RESUMEN
The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacter diazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70 kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333 AA) 6PGDs, compared to the long-chain subfamily (480 AA; e.g., Ovis aries, Homo sapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.
Asunto(s)
Proteínas Bacterianas/metabolismo , Gluconacetobacter/enzimología , Gluconatos/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Ribulosafosfatos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Gluconacetobacter/genética , Gluconatos/química , Humanos , Modelos Químicos , Modelos Moleculares , Estructura Molecular , NAD/metabolismo , NADP/metabolismo , Fosfogluconato Deshidrogenasa/clasificación , Fosfogluconato Deshidrogenasa/genética , Filogenia , Dominios Proteicos , Multimerización de Proteína , Ribulosafosfatos/química , Homología de Secuencia de AminoácidoRESUMEN
The use of plant growth-promoting bacteria represents an alternative to the massive use of mineral fertilizers in agriculture. However, some abiotic stresses commonly found in the environment, like salinity, can affect the efficiency of this approach. Here, we investigated the key mechanisms involved in the response of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus to salt stress by using morphological and cell viability analyses, comparative proteomics, and reverse genetics. Our results revealed that the bacteria produce filamentous cells in response to salt at 100 mM and 150 mM NaCl. However, such a response was not observed at higher concentrations, where cell viability was severely affected. Proteomic analysis showed that salt stress modulates proteins involved in several pathways, including iron uptake, outer membrane efflux, osmotic adjustment, cell division and elongation, and protein transport and quality control. Proteomic data also revealed the repression of several extracytoplasmic proteins, especially those located at periplasm and outer membrane. The role of such pathways in the tolerance to salt stress was analyzed by the use of mutant defectives for Δtbdr (iron uptake), ΔmtlK and ΔotsA (compatible solutes synthesis), and ΔdegP (quality control of nascent extracytoplasmic proteins). ΔdegP presented the highest sensitivity to salt stress, Δtbdr, andΔmtlK also showed increased sensitivity, but ΔotsA was not affected. This is the first demonstration that DegP protein, a protease with minor chaperone activity, is essential for tolerance to salt stress in G. diazotrophicus. Our data contribute to a better understanding of the molecular bases that control the bacterial response/tolerance to salt stress, shedding light on quality control of nascent extracytoplasmic proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Gluconacetobacter/metabolismo , Proteínas de Choque Térmico/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas Periplasmáticas/metabolismo , Serina Endopeptidasas/metabolismo , Cloruro de Sodio/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Gluconacetobacter/enzimología , Gluconacetobacter/genética , Proteínas de Choque Térmico/genética , Hierro/metabolismo , Péptido Hidrolasas/genética , Proteínas Periplasmáticas/genética , Serina Endopeptidasas/genéticaRESUMEN
Gluconacetobacter diazotrophicus is a species of great agronomic potential due to its growth-promotion traits. Its colonization process in different plants has been reported. However, there have been no studies regarding its structural colonization in elephant grass. This is a fast-growing C4-Poaceae plant, and its application in Brazil is mainly aimed at feeding dairy cattle, due to its high nutritional value. Also, in the last decade, this grass has been applied in the production of biofuels. The present study aimed to monitor the colonization process of strain LP343 of G. diazotrophicus inoculated in elephant grass seedlings of PCEA genotype, by using a mCherry-tagged bacterium. Samples of roots and shoots collected at different periods were visualized by confocal laser-scanning microscopy. The colony-counting assay was used to compare the number of cells recovered in different niches and a qPCR was performed for the quantification of endophytic cells in root and shoot tissues. Results suggested that the strain LP343 quickly recognized the PCEA roots as host, attached to the elephant grass roots at 6 h, and 7 days after inoculation were able to colonize the xylem vessels of roots and shoots of elephant grass. This study advances our knowledge about the colonization process of G. diazotrophicus species in elephant grass, contributing to future studies involving the plant-bacteria interaction cultivated under gnotobiotic conditions.
Asunto(s)
Gluconacetobacter/crecimiento & desarrollo , Pennisetum/microbiología , Raíces de Plantas/microbiología , Brotes de la Planta/microbiología , Brasil , Vida Libre de Gérmenes , Gluconacetobacter/genética , Gluconacetobacter/aislamiento & purificación , Pennisetum/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , Brotes de la Planta/crecimiento & desarrollo , Plantones/crecimiento & desarrollo , Plantones/microbiologíaRESUMEN
Microorganisms are constantly challenged by stressful conditions, such as sugar-rich environments. Such environments can cause an imbalance of biochemical activities and compromise cell multiplication. Gluconacetobacter diazotrophicus PAl 5 is among the most sugar-tolerant bacteria, capable of growing in the presence of up to 876 mM sucrose. However, the molecular mechanisms involved in its response to high sucrose remain unknown. The present work aimed to identify sucrose-induced stress resistance genes in G. diazotrophicus PAl 5. Screening of a Tn5 transposon insertion library identified a mutant that was severely compromised in its resistance to high sucrose concentrations. Molecular characterization revealed that the mutation affected the kupA gene, which encodes a K+ uptake transporter (KupA). Functional complementation of the mutant with the wild type kupA gene recovered the sucrose-induced stress resistance phenotype. High sucrose resistance assay, under different potassium concentrations, revealed that KupA acts as a high-affinity K+ transporter, which is essential for resistance to sucrose-induced stress, when extracellular potassium levels are low. This study is the first to show the essential role of the KupA protein for resistance to sucrose-induced stress in bacteria by acting as a high-affinity potassium transporter in G. diazotrophicus PAl 5.
Asunto(s)
Gluconacetobacter/efectos de los fármacos , Gluconacetobacter/fisiología , Proteínas de Transporte de Membrana/metabolismo , Presión Osmótica , Potasio/metabolismo , Estrés Fisiológico , Sacarosa/metabolismo , Elementos Transponibles de ADN , Prueba de Complementación Genética , Gluconacetobacter/genética , Mutagénesis InsercionalRESUMEN
Greater Mexico City is one of the largest urban centers in the world, with an estimated population by 2010 of more than 20 million inhabitants. In urban areas like this, biological material is present at all atmospheric levels including live bacteria. We sampled the low atmosphere in several surveys at different points by the gravity method on LB and blood agar media during winter, spring, summer, and autumn seasons in the years 2008, 2010, 2011, and 2012. The colonial phenotype on blood agar showed α, ß, and γ hemolytic activities among the live collected bacteria. Genomic DNA was extracted and convenient V3 hypervariable region libraries of 16S rDNA gene were high-throughput sequenced. From the data analysis, Firmicutes, Proteobacteria, and Actinobacteria were the more abundant phyla in all surveys, while the genera from the family Enterobacteriaceae, in addition to Bacillus spp., Pseudomonas spp., Acinetobacter spp., Erwinia spp., Gluconacetobacter spp., Proteus spp., Exiguobacterium spp., and Staphylococcus spp. were also abundant. From this study, we conclude that it is possible to detect live airborne nonspore-forming bacteria in the low atmosphere of GMC, associated to the microbial cloud of its inhabitants.
Asunto(s)
Microbiología del Aire , Bacterias/clasificación , Biodiversidad , Filogenia , Actinobacteria/genética , Actinobacteria/aislamiento & purificación , Bacillus/genética , Bacillus/aislamiento & purificación , Bacterias/aislamiento & purificación , Ciudades , Medios de Cultivo , ADN Bacteriano/genética , Genómica , Gluconacetobacter/genética , Gluconacetobacter/aislamiento & purificación , México , Proteobacteria/genética , Proteobacteria/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Gluconacetobacter diazotrophicus is a nitrogen-fixing, endophytic bacterium that has the potential to promote plant growth and increase yield. Genetically modified strains might get more benefits to host plants, including through expression of useful proteins, such as Cry toxins from B. thuringiensis, or enzymes involved in phytohormone production, proteins with antagonistic activity for phytopathogens, or that improve nutrient utilization by the plant. For that, expression systems for G. diazotrophicus are needed, which requires active promoters fused to foreign (or innate) genes. This article describes the construction of a G. diazotrophicus PAL5 promoter library using a promoter-less lacZ-bearing vector, and the identification of six active promoters through ß-galactosidase activity assays, sequencing and localization in the bacterial genome. The characterized promoters, which are located on distinct regions of the bacterial genome and encoding either sense or antisense transcripts, present variable expression strengths and might be used in the future for expressing useful proteins.
Asunto(s)
Técnicas Genéticas , Genoma Bacteriano/genética , Gluconacetobacter/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Biblioteca de Genes , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas/microbiología , beta-Galactosidasa/metabolismoRESUMEN
TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.
Asunto(s)
Proteínas Bacterianas/genética , Gluconacetobacter/genética , Proteínas de la Membrana/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Sideróforos/genética , Transporte Biológico/genética , Medios de Cultivo/química , Prueba de Complementación Genética , Gluconacetobacter/enzimología , Gluconacetobacter/metabolismo , Hierro/metabolismo , Mutagénesis Insercional , Mutación , Nitrogenasa/genética , Fenotipo , Sideróforos/análisis , Sideróforos/metabolismoRESUMEN
The endophytic bacterium Gluconacetobacter diazotrophicus colonizes a broad range of host plants. Its plant growth-promoting capability is related to the capacity to perform biological nitrogen fixation, the biosynthesis of siderophores, antimicrobial substances and the solubilization of mineral nutrients. Colonization of and survival in these endophytic niche requires a complex regulatory network. Among these, quorum sensing systems (QS) are signaling mechanisms involved in the control of several genes related to microbial interactions, host colonization and stress survival. G. diazotrophicus PAL5 possesses a QS composed of a luxR and a luxI homolog, and produces eight molecules from the AHL family as QS signals. In this report data are provided showing that glucose concentration modifies the relative levels of these signal molecules. The activity of G. diazotrophicus PAL5 QS is also altered in presence of other carbon sources and under saline stress conditions. Inactivation of the QS system of G. diazotrophicus PAL5 by means of a quorum quenching strategy allowed the identification of extracellular and intracellular proteins under the control of this regulatory mechanism.
Asunto(s)
Gluconacetobacter/efectos de los fármacos , Gluconacetobacter/fisiología , Glucosa/metabolismo , Percepción de Quorum/efectos de los fármacos , Carbono/metabolismo , Redes Reguladoras de Genes , Gluconacetobacter/genética , Plantas/microbiologíaRESUMEN
Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km(R) -mutant strains in a medium without iron supplementation and in a medium containing 2, 2'-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km(R) -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus.
Asunto(s)
Proteínas Bacterianas/genética , Gluconacetobacter/genética , Hierro/metabolismo , Operón , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/genética , Transporte Biológico , Gluconacetobacter/metabolismo , Proteínas de Transporte de Membrana/genética , Fijación del Nitrógeno , Proteínas de Unión Periplasmáticas/genética , Saccharum/microbiologíaRESUMEN
The phylogenetic position of a cellulose-producing acetic acid bacterium, strain ID13488, isolated from commercially available Colombian homemade fruit vinegar, was investigated. Analyses using nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rRNA gene internal transcribed spacer (ITS) sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated the micro-organism to the genus Gluconacetobacter, and more precisely to the Gluconacetobacter xylinus group. Moreover, the data suggested that the micro-organism belongs to a novel species in this genus, together with LMG 1693(T), a non-cellulose-producing strain isolated from vinegar by Kondo and previously classified as a strain of Gluconacetobacter xylinus. DNA-DNA hybridizations confirmed this finding, revealing a DNA-DNA relatedness value of 81â% between strains ID13488 and LMG 1693(T), and values <70â% between strain LMG 1693(T) and the type strains of the closest phylogenetic neighbours. Additionally, the classification of strains ID13488 and LMG 1693(T) into a single novel species was supported by amplified fragment length polymorphism (AFLP) and (GTG)5-PCR DNA fingerprinting data, as well as by phenotypic data. Strains ID13488 and LMG 1693(T) could be differentiated from closely related species of the genus Gluconacetobacter by their ability to produce 2- and 5-keto-d-gluconic acid from d-glucose, their ability to produce acid from sucrose, but not from 1-propanol, and their ability to grow on 3â% ethanol in the absence of acetic acid and on ethanol, d-ribose, d-xylose, sucrose, sorbitol, d-mannitol and d-gluconate as carbon sources. The DNA G+C content of strains ID13488 and LMG 1693(T) was 58.0 and 60.7 mol%, respectively. The major ubiquinone of LMG 1693(T) was Q-10. Taken together these data indicate that strains ID13488 and LMG 1693(T) represent a novel species of the genus Gluconacetobacter for which the name Gluconacetobacter medellinensis sp. nov. is proposed. The type strain is LMG 1693(T) (â=âNBRC 3288(T)â=âKondo 51(T)).
Asunto(s)
Ácido Acético , Celulosa/biosíntesis , Gluconacetobacter/clasificación , Filogenia , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Técnicas de Tipificación Bacteriana , Composición de Base , Colombia , ADN Bacteriano/genética , Ácidos Grasos/análisis , Genes Bacterianos , Gluconacetobacter/genética , Gluconacetobacter/aislamiento & purificación , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
The mechanisms of cadmium, cobalt and zinc resistance were characterized in the plant-growth-promoting bacterium Gluconacetobacter diazotrophicus PAl 5. The resistance level of the wild-type strain was evaluated through the establishment of minimum inhibitory concentrations (MIC) of the soluble compounds CdCl2·H2O, CoCl2·6H2O and ZnCl2. Gluconacetobacter diazotrophicus PAl 5 was resistant to high concentrations of Cd, Co and Zn, with MICs of 1.2, 20 and 20 mM, respectively. Screening of an insertion library from transposon EZ-Tn5
Asunto(s)
Cadmio/toxicidad , Cobalto/toxicidad , Gluconacetobacter/efectos de los fármacos , Zinc/toxicidad , Secuencia de Aminoácidos , Southern Blotting , Elementos Transponibles de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Gluconacetobacter/genética , Gluconacetobacter/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutagénesis Insercional , FilogeniaRESUMEN
Gluconacetobacter diazotrophicus strain PAL5 is a nitrogen-fixing endophytic bacterium originally isolated from sugarcane and later on was found to colonize other plants such as rice, elephant grass, sweet potato, coffee, and pineapple. Currently, G. diazotrophicus has been considered a plant growth-promoting bacterium due to its characteristics of biological nitrogen fixation, phytohormone secretion, solubilization of mineral nutrients and antagonism to phytopathogens. Reverse transcription followed by quantitative real-time polymerase chain reaction (RT-qPCR) is a method applied for the quantification of nucleic acids because of its specificity and high sensitivity. However, the decision about the reference genes suitable for data validation is still a major issue, especially for nitrogen-fixing bacteria. To evaluate and identify suitable reference genes for gene expression normalization in the diazotrophic G. diazotrophicus, mRNA levels of fourteen candidate genes (rpoA, rpoC, recA, rpoD, fabD, gmk, recF, rho, ldhD, gyrB, gyrBC, dnaG, lpxC and 23SrRNA) and three target genes (matE, omp16 and sucA) were quantified by RT-qPCR after growing the bacteria in different carbon sources. The geNorm and Normfinder programs were used to calculate the expression stabilities. The analyses identified three genes, rho, 23SrRNA and rpoD, whose expressions were stable throughout the growth of strain PAL5 in the chosen carbon sources. In conclusion our results strongly suggest that these three genes are suitable to be used as reference genes for real-time RT-qPCR data normalization in G. diazotrophicus.
Asunto(s)
Carbono/metabolismo , Perfilación de la Expresión Génica/métodos , Gluconacetobacter/genética , Gluconacetobacter/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Perfilación de la Expresión Génica/normas , Genes Bacterianos , Gluconacetobacter/crecimiento & desarrollo , Reacción en Cadena en Tiempo Real de la Polimerasa/normasRESUMEN
The genome of the endophytic diazotrophic bacterial species Gluconacetobacter diazotrophicus PAL5 (PAL5) revealed the presence of a gum gene cluster. In this study, the gumD gene homologue, which is predicted to be responsible for the first step in exopolysaccharide (EPS) production, was insertionally inactivated and the resultant mutant (MGD) was functionally studied. The mutant MGD presented normal growth and nitrogen (N(2)) fixation levels but did not produce EPS when grown on different carbon sources. MGD presented altered colony morphology on soft agar plates (0.3% agar) and was defective in biofilm formation on glass wool. Most interestingly, MGD was defective in rice root surface attachment and in root surface and endophytic colonization. Genetic complementation reverted all mutant phenotypes. Also, the addition of EPS purified from culture supernatants of the wild-type strain PAL5 to the mutant MGD was effective in partially restoring wild-type biofilm formation and plant colonization. These data provide strong evidence that the PAL5 gumD gene is involved in EPS biosynthesis and that EPS biosynthesis is required for biofilm formation and plant colonization. To our knowledge, this is the first report of a role of EPS in the endophytic colonization of graminaceous plants by a nitrogen-fixing bacterium.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Genes Bacterianos/genética , Gluconacetobacter/metabolismo , Oryza/microbiología , Polisacáridos Bacterianos/metabolismo , Endófitos , Prueba de Complementación Genética , Genoma Bacteriano/genética , Gluconacetobacter/genética , Gluconacetobacter/fisiología , Proteínas Fluorescentes Verdes , Hidroponía , Familia de Multigenes , Mutagénesis Insercional , Fijación del Nitrógeno , Raíces de Plantas/microbiología , Polisacáridos Bacterianos/aislamiento & purificación , Plantones/microbiología , SimbiosisRESUMEN
Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long ß(2,6)-linked fructosyl chains with ß(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10(4) fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded ß(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30mgg(-1) of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan.
Asunto(s)
Fructanos/metabolismo , Genes Bacterianos/genética , Gluconacetobacter/enzimología , Gluconacetobacter/genética , Hexosiltransferasas/genética , Nicotiana/genética , Nicotiana/metabolismo , Ácidos/metabolismo , Cromatografía en Capa Delgada , Flores/metabolismo , Hexosiltransferasas/metabolismo , Hidrólisis , Peso Molecular , Fenotipo , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Tallos de la Planta/metabolismo , Plantas Modificadas Genéticamente , Polimerizacion , Nicotiana/crecimiento & desarrollo , Transformación Genética , Vacuolas/metabolismoRESUMEN
Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium that colonizes sugarcane. In order to investigate molecular aspects of the G. diazotrophicus-sugarcane interaction, we performed a quantitative mass spectrometry-based proteomic analysis by (15)N metabolic labeling of bacteria, root samples, and co-cultures. Overall, more than 400 proteins were analyzed and 78 were differentially expressed between the plant-bacterium interaction model and control cultures. A comparative analysis of the G. diazotrophicus in interaction with two distinct genotypes of sugarcane, SP70-1143 and Chunee, revealed proteins with fundamental roles in cellular recognition. G. diazotrophicus presented proteins involved in adaptation to atypical conditions and signaling systems during the interaction with both genotypes. However, SP70-1143 and Chunee, sugarcane genotypes with high and low contribution of biological nitrogen fixation, showed divergent responses in contact with G. diazotrophicus. The SP70-1143 genotype overexpressed proteins from signaling cascades and one from a lipid metabolism pathway, whereas Chunee differentially synthesized proteins involved in chromatin remodeling and protein degradation pathways. In addition, we have identified 30 bacterial proteins in the roots of the plant samples; from those, nine were specifically induced by plant signals. This is the first quantitative proteomic analysis of a bacterium-plant interaction, which generated insights into early signaling of the G. diazotrophicus-sugarcane interaction.
Asunto(s)
Proteínas Bacterianas/análisis , Gluconacetobacter/metabolismo , Proteoma/análisis , Saccharum/microbiología , Simbiosis/fisiología , Adaptación Fisiológica , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Técnicas de Cocultivo , Regulación Bacteriana de la Expresión Génica , Genotipo , Gluconacetobacter/genética , Gluconacetobacter/fisiología , Fijación del Nitrógeno/genética , Isótopos de Nitrógeno/análisis , Isótopos de Nitrógeno/metabolismo , Proteoma/fisiología , Saccharum/genética , Saccharum/crecimiento & desarrollo , Saccharum/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: G. diazotrophicus and A. vinelandii are aerobic nitrogen-fixing bacteria. Although oxygen is essential for the survival of these organisms, it irreversibly inhibits nitrogenase, the complex responsible for nitrogen fixation. Both microorganisms deal with this paradox through compensatory mechanisms. In A. vinelandii a conformational protection mechanism occurs through the interaction between the nitrogenase complex and the FeSII protein. Previous studies suggested the existence of a similar system in G. diazotrophicus, but the putative protein involved was not yet described. This study intends to identify the protein coding gene in the recently sequenced genome of G. diazotrophicus and also provide detailed structural information of nitrogenase conformational protection in both organisms. RESULTS: Genomic analysis of G. diazotrophicus sequences revealed a protein coding ORF (Gdia0615) enclosing a conserved "fer2" domain, typical of the ferredoxin family and found in A. vinelandii FeSII. Comparative models of both FeSII and Gdia0615 disclosed a conserved beta-grasp fold. Cysteine residues that coordinate the 2[Fe-S] cluster are in conserved positions towards the metallocluster. Analysis of solvent accessible residues and electrostatic surfaces unveiled an hydrophobic dimerization interface. Dimers assembled by molecular docking presented a stable behaviour and a proper accommodation of regions possibly involved in binding of FeSII to nitrogenase throughout molecular dynamics simulations in aqueous solution. Molecular modeling of the nitrogenase complex of G. diazotrophicus was performed and models were compared to the crystal structure of A. vinelandii nitrogenase. Docking experiments of FeSII and Gdia0615 with its corresponding nitrogenase complex pointed out in both systems a putative binding site presenting shape and charge complementarities at the Fe-protein/MoFe-protein complex interface. CONCLUSIONS: The identification of the putative FeSII coding gene in G. diazotrophicus genome represents a large step towards the understanding of the conformational protection mechanism of nitrogenase against oxygen. In addition, this is the first study regarding the structural complementarities of FeSII-nitrogenase interactions in diazotrophic bacteria. The combination of bioinformatic tools for genome analysis, comparative protein modeling, docking calculations and molecular dynamics provided a powerful strategy for the elucidation of molecular mechanisms and structural features of FeSII-nitrogenase interaction.
Asunto(s)
Azotobacter vinelandii/enzimología , Gluconacetobacter/enzimología , Modelos Moleculares , Nitrogenasa/metabolismo , Oxígeno/metabolismo , Conformación Proteica , Secuencia de Aminoácidos , Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biología Computacional , Genómica , Gluconacetobacter/genética , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fijación del Nitrógeno , Nitrogenasa/química , Nitrogenasa/genética , Unión Proteica , Electricidad EstáticaRESUMEN
Gluconacetobacter diazotrophicus is a micro-aerobic bacterium able to fix atmospheric nitrogen in endophytic mode. A proteomic approach was used to analyze proteins differentially expressed in the presence and absence of sugarcane plantlets. Two-dimensional gel electrophoresis (2-DE) showed 42 spots with altered levels of expression. Analysis of these spots by matrix-assisted laser desorption ionization time-of-flight in tandem (MALDI-TOF-TOF) identified 38 proteins. Differentially expressed proteins were associated with carbohydrate and energy metabolism, folding, sorting and degradation processes, and transcription and translation. Among proteins expressed in co-cultivated bacteria, four belong to membrane systems; others, like a transcription elongation factor (GreA), a 60 kDa chaperonin (GroEL), and an outer membrane lipoprotein (Omp16) have also been described in other plant-bacteria associations, indicating a common protein expression pattern as a result of symbiosis. A high protein content of 60kDa chaperonin isoforms was detected as non-differentially expressed proteins of the bacteria proteome. These results allow the assessment of the physiological significance of specific proteins to G. diazotrophicus metabolism and to the pathways involved in bacteria-host endophytic interaction.
Asunto(s)
Proteínas Bacterianas/análisis , Gluconacetobacter/genética , Interacciones Huésped-Patógeno/genética , Proteoma/análisis , Saccharum/microbiología , Metabolismo de los Hidratos de Carbono/genética , Técnicas de Cocultivo , Metabolismo Energético/genética , Gluconacetobacter/metabolismo , Simbiosis/genéticaRESUMEN
BACKGROUND: Gluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins. RESULTS: Gluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat. CONCLUSION: The genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.
Asunto(s)
Genoma Bacteriano , Gluconacetobacter/genética , Saccharum/microbiología , Hibridación Genómica Comparativa , ADN Bacteriano/genética , Islas Genómicas , Biblioteca Genómica , Gluconacetobacter/metabolismo , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , Análisis de Secuencia de ADN , SimbiosisRESUMEN
Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium, which is able to colonize sugarcane and other plant species of economic importance. The potentially beneficial effects promoted by this bacterium on plants are nitrogen-fixation, production of phythormones, action against pathogens and mineral nutrient solubilization. In this study, the molecular mechanisms associated with phosphorus and zinc solubilization were analyzed. A transposon mutant library was constructed and screened to select for mutants defective for phosphorous [Ca(5)(PO(4))(3)OH] and zinc (ZnO) solubilization. A total of five mutants were identified in each screen. Both screenings, performed independently, allowed to select the same mutants. The interrupted gene in each mutant was identified by sequencing and the results demonstrate that the production of gluconic acid is a required pathway for solubilization of such nutrients in G. diazotrophicus.
Asunto(s)
Gluconacetobacter/genética , Gluconacetobacter/metabolismo , Mutación , Fósforo/metabolismo , Zinc/metabolismo , Elementos Transponibles de ADN , Eliminación de Gen , Gluconatos/metabolismo , Redes y Vías Metabólicas/genética , Mutagénesis Insercional , Saccharum/microbiologíaRESUMEN
Gluconacetobacter diazotrophicus utilizes plant sucrose with a constitutively expressed levansucrase (LsdA), producing extracellular levan, which may be degraded under energetically unfavored conditions. Reverse transcriptase-PCR analysis revealed that lsdA and the downstream exolevanase gene (lsdB) form an operon. lsdB transcription was induced during growth with low fructose concentrations (0.44 to 33 mM) and repressed by glucose. Transport of LsdB to the periplasm involved N-terminal signal peptide cleavage. Type II secretion mutants failed to transfer LsdB across the outer membrane, impeding levan hydrolysis.