highly resistant to high temperatures. NMR diffusion experiments show that defensin exists in solution in the equilibrium between monomers and dimers. Four types of dimers were constructed using the HADDOCK program and compared to the known dimer structures of other plant defensins. Gaussian network model was used to characterize the internal dynamics of PsDef1 in monomer and dimer states. PsDef1 is a typical representative of P. sylvestris defensins and hence the results of this study are applicable to other members of the family.