Microwave resonator-based sensor system for specific antibody detection.

An antibody-detecting sensor is described that is based on a microwave electrodynamic resonator. A polystyrene film with immobilized bacteria deposited on a lithium niobate plate was placed at one end of the resonator and was used as the sensing element. The second end was electrically shorted. The frequency and depth of the reflection coefficient S11 for three resonances in the range 6.5-8.5 GHz were used as an analytical signal to examine antibody interactions with bacteria and determine the time required for cell immobilization. The sensor distinguished between situations in which bacteria interacted with specific antibodies and those in which no such interaction occurred (control). Although the cell-antibody interaction changed the frequency and depth of the second and third resonance peaks, the parameters of the first resonance peak did not change. The interaction of cells with nonspecific antibodies did not change the parameters of any of the peaks. These results are promising for use in the design of methods to detect specific antibodies, which can supplement the existing methods of antibody analysis.

De novo biosynthesis of 2'-fucosyllactose in a metabolically engineered Escherichia coli using a novel ɑ1,2-fucosyltransferase from Azospirillum lipoferum.

Human milk oligosaccharides are complex, indigestible oligosaccharides that provide ideal nutrition for infant development. Here, 2'-fucosyllactose was efficiently produced in Escherichia coli by using a biosynthetic pathway. For this, both lacZ and wcaJ (encoding ß-galactosidase and UDP-glucose lipid carrier transferase, respectively) were deleted to enhance the 2'-fucosyllactose biosynthesis. To further enhance 2'-fucosyllactose production, SAMT from Azospirillum lipoferum was inserted into the chromosome of the engineered strain, and the native promoter was replaced with a strong constitutive promoter (PJ23119). The titer of 2'-fucosyllactose was increased to 8.03 g/L by introducing the regulators rcsA and rcsB into the recombinant strains. Compared to wbgL-based strains, only 2'-fucosyllactose was produced in SAMT-based strains without other by-products. Finally, the highest titer of 2'-fucosyllactose reached 112.56 g/L in a 5 L bioreactor by fed-batch cultivation, with a productivity of 1.10 g/L/h and a yield of 0.98 mol/mol lactose, indicating a strong potential in industrial production.

Discovery, characterization and engineering of ligases for amide synthesis.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways1-4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants5. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

Identification of the rhizospheric microbe and metabolites that led by the continuous cropping of ramie (Boehmeria nivea L. Gaud).

Continuous cropping lowers the production and quality of ramie (Boehmeria nivea L. Gaud). This study aimed to reveal the metagenomic and metabolomic changes between the healthy- and obstacle-plant after a long period of continuous cropping. After 10 years of continuous cropping, ramie planted in some portions of the land exhibited weak growth and low yield (Obstacle-group), whereas, ramie planted in the other portion of the land grew healthy (Health-group). We collected rhizosphere soil and root samples from which measurements of soil chemical and plant physiochemical properties were taken. All samples were subjected to non-targeted gas chromatograph-mass spectrometer (GS/MS) metabolome analysis. Further, metagenomics was performed to analyze the functional genes in rhizospheric soil organisms. Based on the findings, ramie in Obstacle-group were characterized by shorter plant height, smaller stem diameter, and lower fiber production than that in Health-group. Besides, the Obstacle-group showed a lower relative abundance of Rhizobiaceae, Lysobacter antibioticus, and Bradyrhizobium japonicum, but a higher relative abundance of Azospirillum lipoferum and A. brasilense compared to the Health-group. Metabolomic analysis results implicated cysteinylglycine (Cys-Gly), uracil, malonate, and glycerol as the key differential metabolites between the Health- and Obstacle-group. Notably, this work revealed that bacteria such as Rhizobia potentially synthesize IAA and are likely to reduce the biotic stress of ramie. L. antibioticus also exerts a positive effect on plants in the fight against biotic stress and is mediated by metabolites including orthophosphate, uracil, and Cys-Gly, which may serve as markers for disease risk. These bacterial effects can play a key role in plant resistance to biotic stress via metabolic and methionine metabolism pathways.

Analysis of Microbial Cell Viability in a Liquid Using an Acoustic Sensor.

A method was developed for the rapid analysis and evaluation of the viability of bacteriophage-infected Escherichia coli (E.coli) XL-1 directly in a conducting suspension by using a slot-mode sensor. The method is based on recording the changes in the depth and frequency of resonant absorption peaks in the frequency dependence of the insertion loss of the sensor before and after the biologic interaction of E. coli with specific bacteriophages. The possibility was shown of recording the infection of E. coli with specific bacteriophages and assessing its viability in suspensions with a conductivity of 4.5-30 µS/cm. Сontrol experiments were carried out with non-specific interactions of E. coli cells with bacteriophages, in which no changes in the sensor variables were observed. The optimal informational variable for estimating the number of viable cells was the degree of change in the depth of the resonant peaks in the frequency dependence of the insertion loss of the sensor. The limit of cell detection was ∼102-103 cells mL-1, with an analysis time of about 5 min. An additional advantage of the sensor was the availability of a removable liquid container, which allows one to use it repeatedly and to facilitate the cleaning of the container from spent samples. The results are promising for the detection of bacteria and assessment of their viability in solutions with conductivity in the range 4.5-30 µS/cm.

Nutritional value of Sesamum indicum L. was improved by Azospirillum and Azotobacter under low input of NP fertilizers.

BACKGROUND: Sesame (Sesame indicum L.) is well-known as a versatile industrial crop having various usages and contains 50-55% oil, 20% protein, 14-20% carbohydrate and 2-3% fiber. Several environmental factors are known to adversely affect yield and productivity of sesame. Our overall aim was to improve the growth, yield and quality of sesame cv. TS-3 using plant growth promoting rhizobacteria (PGPR) and saving the nitrogen and phosphate fertilizers (NP) by 50%. Field experiment (randomized complete block design) was conducted during the months of July to October of two consecutive years 2012-2013. Azospirillum (AL) and Azotobacter (AV) were applied as seed inoculation alone as well as along with half of the recommended dose of nitrogen (N) and phosphate (P) fertilizers (urea and diammonium phosphate) at the rate of 25 kg/ha and 30 kg/ha respectively. RESULTS: Here we report that A. lipoferum along with half dose of NP fertilizers (ALCF) were highly effective in increasing the agronomic and yield traits of sesame as compared to the control. A. vinelandii plus NP fertilizers (AVCF) exhibited higher seed oil content. Minimum acid value, optimum specific gravity and modified fatty acid composition were observed in ALCF treatment. Increase in oleic acid by ALCF is directly linked with improved oil quality for health benefits as oleic acid is the fatty acid which creates a balance between saturation and unsaturation of oil and for the hypotensive (blood pressure reducing) effects. CONCLUSION: It is inferred that ALCF treatment improved plant growth, seed yield and oil quality of sesame pertaining to good quality edible oil production.

Biopriming of maize germination by the plant growth-promoting rhizobacterium Azospirillum lipoferum CRT1.

Plant growth-promoting rhizobacteria (PGPR) naturally aid plant growth, development and tolerance to stress. Yield increase by the commercial isolate Azospirillum lipoferum CRT1 was recently attributed to an enhanced sprouting success. In order to provide the first biochemical and physiological analysis of sprouting enhancement by PGPR, seed germination and metabolism were followed by time-lapse photography and GC/MS-based metabolomics, respectively, after inoculating two differentially-responding maize cultivars with A. lipoferum CRT1. Bacterial growth on the seeds and plantlet development were also determined. Bacterial inoculation of the seeds of one cultivar led to a 6-8 h hastening of radicle emergence, increased surface bacterial counts, lower contents of energetic primary metabolites before radicle emergence and increased photosynthetic yield, and root surface area, in 3-leaf plantlets. None of these changes were observed on the other maize cultivar that rather accumulated greater levels of stress-related metabolites shortly after radicle emergence. Bacterial counts and cell division-driven central root growth increased in parallel and similarly on both cultivars. A. lipoferum CRT1 stimulated pre-germinating or defense events in a cultivar-dependent manner in maize after rapid (less than 24 h) recognition with initially resting seeds. This PGPR isolate therefore bears agronomic potential as a biopriming agent.

Structural characterization and applications of a novel polysaccharide produced by Azospirillum lipoferum MTCC 2306.

Azospirillum lipoferum MTCC 2306, a free-living nitrogen fixing bacteria, has a doubling time of 1.7 h in MPSS media. At the end of 28 h at a pH of 7 and temperature of 30 °C it produces 1.8 ± 0.013 g/L biomass and 2.1 ± 0.018 g/L of cyclic beta glucan (CßG) in MPSS medium with a yield coefficient (YP/S) of 2.1. This novel polysaccharide is a water-soluble cyclic biopolymer and is generally produced by Rhizobiaceae and predominantly made up of glucose. The CßG has a degree of polymerisation varying between 10 and 13 and has both α- and ß-glycosidic linkages. It is not substituted with any functional groups such as acetates or succinates. Its ability to bind to aniline blue suggests that it can be a potential candidate for being used as carrier in medical imaging as well as in reducing toxicity of textile effluents. It is able to encapsulate rifampicin, a hydrophobic drug and increase its aqueous solubility by 71%. So, CßG appears to have promising applications in the field of drug, food, cosmetic and nutraceutical industries.

Analysis of the microbial cell-Ab binding in buffer solution by the piezoelectric resonator.

The possibility of the registration of the interaction of the cells Azospirillum lipoferum Sp59b with the specific antibodies directly in the conducting suspensions by using an acoustic sensor was shown. The main element of the sensor is a piezoelectric resonator with a lateral electric field. The analysis is based on a comparison of the resonator's electrical impedance before and after the specific biological interaction between the cells and antibodies. By using this sensor one can detect and identify the bacterial cells directly in the buffer solution with the conductivity between 2.4 and 20 µS/cm. The minimum detectable concentration of the bacterial cells turned out to be ∼103 cells/ml and for a short time (less than 10 min). Also the possibility of the detection of the cells in the presence of the extraneous microflora was shown. The results provide the opportunities for the development of a new class of the methods for the analysis of the microbial cells in real-time directly in the buffer solution.

Field-based assessment of the mechanism of maize yield enhancement by Azospirillum lipoferum CRT1.

Plant Growth-Promoting Bacteria (PGPB) of the genus Azospirillum are known to enhance root growth and yield in many plant species including cereals. To probe the underlying mechanisms, correlations between modifications of yield and 6-leaf plantlet characteristics were estimated on maize in four fields with contrasting soil properties over two consecutive years using the commercial isolate A. lipoferum CRT1. In both years, plantlet metabolome, photosynthetic potential and organ morphology were found to display field- and inoculation-specific signatures. Metabolomic analyses revealed that A. lipoferum CRT1 mostly affected sugar metabolism with no suggested impact on N and P assimilation. Mineral nitrogen feeding increased yield but did not affect yield enhancement by the bacterial partner. However, greater improvements of leaf photosynthetic potential correlated with yield diminutions and larger plantlets in all of their proportions correlated with yield enhancements. Bacterial inoculation restored proper seed-to-adult plant ratio when it accidentally dropped below 80%. Only in these cases did it raise yield. All in all, securing mature plant density is hypothesized as being the primary driver of A. lipoferum CRT1-mediated yield enhancement in maize fields.