Displacers improve the selectivity of phosphopeptide enrichment by metal oxide affinity chromatography / Uso de desplazadores para mejorar la selectividad de los fosfopéptidos enriquecidos por cromatografía de afinidad con óxidos de metales

Abstract: Background: A key process in cell regulation is protein phosphorylation, which is catalyzed by protein kinases and phosphatases. However, phosphoproteomics studies are difficult because of the complexity of protein phosphorylation and the number of phosphorylation sites. Methods: We describe an efficient approach analyzing phosphopeptides in single, separated protein by two-dimensional gel electrophoresis. In this method, a titanium oxide (TiO2)-packed NuTip is used as a phosphopeptide trap, together with displacers as lactic acid in the loading buffer to increase the efficiency of the interaction between TiO2 and phosphorylated peptides. Results: The results were obtained from the comparison of mass spectra of proteolytic peptides of proteins with a matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) instrument. Conclusions: This method has been applied to identifying phosphoproteins involved in the symbiosis Rhizobium etli-Phaseolus vulgaris.

Resumen: Introducción: Un proceso clave en la regulación celular es la fosforilación de proteínas, que se lleva a cabo por cinasas y fosfatasas. Sin embargo, los estudios de fosfoproteómica son difíciles debido a la complejidad de la fosforilación proteica y el número de sitios de fosforilación. Métodos: En el presente trabajo se describe una eficiente estrategia metodológica para analizar fosfopéptidos de proteínas separadas mediante electroforesis bidimensional. En este método, una columna con microesferas de dióxido de titanio (TiO2/NuTip) se utilizó para atrapar los fosfopéptidos en la superficie del TiO2 previamente empacado en una punta. El uso de desplazadores en el buffer de carga, como el ácido láctico, mejoró significativamente la selectividad. Resultados: Los resultados se obtuvieron mediante la comparación de los espectros de masas de péptidos proteolíticos de proteínas analizados utilizando un instrumento de desorción/ionización láser asistida por matriz-tiempo de vuelo (MALDI-TOF). Conclusiones: Este método se ha aplicado para la identificación de fosfoproteínas involucradas en la simbiosis del Rhizobium etli con Phaseolus vulgaris.

Symbiotic potential and survival of native rhizobia kept on different carriers

Native rhizobia are ideal for use as commercial legume inoculants. The characteristics of the carrier used to store the inoculants are important for the survival and symbiotic potential of the rhizobia. The objective of this study was to investigate the effects of peat (PEAT), perlite sugarcane bagasse (PSB), carboxymethyl cellulose plus starch (CMCS), and yeast extract mannitol supplemented with mannitol (YEMM) on the survival, nodulation potential and N₂ fixation capacity of the native strains Sinorhizobium mexicanum ITTG R7^T and Rhizobium calliandrae LBP2-1^T and of the reference strain Rhizobium etli CFN42^T. A factorial design (4 × 3) with four repetitions was used to determine the symbiotic potential of the rhizobial strains. The survival of the strains was higher for PEAT (46% for strain LBP2-1^T, 167% for strain CFN42^T and 219% for strain ITTG R7^T) than for the other carriers after 240 days, except for CFN42^T kept on CMCS (225%). All the strains kept on the different carriers effectively nodulated common bean, with the lowest number of nodules found (5 nodules) when CFN42^T was kept on CMCS and with the highest number of nodules found (28 nodules) when ITTG R7^T was kept on PSB. The nitrogenase activity was the highest for ITTG R7^T kept on PEAT (4911 μmol C₂H₄ per fresh weight nodule h⁻¹); however, no activity was found when the strains were kept on YEMM. Thus, the survival and symbiotic potential of the rhizobia depended on the carrier used to store them.

Developing novel bacterial based bioformulation having PGPR properties for enhanced production of agricultural crops.

Plant growth promoting rhizobacteria (PGPR) are beneficial rhizobacteria which enhance plant growth as well as the productivity by a variety of mechanisms. PGPR were isolated from the rhizosphere region of som plants (Machilus bombycina King) maintained at the Central Muga Eri Research and Training Institute, Lahdoigarh, Jorhat. A bacterial based bioformulation was prepared and sprayed over the experimental crops including tomato (Solanum lycopersicum), cauliflower (Brassica oleracea var botrytis), chili (Capsicum annuum) and brinjal (Solanum melongena). Biochemical analysis was done on these PGPR treated crops as well as the untreated crops. The bioformulations prepared from Bacillus cereus (MTCC 8297), Pseudomonas rhodesiae (MTCC 8299) and Pseudomonas rhodesiae (MTCC 8300) was found to be the most effective in increasing the shoot height, number of leaves, early fruiting and total biomass content of the plants after treatment.

Phenotypic and Genotypic Characteristics of Rhizobia Isolated from Meknes-tafilalet Soils and Study of Their Ability to Nodulate Bituminaria bituminosa.

Aims: The objectives were to isolate and characterize phenotypically and genotypically the rhizobial strains from the soils belonging to the Meknes-Tafilalet region in order to select strains that are able to nodulate Bituminaria bituminosa. Study Design: An experimental study. Place and Duration of Study: Department of biology (Soil & Environment Microbiology Unit) Faculty of Sciences, Moulay Ismail University and Technical Support Unit for Scientific Research, CNRST in Rabat; between January and August 2010. Methodology: Samples from 23 different sites belonging to the Meknes-Tafilalet region were collected in order to select rhizobial strains that are able to nodulate Bituminaria bituminosa. The morphological, cultural and phenotypic parameters of isolated strains were studied. The phenotypic characteristics include colony morphology, growth speed, tolerances to temperature, salt and pH. To assess the genotypic diversity among the isolates, molecular characteristics based on 16S rDNA gene sequencing were performed. Results: The majority of the isolated strains showed fast-growing capacity (75%). Most strains tolerate neutral to alkaline pH, however some strains (18%) showed weak growth capacity at pH 4. All isolates were tolerant to high salt stress ([NaCl] = 3%). The genotypic characterization based on16S rDNA gene sequencing of the twelve strains showed a high diversity between the isolates. Conclusion: Taken together, our results highlight the important biodiversity of the isolated rhizobial strains and open opportunities for the development of new bio-fertilizer.

Biological control of potato black scurf by rhizosphere associated bacteria

The present work was carried out to study the potential of plant rhizosphere associated bacteria for the biocontrol of potato black scurf disease caused by Rhizoctonia solani Khun AG-3. A total of twenty-eight bacteria isolated from diseased and healthy potato plants grown in the soil of Naran and Faisalabad, Pakistan were evaluated for their antagonistic potential. Nine bacterial strains were found to be antagonistic in vitro, reduced the fungal growth and caused the lysis of sclerotia of R. solani in dual culture assay as well as in extracellular metabolite efficacy test. The selected antagonistic strains were further tested for the production and efficacy of volatile and diffusible antibiotics, lytic enzymes and siderophores against R. solani. Selected antagonistic bacteria were also characterized for growth promoting attributes i.e., phosphate solubilization, nitrogen fixation and indole acetic acid production. Biocontrol efficacy and percent yield increase by these antagonists was estimated in greenhouse experiment. Statistical analysis showed that two Pseudomonas spp. StT2 and StS3 were the most effective with 65.1 and 73.9 percent biocontrol efficacy, as well as 87.3 and 98.3 percent yield increase, respectively. Potential antagonistic bacterial strain StS3 showed maximum homology to Pseudomonas sp. as determined by 16S rRNA gene sequencing. These results suggest that bacterial isolates StS3 and StT2 have excellent potential to be used as effective biocontrol agents promoting plant growth with reduced disease incidence.

Nodulation competitiveness between contrasting phage phenotypes of pigeonpea rhizobial strains.

Competitiveness between (I) lysogenic vs. phage-indicator strains, (II) phage-resistant vs phage-sensitive strains, and (III) large plaque vs. small plaque developing strains was examined under laboratory and field conditions in order to study the involvement of these crucial phage sensitivity patterns in the competition for nodule occupancy of pigeonpea rhizobia. The phage-indicator strain (A039) exhibited higher competitiveness over the lysogenic strain (A025 Sm(r)); the phage sensitive strain (IHP-195) over the phage resistant strain (IHP 195 Sm(r)V(r)); and the large plaque developing strain (A059) over the small plaque developing strain (IHP195 Sm(r)) in association with pigeonpea cv. bahar both under laboratory and field conditions. Dual inoculation of A025 Sm(r) + A039 and A059 + IHP195 Sm(r) (mixed in equal proportion just before treatment) improved the nodule occupancy by inoculant strains against native rhizobia and resulted into higher plant dry weight and yield as compared to their application as single inoculum. The phage-resistant mutant IHP195 Sm(r)V(r) showed reduced competitiveness against native rhizobia, compared to its parental strain. The dual inoculation of parental strain and phage-resistant mutant gave the same result as the inoculation of parental strain alone.

Role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and vitamins in symbiosis.

Rhizobia require the availability of 20 amino acids for the establishment of effective symbiosis with legumes. Some of these amino acids are synthesized by rhizobium, whereas the remaining are supplied by the host plant. The supply from plant appears to be plant-type specific. Alfalfa provides arginine, cysteine, isoleucine, valine and tryptophan, and cowpea and soybean provide histidine. The production of ornithine and anthranilic acid, the intermediates in the biosynthetic pathways of arginine and tryptophan, respectively, seems to be essential for effective symbiosis of Sinorhizobium meliloti with alfalfa. The expression of ilvC gene of S. meliloti is required for induction of nodules on the roots of alfalfa plants. An undiminished metabolic flow through the rhizobial pathways for the synthesis of purines and pyrimidines and the synthesis of biotin, nicotinic acid, riboflavin and thiamine by rhizobium appear to be requirements for normal symbiosis. To the best of our knowledge, this is the first review article on the role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and vitamins in rhizobium-legume symbiosis. The scientific developments of about 35 years in this field have been reviewed.

Symbiotic effectiveness of bacteriocin producing and non-producing strains of Rhizobium in green gram (Vigna radiata).

Rhizobium strains nodulating green gram [Vigna radiata (L.) Wilczek] were found to produce bacteriocin on modified Bergersen's medium and inhibited the growth of homologous Rhizobium strains. Four bacteriocin producing and four bacteriocin non-producing strains were compared for their effect on nodulation, in planta nitrogenase activity and plant dry weight of green gram. The bacteriocin producers formed more nodules in comparison to non-bacteriocin producers. However, the symbiotic effectiveness of bacteriocin producers was less in terms of plant dry weight in comparison to non-bacteriocin producers.