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    Here, we describe a simple, non-time consuming and inexpensive method for monitoring of Calcofluor white M2R-binding exopolysaccharides in individual bacterial cells. This method was demonstrated by time-lapse microscopy of succinoglycan-producing cells of the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti. The method is most likely applicable to other bacteria producing β-(1. →. 3) and β-(1. →. 4) linked polysaccharides.
    Bacillus amyloliquefaciens MEP218 is a native isolate with a broad spectrum of antifungal activity against plant pathogenic fungi. The ability of strain MEP218 to antagonize phytopathogens is due to the production of cyclic lipopeptides (CLPs). In this work, different carbon (C) and nitrogen (N) sources and C to N ratios were evaluated in order to improve both, biomass and CLPs production by strain MEP218. Among the C and N sources and C to N ratios tested, glucose and NH4NO3 at the C to N ratio of 10:1 enhanced significantly biomass and CLPs yield. Moreover, CLPs produced in this medium exhibited higher antibacterial activity against Xanthomonas axonopodis pv. vesicatoria (Xav) than those obtained in the recommended medium for CLPs production. Interestingly, CLPs addition influenced the development of Xav biofilm on biotic and abiotic surfaces. A comparison of HPLC-chromatograms of CLPs obtained in the optimized medium versus the ones obtained in the recommended medium showed a notable increase of surfactin in the CLPs obtained in the optimized medium. Furthermore, two peaks with antibacterial activity against Xav, identified by mass spectrometry analysis as fengycins A and B, were detected in the CLPs from the strain MEP218, grown in the optimized culture medium. The results obtained in this work suggest that changes in C and N sources and C to N ratios affect the yield and type of CLPs produced by B. amyloliquefaciens MEP218. To the best of our knowledge, this is the first study to report the finding of fengycins with antibacterial activity. CLPs produced by the strain MEP218 are potential candidates for controlling bacterial spot disease in tomato and pepper.
    Azospirillum brasilense is a soil bacterium capable of promoting plant growth. Several surface components were previously reported to be involved in the attachment of A. brasilense to root plants. Among these components are the exopolysaccharide (EPS), lipopolysaccharide (LPS) and the polar flagellum. Flagellin from polar flagellum is glycosylated and it was suggested that genes involved in such a posttranslational modification are the same ones involved in the biosynthesis of sugars present in the O-antigen of the LPS. In this work, we report on the characterization of two homologs present in A. brasilense Cd, to the well characterized flagellin modification genes, flmA and flmB, from Aeromonas caviae. We show that mutations in either flmA or flmB genes of A. brasilense resulted in non-motile cells due to alterations in the polar flagellum assembly. Moreover, these mutations also affected the capability of A. brasilense cells to adsorb to maize roots and to produce LPS and EPS. By generating a mutant containing the polar flagellum affected in their rotation, we show the importance of the bacterial motility for the early colonization of maize roots.
    In Gram-negative bacteria, tyrosine phosphorylation has been shown to play a role in the control of exopolysaccharide (EPS) production. This report demonstrates that the chromosomal open reading frame SMc02309 from Sinorhizobium meliloti 2011, encodes a protein with significant sequence similarity to low molecular weight protein tyrosine phosphatases (LMW-PTPs), such as the Escherichia coli Wzb. Unlike other well-characterized EPS biosynthesis gene clusters, which contain neighboring LMW-PTPs and kinase, the S. meliloti succinoglycan (EPS I) gene cluster located on megaplasmid pSymB does not encode a phosphatase. Biochemical assays revealed that the SMc02309 protein hydrolyzes p-nitrophenyl phosphate (p-NPP) with kinetic parameters similar to other bacterial LMW-PTPs. Furthermore, we show evidence that SMc02309 is not the LMW-PTP of the bacterial tyrosine-kinase (BY-kinase) ExoP. Nevertheless ExoN, a UDP-glucose pyrophosphorylase involved in the first stages of EPS I biosynthesis, is phosphorylated at tyrosine residues and constitutes an endogenous substrate of the SMc02309 protein. Additionally, we show that the UDP-glucose pyrophosphorylase activity is modulated by SMc02309-mediated tyrosine dephosphorylation. Moreover, a mutation in the SMc02309 gene decreases EPS I production and delays nodulation on M. sativa roots.
    The aim of this work was to clarify the mechanism of monounsaturated fatty acid (MUFA) synthesis in Bradyrhizobium TAL1000 and the effect of high temperature on this process. Bradyrhizobium TAL1000 was exposed to a high growth temperature and heat shock, and fatty acid composition and synthesis were tested. To determine the presence of a possible desaturase, a gene was identify and overexpressed in Escherichia coli. The desaturase expression was detected by RT-PCR and Western blotting. In B. TAL1000, an aerobic mechanism for MUFA synthesis was detected. Desaturation was decreased by high growth temperature and by heat shock. Two hours of exposure to 37°C were required for the change in MUFA levels. A potential ∆9 desaturase gene was identified and successfully expressed in E. coli. A high growth temperature and not heat shock reduced transcript and protein desaturase levels in rhizobial strain. In B. TAL1000, the anaerobic MUFA biosynthetic pathway is supplemented by an aerobic mechanism mediated by desaturase and is down-regulated by temperature to maintain membrane fluidity under stressful conditions. This knowledge will be useful for developing strategies to improve a sustainable practice of this bacterium under stress and to enhance the bioprocess for the inoculants' manufacture.
    Legumes are able to fix nitrogen because of the bacterial symbionts (rhizobia) that inhabit nodules on their roots. The amount of ammonia produced by rhizobial fixation of nitrogen rivals that of the world's entire fertilizer industry. Consequently, this symbiotic relationship between legumes and rhizobia is of great agronomic and ecological importance. Typical environmental stresses faced by the legume and their symbiotic partner may include, water stress, salinity and temperature and influence the survival in the soil. In the Rhizobia-legume symbiosis, the host plant also influence rhizobial survival. In Arachis hypogaea rhizobia symbiosis is known that different abiotic stresses affect the viability, trehalose and membrane components content of rhizobia. Also, the attachment ability of peanut rhizobia is affected under abiotic stresses. This chapter addresses the idea that the rhizobia and the plants must be able to adapt to survive to the environmental conditions. Our hypothesis is that rhizobia survival in the soilenvironmental because they are able to modify fatty acid and phospholipid components of their membranes, as well as other molecules with important roles in stress tolerance.
    The rhizosphere is a multiple interface between soils, plant roots, microbes and fauna, where different biological components interact strongly. Rhizosphere interactions are based on complex exchanges that take place around plant roots. Beneficial, detrimental and neutral relationships between plant roots and microorganisms are all regulated by complex molecular signalling. Plants exude a variety of organic compounds (e.g. carbohydrates, carboxylic acids, phenolics, amino acids, flavonoids) as well as inorganic ions (protons and other ions) into the rhizosphere which change the chemistry and biology of the root microenvironment. All chemical compounds secreted by plants are collectively named rhizodepositions. In the rhizosphere, bacteria that exert beneficial effects on plant development are referred to as plant growth-promoting rhizobacteria (PGPR) because their application is often associated with increased rates of plant growth. On the other hand, although many technologies have been used in the improvement of stress tolerance in plants, fewer reports have been published on how PGPR can exert tolerance to salt, drought or heavy metals. In addition, the industrial use and technological application of compounds from plants and rhizobacteria are required to be successful in attaining sustainable microbial-based agrotechnologies. Among crops, legumes are a good source of starch, dietary fibre, protein and minerals. It has long been recognized that legumes are functional foods that promote good health and have therapeutic properties. This chapter shows the significance of some biochemical and biological compounds derived from legumes and rhizobacteria with potential in biotechnology.
    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.
    The rhizosphere is the volume of soil under the influence of plants roots, where very important and intensive microbe–plant interactions take place. These interactions can both significantly influence plant growth and crop yields and have biotechnological applications. The rhizosphere harbors a diverse community of microorganisms that interact and compete with each other and with the plant root. The activity of some of the members of this community affects the growth and the physiology of the others, as well as the physical and chemical properties of the soil. Among all these interactions, those resulting in symbiotic and non-symbiotic nitrogen fixation are considerably important. In recent years, the use of bacteria (rhizobacteria) to promote plant growth has increased in several regions of the world and has acquired relevant importance in developing countries that are the producers of raw materials for food. Rhizobacteria can affect plant growth by producing and releasing secondary metabolites, which either decrease or prevent the deleterious effects of phytopathogenic organisms in the rhizosphere, and/or by facilitating the availability and uptake of certain nutrients from the root environment. Significant increases in the growth and yield of agriculturally important crops in response to inoculation with rhizobacteria have been reported. This practical application of plant growth-promoting rhizobacteria is the main focus of this chapter.
    The legume–rhizobia symbiosis is considered the most important nitrogen-fixing interaction from an agricultural point of view. However, biotic and abiotic factors can modify critical parameters of both the legumes and the rhizobia. These changes may lead to differences in the molecular dialogue, consequently reducing the symbiotic effectiveness. Therefore, optimal performance of the N-fixing symbiosis will be guaranteed by selection of both symbiotic partners for adaptation to the target environment. The symbiotic process can be negatively affected by many other rhizosphere interactions, resulting in important ecological, economic, and nutritional losses. The application of agricultural techniques that are friendly with the environment, based on the use of plant growth promoting rhizobacteria (PGPR), can increase the efficiency of the symbiotic process. The use of these beneficial microorganisms could reduce the use of polluting chemicals allowing sustainable production of legumes. Co-inoculations of appropriate rhizobia together with PGPR may profoundly increase the crop yield by different mechanisms. The negative effects of environmental stresses on the legume–rhizobia symbiosis may further be significantly diminished by applying mixtures of rhizobia and PGPR.
    Phosphatidylcholine, the major phospholipid in eukaryotes, is found in rhizobia and in many other bacteria interacting with eukaryotic hosts. Phosphatidylcholine has been shown to be required for a successful interaction of Bradyrhizobium japonicum USDA 110 with soybean roots. Our aim was to study the role of bacterial phosphatidylcholine in the Bradyrhizobium-peanut (Arachis hypogaea) symbiosis. Phospholipid N-methyltransferase (Pmt) and minor phosphatidylcholine synthase (Pcs) activities were detected in crude extracts of the peanut-nodulating strain Bradyrhizobium sp. SEMIA 6144. Our results suggest that phosphatidylcholine formation in Bradyrhizobium sp. SEMIA 6144 is mainly due to the phospholipid methylation pathway. Southern blot analysis using pmt- and pcs-probes of B. japonicum USDA 110 revealed a pcs and multiple pmt homologues in Bradyrhizobium sp. SEMIA 6144. A pmtA knockout mutant was constructed in Bradyrhizobium sp. SEMIA 6144 that showed a 50% decrease in the phosphatidylcholine content in comparison with the wild-type strain. The mutant was severely affected in motility and cell size, but formed wild-type-like nodules on its host plant. However, in coinoculation experiments, the pmtA-deficient mutant was less competitive than the wild type, suggesting that wild-type levels of phosphatidylcholine are required for full competitivity of Bradyrhizobium in symbiosis with peanut plants.
    The effects of saline and osmotic stress on four peanut rhizobia, plant growth and symbiotic N2-fixation inArachis hypogaea were studied. Abiotic stress was applied by adding either 100 mM NaCl or 20 mM PEG6000. At the rhizobial level,Bradyrhizobium ATCC10317 and TAL1000 showed stronger tolerance to stress than TAL1371 and SEMIA6144. The effect of salinity on the bacterium-plant association was studied by using the variety Blanco Manfredi M68. In the absence of stresses, all the strains induced a significantly higher number of nodules on the roots, although TAL1371 and SEMIA6144 were more effective. Both stresses affected the interaction process, while TALl371 was the best partner. KeywordsPeanut rhizobia- Arachis hypogaea -abiotic stress
    Phospholipids provide the membrane with its barrier function and play a role in a variety of processes in the bacterial cell, as responding to environmental changes. The aim of the present study was to characterize the physiological and metabolic response of Bradyrhizobium SEMIA 6144 to saline and temperature stress. This study provides metabolic and compositional evidence that nodulating peanut Bradyrhizobium SEMIA 6144 is able to synthesize fatty acids, to incorporate them into its phospholipids (PL), and then modify them in response to stress conditions such as temperature and salinity. The fatty acids were formed from [1-(14)C]acetate and mostly incorporated in PL (95%). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) were found to be the major phospholipids in the bacteria analyzed. The amount and the labeling of each individual PL was increased by NaCl, while they were decreased by temperature stress. The amount of PC, PE, and PG under the combined stresses decreased, as in the temperature effect. The results indicate that synthesized PL of Bradyrhizobium SEMIA 6144 are modified under the tested conditions. Because in all conditions tested the PC amount was always modified and PC was the major PL, we suggest that this PL may be involved in the bacteria response to environmental conditions.
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