New Inoculation Strategy for Legume Based on Rhizobium-Metabolite Co-encapsulation.

The new strategies that are trying to be developed to protect microorganisms for a successful application have generated various types of granulated, powdered, or liquid formulations. In this work, we have developed a rhizobial encapsulation system for legumes accompanied by metabolites to enhance microorganism-plant communication. This novel way of producing a biofertilizer for legumes was developed based on alginate, a degradable compound that allows environmentally friendly use. This way of generating an inoculant allows it designing by making different molecular combinations for different purposes, being a double inoculant, biological and molecular.

Immobilization of Bradyrhizobium and Azospirillum in alginate matrix for long time of storage maintains cell viability and interaction with peanut.

Immobilizarion of PGPR for agricultural applications aims to provide temporary physical protection from stressful environmental conditions and the gradual release of cells for successful root colonization, release the cells gradually. In this work, we immobilized Bradyrhizobium sp. SEMIA6144 or Azospirillum brasilense Az39 cells in 2% alginate beads prepared by ionic gelation process, and then stored up to 12 months at 4 °C. Alginate matrix showed interaction with the immobilized bacteria (FTIR), allowed a constant release of cells, and improved their viability and capability to interact with Arachis hypogaea. Cell number into beads reached 107 CFU.bead-1; however, viability decreased from 4 months of storage for Az39, while it was maintained up to 12 months for SEMIA6144, showing a low metabolic activity measured by the MTT assay. Adhesion of SEMIA6144 and Az39 from new beads to peanut root was 11.5% and 16%, respectively, higher than non-immobilized bacteria. Peanut inoculation with 12 months storage SEMIA6144 beads significantly increased root length and biomass at 30 days of growth, and under restrictive water condition (RWC), nodulation and total plant N content increased compared with liquid inoculation. Our results demonstrate that immobilization of SEMIA6144 and Az39 in alginate matrix is a potential alternative to enhance peanut growth even under RWC. KEY POINTS: • Alginate encapsulation enhances viability of SEMIA6144 or Az39 under storage at 4 °C for 1 year. • Alginate beads 2% ensure the gradual release of the microorganisms. • Cells from beads stored for long periods present chemotaxis and adhesion to peanut root. • Peanut inoculation with 1-year-old SEMIA6144 beads improves nodulation and growth in RWC.

Morphological and structural response of Bacillus sp. SFC 500-1E after Cr(VI) and phenol treatment.

Bacillus sp. SFC 500-1E, a bacterial strain isolated from tannery sediments, is able to remove Cr(VI) and simultaneously tolerate high concentrations of phenol. In this study, we used high-resolution microscopies, fluorescence polarization techniques, and several biochemical approaches to improve our understanding about the adaptive mechanisms of this strain to survive in the presence of Cr(VI) and phenol, both individually and simultaneously. Among adaptive strategies developed by Bacillus sp. SFC 500-1E, an increase in bacterial size, such as length, width, and height, and ultrastructural alterations, such as electron-dense precipitates, the presence of exopolymers, and cell lysis, are noteworthy. The exopolymers observed were consistent with the extensive biofilm formation and exopolysaccharides and extracellular protein quantification. At the cell membrane level, a rapid rigidity was induced in Cr(VI) + phenol treatment. This effect was counteracted after 16 h by changes at the level of phospholipids, mainly in the composition of fatty acids (FAs); in particular, an increase in the unsaturated fatty acid/saturated fatty acid ratio was detected. This study shows evidence of some adaptive responses displayed by Bacillus sp. SFC 500-1E, which allows it to survive in stressful conditions.

Membrane Rigidity and Phosphatidic Acid (PtdOH) Signal: Two Important Events in Acinetobacter guillouiae SFC 500-1A Exposed to Chromium(VI) and Phenol.

The remodeling of membrane lipids is a mechanism that allows microorganisms to survive in unfavorable environments such as industrial effluents, which often contain inorganic and organic pollutants, like chromium and phenol. In the present work, we evaluated the effect of Cr(VI) and phenol on the membrane of Acinetobacter guillouiae SFC 500-1A, a bacterial strain isolated from tannery sediments where such pollutants can be found. The presence of lipid kinases and phospholipases and the changes in their activities under exposure to these pollutants were determined. Cr(VI) and Cr(VI) + phenol caused the membrane to become more rigid for up to 16 h after exposure. This could be due to an increase in cardiolipin (Ptd2 Gro) and a decrease in phosphatidylethanolamine (PtdEtn), which are indicative of more order and rigidity in the membrane. Increased phospholipase A activity (PLA, EC 3.1.1.4) could be responsible for the decrease in PtdEtn levels. Moreover, our results indicate that Cr(VI) and Cr(VI) + phenol trigger the phosphatidic acid (PtdOH) signal. The finding of significantly increased phosphatidylinositol-4-phosphate (PtdIns-4-P) levels means this is likely achieved via PtdIns-PLC/DGK. This report provides the first evidence that A. guillouiae SFC 500-1A is able to sense Cr(VI) and phenol, transduce this signal through changes in the physical state of the membrane, and trigger lipid-signaling events.

Changes in the lipid composition of Bradyrhizobium cell envelope reveal a rapid response to water deficit involving lysophosphatidylethanolamine synthesis from phosphatidylethanolamine in outer membrane.

We evaluate the behavior of the membrane of Bradyrhizobium sp. SEMIA6144 during adaptation to polyethylene glycol (PEG). A dehydrating effect on the morphology of the cell surface, as well as a fluidizing effect on the membrane was observed 10 min after PEG shock; however, the bacteria were able to restore optimal membrane fluidity. Shock for 1 h caused an increase of lysophosphatidylethanolamine in the outer membrane at the expense of phosphatidylcholine and phosphatidylethanolamine (PE), through an increase in phospholipase activity. The amount of lysophosphatidylethanolamine did not remain constant during PEG shock, but after 24 h the outer membrane was composed of large amounts of phosphatidylcholine and less amount of lysophosphatidylethanolamine similar to the control. The inner membrane composition was also modified after 1 h of shock, observing an increase of phosphatidylcholine at the expense of PE, the proportions of these phospholipids were then modified to reach 24 h of shock values similar to the control. Vesicles prepared with the lipids of cells exposed to 1 h shock presented higher rigidity compared to the control, indicating that changes in the composition of phospholipids after 1 h of shock restoring fluidity after the PEG effect and would allow cells to maintain surface morphology.

Δ9 desaturase from Trypanosoma cruzi: Key enzyme in the parasite metabolism. Cloning and overexpression.

Desaturases, key enzymes in the metabolism of fatty acids, regulate the physical and biochemical properties of membranes. They adjust the composition of saturated and unsaturated fatty acids in response to changes in the environmental. We demonstrated the existence of Δ9 desaturase activity in epimastigotes of the Trypanosoma cruzi Tulahuen strain. In the present study, showed that this enzyme has an approximate molecular mass of 50kDa and a pI value of approximately 9. In order to characterize the Δ9 desaturase of Trypanosoma cruzi, (TcΔ9DES) we have cloned, sequenced and expressed in Escherichia coli. The gene consists of 1300bp and encodes a peptide of 433 amino acids with a molecular weight of 50kDa. Analysis of the amino acid sequence revealed three clusters of histidine and two hydrophobic regions, characteristic of membrane-bound desaturases. Gene expression studies showed that TcΔ9DES was overexpressed as an active protein. Fatty acid analysis showed that the expressed protein was confirmed to be functional with Δ9 desaturase activity. This enzyme changed the fatty acid profile of TcΔ9DES-expressing E. coli, decreasing the levels of palmitic (16:0) and stearic (18:0) acids and enhancing palmitoleic (16:1Δ9) and monounsaturated 18 carbons fatty acids. When [1-14C]palmitic or [1-14C]stearic acid was used as substrate, TcΔ9DES-expressing E. coli exhibited high desaturase activity associated with increased levels of monounsaturated fatty acids, suggesting that the TcΔ9DES enzyme was actively expressed in E. coli. To check the commitment of TcΔ9DES against sterol biosynthesis inhibitors we tested the activity under ketoconazole effect. Native TcΔ9DES, showed a significant activity inhibition. Since TcΔ9DES has shown active participation under different environmental factors, among them, ketoconazole, we consider that it plays a critical role in the metabolism of the parasite.

Arachis hypogaea PGPR isolated from Argentine soil modifies its lipids components in response to temperature and salinity.

The aim of this work was to clarify the mechanism related to plant growth promoting of a bacterial strain (L115) isolated from Arachis hypogaea rhizospheres and the effects of high growth temperature and salinity on phospholipids and fatty acids composition. L115 was isolated from peanut rhizospheres and identified according to the sequence analysis of the 16S rRNA gene. Phenotypic, metabolic and plant growth promoting rhizobacteria (PGPR) characteristics of L115 were tested. Inoculation test in plant growth chamber was performed. In addition, L115 was exposed to a 37 °C and 300 mM NaCl and phospholipids and fatty acid composition were evaluated. L115 strain was identified as Ochrobactrum intermedium and was able to increase the peanut shoot and root length as well as dry weight, indicating a PGPR role by being able to produce indole acetic acid and siderophores and present ACC deaminase activity. In addition, L115 showed tolerance to both high growth temperature and 300 mM NaCl. The most striking change was a decreased percentage of 18:1 fatty acid and an increase in 16:0 and 18:0 fatty acids, under high growth temperature or a combination of increased temperature and salinity. The most important change in phospholipid levels was an increase in phosphatidylcholine biosynthesis in all growth conditions. L115 can promote the growth of peanut and can tolerate high growth temperature and salinity modifying the fatty acid unsaturation degree and increasing phosphatidylcholine levels. This work is the first to report the importance of the genus Ochrobactrum as PGPR on peanut growth as well as on the metabolic behaviour against abiotic stresses that occur in soil. This knowledge will be useful for developing strategies to improve the growth of this bacterium under stress and to enhance its bioprocess for the production of inoculants.

Biochemical and molecular evidence of a Δ9 fatty acid desaturase from Ensifer meliloti 1021.

It has been reported that Ensifer meliloti presents a high proportion of monounsaturated fatty acids and has a putative desaturase gene designated as PhFAD12 (National Centre for Biotechnology Information), encoding a putative Δ12 desaturase-like protein. In this work, we report the desaturation capacity and characterisation of this gene encoding the putative fatty acid desaturase of E. meliloti 1021. This gene was also isolated from the rhizobial strain and overexpressed in Escherichia coli. Compared to a control, the expression of this gene in the transformed strain decreased the levels of palmitic and stearic acids, enhanced palmitoleic and cis-vaccenic levels, and allowed for the detection of oleic acid. E. coli overexpressing the putative desaturase gene was capable of desaturating palmitic and stearic acids to monounsaturated fatty acids, similarly to the rhizobial strain. Our studies show that AAK64726 encodes a Δ9 desaturase instead of a Δ12 desaturase as previously indicated. This work describes evidence for the presence of a desaturase-mediated mechanism in monounsaturated fatty acid synthesis in E. meliloti 1021, which is modified by high growth temperature. This mechanism supplements the anaerobic mechanism for unsaturated fatty acid synthesis.

Growth temperature and salinity impact fatty acid composition and degree of unsaturation in peanut-nodulating rhizobia.

Growth and survival of bacteria depend on homeostasis of membrane lipids, and the capacity to adjust lipid composition to adapt to various environmental stresses. Membrane fluidity is regulated in part by the ratio of unsaturated to saturated fatty acids present in membrane lipids. Here, we studied the effects of high growth temperature and salinity (NaCl) stress, separately or in combination, on fatty acids composition and de novo synthesis in two peanut-nodulating Bradyrhizobium strains (fast-growing TAL1000 and slow-growing SEMIA6144). Both strains contained the fatty acids palmitic, stearic, and cis-vaccenic + oleic. TAL1000 also contained eicosatrienoic acid and cyclopropane fatty acid. The most striking change, in both strains, was a decreased percentage of cis-vaccenic + oleic (≥ 80% for TAL1000), and an associated increase in saturated fatty acids, under high growth temperature or combined conditions. Cyclopropane fatty acid was significantly increased in TAL1000 under the above conditions. De novo synthesis of fatty acids was shifted to the synthesis of a higher proportion of saturated fatty acids under all tested conditions, but to a lesser degree for SEMIA6144 compared to TAL1000. The major adaptive response of these rhizobial strains to increased temperature and salinity was an altered degree of fatty acid unsaturation, to maintain the normal physical state of membrane lipids.