Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress.

Reference genes, also referred to as housekeeping genes (HKGs), play an important role in gene expression analysis by serving as an internal control. These HKGs are usually involved in basic cellular functions and their expression should remain at relatively constant levels. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been used to measure gene expression. Since the normalization of gene expression data depends on baseline expression of HKGs, it is important to identify and verify true HKGs for the qRT-PCR analysis. The goal of this study is to identify and confirm HKGs in Bradyrhizobium diazoefficiens, a nitrogen fixing bacterium which forms a symbiotic relationship with soybean. By revealing such HKGs, the normalization of gene expression would be more robust, reliable, and consistent. Here, we analyzed previous gene expression data for B. diazoefficiens under multiple environmental conditions. As a result, we identified seven constitutively expressed genes among 8453 genes across all conditions. Their fold-change values were within a range of −1.25-fold < x < 1.25-fold. We adopted GeNorm, NormFinder, and comparative ∆Ct methods to rank the seven candidate genes based on their expression stability. To validate these potential HKGs, we measured their expression in various experimental conditions, such as heat, pH, and heavy metal stress. The HKGs that were found in B. diazoefficiens were also applied in closely related species by identifying their homologs.

Microbial co-occurrence network in the rhizosphere microbiome: its association with physicochemical properties and soybean yield at a regional scale.

Microbial communities in the rhizosphere play a crucial role in determining plant growth and crop yield. A few studies have been performed to evaluate the diversity and co-occurrence patterns of rhizosphere microbiomes in soybean (Glycine max) at a regional scale. Here, we used a culture-independent method to compare the bacterial communities of the soybean rhizosphere between Nebraska (NE), a high-yield state, and Oklahoma (OK), a low-yield state. It is well known that the rhizosphere microbiome is a subset of microbes that ultimately get colonized by microbial communities from the surrounding bulk soil. Therefore, we hypothesized that differences in the soybean yield are attributed to the variations in the rhizosphere microbes at taxonomic, functional, and community levels. In addition, soil physicochemical properties were also evaluated from each sampling site for comparative study. Our result showed that distinct clusters were formed between NE and OK in terms of their soil physicochemical property. Among 3 primary nutrients (i.e., nitrogen, phosphorus, and potassium), potassium is more positively correlated with the high-yield state NE samples. We also attempted to identify keystone communities that significantly affected the soybean yield using co-occurrence network patterns. Network analysis revealed that communities formed distinct clusters in which members of modules having significantly positive correlations with the soybean yield were more abundant in NE than OK. In addition, we identified the most influential bacteria for the soybean yield in the identified modules. For instance, included are class Anaerolineae, family Micromonosporaceae, genus Plantomyces, and genus Nitrospira in the most complex module (ME9) and genus Rhizobium in ME23. This research would help to further identify a way to increase soybean yield in low-yield states in the U.S. as well as worldwide by reconstructing the microbial communities in the rhizosphere.

Draft Genome Sequences of Two Desiccation-Tolerant Strains, Bradyrhizobium japonicum TXVA and TXEA, Isolated from the Root Nodules of Soybean Grown in Texas.

Two Bradyrhizobium japonicum strains, TXVA and TXEA, were isolated for their desiccation tolerance and symbiotic performance with soybean as biofertilizers. Their genomes were sequenced and annotated using the Department of Energy Joint Genome Institute annotation pipeline. Sequencing yielded chromosomes of 9,193,770 and 9,339,455 bp for TXVA and TXEA, respectively.

Triphenylphosphonium-conjugated glycol chitosan microspheres for mitochondria-targeted drug delivery.

To develop an efficient vector for mitochondria-targeted drug delivery, we synthesized triphenylphosphonium (TPP)-modified glycol chitosan polymeric microspheres that had a unique chemical structure with both lipophilic phenyl groups and cationic phosphonium. Notably, TPP can easily pass through the phospholipid bilayer of mitochondria, thereby resulting in specific accumulation of a combined drug molecule in the mitochondria due to the membrane potential between TPP and its membrane. Therefore, TPP has been widely used as a mitochondria-targeting moiety. Triphenylphosphonium-glycol chitosan derivatives (GC-TPP and GME-TPP) with two different degrees of substitution (11% and 36%) were prepared by amidation and Michael addition. The chemical structures of GC-TPP and GME-TPP were characterized by 1H nuclear magnetic resonance and Fourier-transform infrared spectroscopy, and their sizes were measured via field emission scanning electron microscopy and dynamic light scattering. Cellular uptake through flow cytometric analysis and confocal microscopy confirmed that both GC-TPP and GME-TPP were well introduced into cells, targeting the mitochondria. In addition, cytotoxicity testing of the most common cell lines, such as HEK293, HeLa, NIH3T3, and HepG2, indicated the absence of polymer toxicity. To evaluate the carrier effectiveness of TPP for drug delivery, doxorubicin (Dox) was used as an anticancer drug. Confocal microscopy images showed that Dox-loaded GME-TPP accumulated inside cells more than Dox-loaded GC-TPP. The anticancer effects of Dox were also determined by MTT assay, apoptosis/necrosis assay, and three-dimensional spheroids. In summary, the results indicate that GC-TPP and GME-TPP microspheres possess great potential as effective drug delivery carriers.

Genome Sequence of Bacillus subtilis natto VK161, a Novel Strain That Produces Vitamin K2.

Bacillus subtilis strain natto VK161 was selected for its high production of vitamin K2 Its genome was sequenced and annotated in the Department of Energy-Joint Genome Institute (DOE-JGI) annotation pipeline. It resulted in a chromosome of 4,073,396 bp, which is composed of 4,332 protein-coding genes, 23 rRNA genes, and 77 tRNA genes.

Photo-fermentation of purple sweet potato (Ipomoea batatas L.) using probiotic bacteria and LED lights to yield functionalized bioactive compounds.

The purpose of this study was to examine if fermentation of purple sweet potato (Ipomoea batatas L.) powder (PSP) by Lactobacillus brevis under green, red, blue, white light-emitting diode (LED) illumination or sunlight might yield functionalized products with good antibacterial, antioxidant activity, and/or cytotoxic activity. The Purple sweet potato (PSP) powder fermented with probiotic bacteria L. brevis under white LED light (1.9 ± 1.80/1.6 ± 0.52), blue LED light (1.4 ± 1.32/1.8 ± 0.83), or sunlight (1.2 ± 1.26/1.5 ± 1.83) for Propionibacterium acne and Staphylococcus epidermidis displayed good to moderate antibacterial activity based on minimum inhibitory concentration (MIC) red, blue, white LED lights and sunlight (80 µg/mL) for P. acne and S. epidermidis, minimum bactericidal concentration red, blue LED lights and sunlight shows (46/48, 61/70, 50/48 µg/mL) for P. acne and S. epidermidis. Antioxidant activity for dark, white, blue and green LED lights for ABTS and white, blue and green Led for DPPH assay resulted in lower activity. Fourier transform infrared spectroscopy was performed to determine the functional groups in the non-fermented (control) and fermented products of PSP powders obtained using different light sources. Sunlight, white, and blue LED light-fermented extracts contained alcohol, acid, and phenol groups, as well as aliphatic amines. The results of this study clearly indicate that fermentation of purple sweet potato with probiotic bacteria under various LED light sources can yield compounds that can be used in cosmetic and value-added food products.

Characterization and assessment of two biocontrol bacteria against Pseudomonas syringae wilt in Solanum lycopersicum and its genetic responses.

Pseudomonas and Bacillus species are attractive due to their potential bio-control application against plant bacterial pathogens. Pseudomonas aeruginosa strain D4 and Bacillus stratosphericus strain FW3 were isolated from mine tailings in South Korea. In these potent bacterial strains, we observed improved antagonistic activity against Pseudomonas syringae DC3000. These strains produced biocatalysts for plant growth promotion, and in vivo examination of Solanum lycopersicum included analysis of disease severity, ion leakage, chlorophyll content, and H2O2 detection. In addition, regulation of the defense genes pathogen-related protein 1a (PR1a) and phenylalanine ammonia lyase (PAL) was compared with treated plants and untreated control plants. The results suggest that these two bacterial strains provide protection against plant pathogens via direct and indirect modes of action and could be used as a bio-control agent.

Potential for plant biocontrol activity of isolated Pseudomonas aeruginosa and Bacillus stratosphericus strains against bacterial pathogens acting through both induced plant resistance and direct antagonism.

Phytopathogenic bacteria have caused significant damage to agricultural crops in both controlled and open cultivation practices, imposing heavy losses to farmers. Thereby, the goal of this study was to evaluate Pseudomonas aeruginosa and Bacillus stratosphericus isolated from soil has antagonistic activity against bacterial phytopathogens with the potential to control plant diseases. Isolated novel strains of P. aeruginosa and B. stratosphericus showed broad spectrum of antagonistic activity against five bacterial phytopathogens. Antagonistic activity was examined under optimized pH (8 and 7), carbon sources (lactose and starch), nitrogen sources (ammonium chloride, peptone and ammonium nitrate) for P. aeruginosa and B. stratosphericus, respectively, and biocatalyst production (chitinase, protease and amylase) was studied. Additionally, up-regulation of defense-related genes (PR-1a and PAL) was studied in tomato plants treated with P. aeruginosa and B. stratosphericus. The induction of defense-related genes in tomato plant was triggered after 12 h treatment with a cell concentration of 0.20 O.D. for P. aeruginosa and 0.21 O.D. for B. stratosphericus during treatment period. Broad spectrum antagonistic activity was observed due to antibiotic and siderophore production by P. aeruginosa and B. stratosphericus.

Extraction of natural colorant from purple sweet potato and dyeing of fabrics with silver nanoparticles for augmented antibacterial activity against skin pathogens.

The main objective of this study was to extract natural colorant from purple sweet potato powder (PSPP) via a water bath and ultrasound water bath using acidified ethanol (A. EtOH) as the extraction solvent. When optimizing the colorant extraction conditions of the solvents, acidified ethanol with ultrasound yielded a high extraction capacity and color intensity at pH2, temperature of 80°C, 20mL of A. EtOH, 1.5g of PSPP, time of 45min, and ultrasonic output power of 75W. Subsequently, the colorant was extracted using the optimized conditions for dyeing of textiles (leather, silk, and cotton). This natural colorant extraction technique can avoid serious environmental pollution during the extraction and is an alternative to synthetic dyes, using less solvent and simplified abstraction procedures. The extracted purple sweet potato natural colorant (PSPC) was used to dye leather, silk, and cotton fabrics in an eco-friendly approach with augmented antibacterial activity by in situ synthesis of silver nanoparticles (AgNPs) and dyeing. The optimal dyeing conditions for higher color strength (K/S) values were pH2 and 70°C for 45min. The colorimetric parameters L∗, a∗, b∗, C, and H were measured to determine the depth of the color. The Fourier transform infrared spectroscopy (FTIR) spectra of undyed control, dyed with PSPC and dyed with blend of PSPC and AgNPs treated leather, silk and cotton fabric were investigated to study the interaction among fiber type, nanoparticles, and dye. The structural morphology of leather and silk and cotton fabrics and the anchoring of AgNPs with elemental compositions were investigated by scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS). The dry and wet rubbing fastness for dye alone and dye with nanoparticles were grade 4-5 and 4, respectively. Thus, the results of the present study clearly suggest that in situ synthesis of AgNPs along with dyeing should be considered in the development of antimicrobial textile finishes.

Fabrication, optimization, and characterization of noble silver nanoparticles from sugarcane leaf (Saccharum officinarum) extract for antifungal application.

Metal nanoparticles obtained from green route are gaining significant prominence as a result of their potential applications in nanomedicine and material engineering. Overall metal nanoparticles studied, silver nanoparticles (AgNPs) clutch prominent place in nanoparticles research field. Herein, we have reported the green synthesis of Saccharum officinarum leaf biomass extract-mediated synthesis of AgNPs. Initial nanoparticle production was confirmed by visual observation as color change from light yellow to bright brown color with yellow shade and spectrophotometrically at 450 nm and the various reaction conditions were optimized. The FTIR spectra of the biomass extract and synthesized AgNPs authorized the presence of phyto constituents as capping agent. The High Resolution-Transmission Electron Microscopy (HR-TEM) analyses confirm the morphology and the average particle size of AgNPs as ~28.2 nm. The crystalline nature oxide state and mean particle diameter of AgNPs were confirmed by X-ray diffraction (XRD) analysis, Selected Area Electron Diffraction (SAED) pattern and face-centered cubic (FCC). The obtained AgNPs show moderate to good antifungal activity against Phytophthora capsici, Colletotrichum acutatum and Cladosporium fulvum as 10, 12 and 14 mm zones of inhibition against synthesized AgNPs at 250 µg/well, respectively.

Green synthesis of silver oxide nanoparticles and its antibacterial activity against dental pathogens.

In the present study, the use of silver oxide nanoparticles (Ag2O NPs) synthesized using Ficus benghalensis prop root extract (FBPRE) as a reducing and stabilizing agent is reported and evaluated for its antibacterial activity against dental bacterial strains. The effects of pH, extract concentration, metal ion concentration, and contact time were studied to confirm the optimum production of Ag2O NPs. Our results suggest that, by increasing the extract concentration and the time frame, there will be a significant increase in the formation of nanoparticles. The UV-vis adsorption spectra show the absorbance peak in the range of 430 nm, and FTIR spectral peaks indicate that the phytochemicals in the extract are responsible for the formation of the nanoparticles. The HR-TEM image, SAED, and XRD pattern confirmed the morphology (spherical), silver oxide 42.7 nm and silver 51.4 nm, and crystalline nature of the obtained nanoparticles, respectively. The blend of FBPRE and Ag2O NPs showed excellent antibacterial activities against the two-dental bacteria Streptococcus mutans and Lactobacilli sp. The study results suggest that the blend of synthesized Ag2O NPS and FBPRE will be useful in tooth paste as a germicidal agent after extensive investigation with animal models.

Characterization of MocR, a GntR-like transcriptional regulator, in Bradyrhizobium japonicum: its impact on motility, biofilm formation, and soybean nodulation.

Bradyrhizobium japonicum is a Gram-negative soil bacterium that can fix nitrogen into ammonia by developing a symbiotic relationship with the soybean plant. MocR proteins make up a subfamily of GntR superfamily, one of the most widely distributed and prolific groups of the helix-turn-helix transcription factors. In this study, we constructed a mutant strain for mocR (blr6977) to investigate its role in cellular processes and symbiosis in B. japonicum. Although growth rate and morphology of the mutant were indistinguishable from those of the wild type, the mutant showed significant differences in motility and attachment (i.e., biofilm formation) from the wild type. The mutant displayed a decrease in biofilm formation, but was more motile than the wild type. The inactivation of mocR did not affect the number of nodules on soybean roots, but caused delayed nodulation. Delayed nodulation intrigued us to study competitiveness of the mutant infecting soybeans. The mutant was less competitive than the wild type, indicating that delayed nodulation might be due to competitiveness. Gene expressions of other MocR subfamily members were also compared between the wild type and mutant strains. None of the mocR-like genes examined in this study were differentially expressed between both strains.

Role of the extracytoplasmic function sigma factor CarQ in oxidative response of Bradyrhizobium japonicum.

As a nitrogen-fixing bacterium, Bradyrhizobium japonicum can establish a symbiotic relationship with the soybean plant (Glycine max). To be a successful symbiont, B. japonicum must deal with plant defense responses, such as an oxidative burst. Our previous functional genomics study showed that carQ (bll1028) encoding extracytoplasmic function (ECF) sigma factor was highly expressed (107.8-fold induction) under oxidative stress. Little is known about the underlying mechanisms of how CarQ responds to oxidative stress. In this study, a carQ knock-out mutant was constructed using site-specific mutagenesis to identify the role of carQ in the oxidative response of B. japonicum. The carQ mutant showed a longer generation time than the wild type and exhibited significantly decreased survival at 10 mM H(2)O(2) for 10 min of exposure. Surprisingly, there was no significant difference in expression of oxidative stress-responsive genes such as katG and sod between the wild type and carQ mutant. The mutant also showed a significant increase in susceptibility to H(2)O(2) compared to the wild type in the zone inhibition assay. Nodulation phenotypes of the carQ mutant were distinguishable compared to those of the wild type, including lower numbers of nodules, decreased nodule dry weight, decreased plant dry weight, and a lower nitrogen fixation capability. Moreover, desiccation of mutant cells also resulted in significantly lower percent of survival in both early (after 4 h) and late (after 24 h) desiccation periods. Taken together, this information will provide an insight into the role of the ECF sigma factor in B. japonicum to deal with a plant-derived oxidative burst.

Characterization of a Functional Role of the Bradyrhizobium japonicum Isocitrate Lyase in Desiccation Tolerance.

Bradyrhizobium japonicum is a nitrogen-fixing symbiont of soybean. In previous studies, transcriptomic profiling of B. japonicum USDA110, grown under various environmental conditions, revealed the highly induced gene aceA, encoding isocitrate lyase (ICL). The ICL catalyzes the conversion of isocitrate to succinate and glyoxylate in the glyoxylate bypass of the TCA cycle. Here, we evaluated the functional role of B. japonicum ICL under desiccation-induced stress conditions. We purified AceA (molecular mass = 65 kDa) from B. japonicum USDA110, using a His-tag and Ni-NTA column approach, and confirmed its ICL enzyme activity. The aceA mutant showed higher sensitivity to desiccation stress (27% relative humidity (RH)), compared to the wild type. ICL activity of the wild type strain increased approximately 2.5-fold upon exposure to 27% RH for 24 h. The aceA mutant also showed an increased susceptibility to salt stress. Gene expression analysis of aceA using qRT-PCR revealed a 148-fold induction by desiccation, while other genes involved in the glyoxylate pathway were not differentially expressed in this condition. Transcriptome analyses revealed that stress-related genes, such as chaperones, were upregulated in the wild-type under desiccating conditions, even though fold induction was not dramatic (ca. 1.5-2.5-fold).

Effects of the Bradyrhizobium japonicum waaL (rfaL) Gene on Hydrophobicity, Motility, Stress Tolerance, and Symbiotic Relationship with Soybeans.

We cloned and sequenced the waaL (rfaL) gene from Bradyrhizobium japonicum, which infects soybean and forms nitrogen-fixing nodules on soybean roots. waaL has been extensively studied in the lipopolysaccharide (LPS) biosynthesis of enteric bacteria, but little is known about its function in (brady)rhizobial LPS architecture. To characterize its role as O-antigen ligase in the LPS biosynthesis pathway, we constructed a waaL knock-out mutant and its complemented strain named JS015 and CS015, respectively. LPS analysis showed that an LPS structure of JS015 is deficient in O-antigen as compared to that of the wild type and complemented strain CS015, suggesting that WaaL ligates the O-antigen to lipid A-core oligosaccharide to form a complete LPS. JS015 also revealed increased cell surface hydrophobicity, but it showed decreased motility in soft agar plates. In addition to the alteration in cell surface properties, disruption of the waaL gene caused increased sensitivity of JS015 to hydrogen peroxide, osmotic pressure, and novobiocin. Specifically, plant tests revealed that JS015 failed to nodulate the host plant soybean, indicating that the rhizobial waaL gene is responsible for the establishment of a symbiotic relationship between soybean and B. japonicum.

Nitrospirillum irinus sp. nov., a diazotrophic bacterium isolated from the rhizosphere soil of Iris and emended description of the genus Nitrospirillum.

A polyphasic approach was used to characterize a novel nitrogen-fixing bacterial strain, designated YC6995(T), isolated from the rhizosphere soil of Iris ensata var. spontanea (Makino) Nakai inhabiting a wetland located at an altitude of 960 m on Jiri Mountain, Korea. Strain YC6995(T) cells were Gram-negative, and rod-shaped, with motility provided by a single polar flagellum. Optimal growth conditions were 30 °C and pH 7.0. The major fatty acids of strain YC6995(T) were C18:1 ω7c, C18:1 2-OH and C16:0 3-OH. The major respiratory quinone was ubiquinone-10 (Q-10). The polar lipids were phosphatidylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine, phosphatidylglycerol and unidentified glycolipids. The genomic DNA G+C content was 64.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed strain YC6995(T) to form a phyletic lineage with Nitrospirillum amazonense DSM 2787(T) with a high sequence similarity (97.2 %), but it displayed low sequence similarity with other remotely related genera, including Azospirillum (<93 %), Rhodocista (93.1-93.4 %), and Skermanella (91.2-93.3 %) in the family Alphaproteobacteria. Based on the phenotypic, chemotaxonomic, and phylogenetic evidences, strain YC6995(T) represents a novel species within the genus Nitrospirillum, for which the name Nitrospirillum irinus sp. nov. is proposed. The type strain is YC6995(T) (= KACC 13777(T) = DSM 22198(T)). An emended description of the genus Nitrospirillum is also proposed.

DNA Microarray-Based Identification of Genes Regulated by NtrC in Bradyrhizobium japonicum.

The Bradyrhizobium japonicum NtrBC two-component system is a critical regulator of cellular nitrogen metabolism, including the acquisition and catabolism of nitrogenous compounds. To better define the roles of this system, genome-wide transcriptional profiling was performed to identify the NtrC regulon during the response to nitrogen limitation. Upon cells perceiving low intracellular nitrogen, they stimulate the phosphorylation of NtrC, which induces genes responsible for alteration of the core glutamine synthetase/glutamate synthase nitrogen assimilation pathway, including the genes for the glutamine synthetases and PII proteins. In addition, genes responsible for the import and utilization of multiple nitrogen sources, specifically nitrate and nitrite, were upregulated by NtrC activation. Mutational analysis of a candidate nitrite reductase revealed a role for NtrC in regulating the assimilation of nitrite, since mutations in both ntrC and the gene encoding the candidate nitrite reductase abolished the ability to grow on nitrite as a sole nitrogen source.

Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes.

Lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria and are essential for their growth and survival. They act as a structural barrier and play an important role in the interaction with eukaryotic hosts. Here we demonstrate that a photosynthetic Bradyrhizobium strain, symbiont of Aeschynomene legumes, synthesizes a unique LPS bearing a hopanoid covalently attached to lipid A. Biophysical analyses of reconstituted liposomes indicate that this hopanoid-lipid A structure reinforces the stability and rigidity of the outer membrane. In addition, the bacterium produces other hopanoid molecules not linked to LPS. A hopanoid-deficient strain, lacking a squalene hopene cyclase, displays increased sensitivity to stressful conditions and reduced ability to survive intracellularly in the host plant. This unusual combination of hopanoid and LPS molecules may represent an adaptation to optimize bacterial survival in both free-living and symbiotic states.

Differential responses of B vitamins in black soybean seeds.

This study was aimed to determine the contents and the association of B vitamins from seeds of 10 black and one yellow soybean (Glycine max (L.) Merr.) varieties with either green or yellow cotyledon. Thiamine, flavin adenine dinucleotide (FAD), riboflavin and total riboflavin were found highest in 'Chengjakong', while flavin mononucleotide (FMN) was greatest in 'Mirang'. Nicotinic acid and total vitamin B3 were highest in 'Shingi' as a yellow soybean variety but pantothenic acid and pyridoxine contents were greatest in 'Tawon' and 'Mirang', respectively. These content variations of B vitamins directly reflected the wide segregation of soybean varieties on the principal component analysis (PCA) scores plot, indicating that these 4 soybean varieties appeared to be least associated with other soybean varieties based on the different responses of B vitamins. The results of cluster and correlation analyses presented that the cotyledon colour of soybean seed contributed to a variation of B vitamin contents. Overall, the results suggest that a wide range of B vitamin contents would be affected by genotypic factors alongside the difference of cotyledon colour.