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.

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.

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.

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).

Alteration of the exopolysaccharide production and the transcriptional profile of free-living Frankia strain CcI3 under nitrogen-fixing conditions.

We investigated the effect of different nitrogen (N) sources on exopolysaccharide (EPS) production and composition by Frankia strain CcI3, a N2-fixing actinomycete that forms root nodules with Casuarina species. Frankia cells grown in the absence of NH4Cl (i.e., under N2-fixing conditions) produced 1.7-fold more EPS, with lower galactose (45.1 vs. 54.7 mol%) and higher mannose (17.3 vs. 9.7 mol%) contents than those grown in the presence of NH4Cl as a combined N-source. In the absence of the combined N-source, terminally linked and branched residue contents were nearly twice as high with 32.8 vs. 15.1 mol% and 15.1 vs. 8.7 mol%, respectively, than in its presence, while the content of linearly linked residues was lower with 52.1 mol% compared to 76.2 mol%. To find out clues for the altered EPS production at the transcriptional level, we performed whole-gene expression profiling using quantitative reverse transcription PCR and microarray technology. The transcription profiles of Frankia strain CcI3 grown in the absence of NH4Cl revealed up to 2 orders of magnitude higher transcription of nitrogen fixation-related genes compared to those of CcI3 cells grown in the presence of NH4Cl. Unexpectedly, microarray data did not provide evidence for transcriptional regulation as a mechanism for differences in EPS production. These findings indicate effects of nitrogen fixation on the production and composition of EPS in Frankia strain CcI3 and suggest posttranscriptional regulation of enhanced EPS production in the absence of the combined N-source.

Effect of soybean coumestrol on Bradyrhizobium japonicum nodulation ability, biofilm formation, and transcriptional profile.

Flavonoids, secondary plant metabolites which mainly have a polyphenolic structure, play an important role in plant-microbe communications for nitrogen-fixing symbiosis. Among 10 polyphenolic compounds isolated from soybean roots in our previous study, coumestrol showed the highest antioxidant activity. In this study, its effect on the soybean nodulation was tested. The soybean symbiont Bradyrhizobium japonicum USDA110 pretreated with 20 µM coumestrol enhanced soybean nodulation by increasing the number of nodules 1.7-fold compared to the control. We also tested the effect of coumestrol on B. japonicum biofilm formation. At a concentration of 2 µM, coumestrol caused a higher degree of biofilm formation than two major soybean isoflavonoids, genistein and daidzein, although no biofilm formation was observed at a concentration of 20 µM each compound. A genome-wide transcriptional analysis was performed to obtain a comprehensive snapshot of the B. japonicum response to coumestrol. When the bacterium was incubated in 20 µM coumestrol for 24 h, a total of 371 genes (139 upregulated and 232 downregulated) were differentially expressed at a 2-fold cutoff with a q value of less than 5%. No common nod gene induction was found in the microarray data. However, quantitative reverse transcription-PCR (qRT-PCR) data showed that incubation for 12 h resulted in a moderate induction (ca. 2-fold) of nodD1 and nodABC, indicating that soybean coumestrol is a weak inducer of common nod genes. In addition, disruption of nfeD (bll4952) affected the soybean nodulation by an approximate 30% reduction in the average number of nodules.

A cloning vector employing a versatile ß-glucosidase as an indicator for recombinant clones.

A mutant glucosidase, cpGluT, with activity toward chromogenic substrates (X-gal [5-bromo-4-chloro-3-idolyl-ß-d-galactoside] and indican) and a fluorogenic 4-methylumbeliferyl-ß-d-glucopyranoside (MUG) was constructed by replacing the monomeric ß-glucosidase region (E314-N326) with designed multiple cloning sites. When expressed in hosts (lacZ+ and lacZ-), a vector containing the cpGluT produced a colored or fluorescent phenotype according to the substrate supplemented on LB plates without any inducer. cpGluT is readily incorporable into customized vectors and does not require special hosts to detect recombinant plasmids, thereby making screening recombinants more effective and less expensive.

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.

Whole-genome expression profiling of Bradyrhizobium japonicum in response to hydrogen peroxide.

Bradyrhizobium japonicum, a nitrogen-fixing bacterium in soil, establishes a symbiotic relationship with the leguminous soybean plant. Despite a mutualistic association between the two partners, the host plant produces an oxidative burst to protect itself from the invasion of rhizobial cells. We investigated the effects of H(2)O(2)-mediated oxidative stress on B. japonicum gene expression in both prolonged exposure (PE) and fulminant shock (FS) conditions. In total, 439 and 650 genes were differentially expressed for the PE and FS conditions, respectively, at a twofold cut-off with q < 0.05. A number of genes within the transport and binding proteins category were upregulated during PE and a majority of those genes are involved in ABC transporter systems. Many genes encoding ? factors, global stress response proteins, the FixK(2) transcription factor, and its regulatory targets were found to be upregulated in the FS condition. Surprisingly, catalase and peroxidase genes which are typically expressed in other bacteria under oxidative stress were not differentially expressed in either condition. The isocitrate lyase gene (aceA) was induced by fulminant H(2)O(2) shock, as was evident at both the transcriptional and translational levels. Interestingly, there was no significant effect of H(2)O(2) on exopolysaccharide production at the given experimental conditions.

Genome-wide transcriptional and physiological responses of Bradyrhizobium japonicum to paraquat-mediated oxidative stress.

The rhizobial bacterium Bradyrhizobium japonicum functions as a nitrogen-fixing symbiont of the soybean plant (Glycine max). Plants are capable of producing an oxidative burst, a rapid proliferation of reactive oxygen species (ROS), as a defense mechanism against pathogenic and symbiotic bacteria. Therefore, B. japonicum must be able to resist such a defense mechanism to initiate nodulation. In this study, paraquat, a known superoxide radical-inducing agent, was used to investigate this response. Genome-wide transcriptional profiles were created for both prolonged exposure (PE) and fulminant shock (FS) conditions. These profiles revealed that 190 and 86 genes were up- and downregulated for the former condition, and that 299 and 105 genes were up- and downregulated for the latter condition, respectively (>2.0-fold; P < 0.05). Many genes within putative operons for F(0)F(1)-ATP synthase, chemotaxis, transport, and ribosomal proteins were upregulated during PE. The transcriptional profile for the FS condition strangely resembled that of a bacteroid condition, including the FixK(2) transcription factor and most of its response elements. However, genes encoding canonical ROS scavenging enzymes, such as superoxide dismutase and catalase, were not detected, suggesting constitutive expression of those genes by endogenous ROS. Various physiological tests, including exopolysaccharide (EPS), cellular protein, and motility characterization, were performed to corroborate the gene expression data. The results suggest that B. japonicum responds to tolerable oxidative stress during PE through enhanced motility, increased translational activity, and EPS production, in addition to the expression of genes involved in global stress responses, such as chaperones and sigma factors.

A unique bicyclic monosaccharide from the Bradyrhizobium lipopolysaccharide and its role in the molecular interaction with plants.

Sugar coat: The nitrogen-fixing soil bacterium Bradyrhizobium sp. BTAi1 is coated with a unique lipopolysaccharide that does not induce innate immune responses in its host plant Aeschynomene indica or in different plant families. The chemical nature of the monosaccharide forming the polymer (see picture) is unprecedented in nature, which helps to avoid "harmful" recognition by its symbiotic host.

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.

In situ monitoring of antibiotic susceptibility of bacterial biofilms in a microfluidic device.

Antibiotic resistance of biofilms is a growing public health concern due to overuse and improper use of antibiotics. Thus, determining an effective minimal concentration of antibiotics to eradicate bacterial biofilms is crucial. Here we present a simple, novel one-pot assay for the analysis of antibiotic susceptibility of bacterial biofilms using a microfluidics system where continuous concentration gradients of antibiotics are generated. The results of minimal biofilm eradication concentration (MBEC) clearly confirm that the concentration required to eradicate biofilm-grown Pseudomonas aeruginosa is higher than the minimal inhibitory concentration (MIC) that has been widely used to determine the lowest concentration of antibiotics against planktonically grown bacteria.

Influence of water limitation on endogenous oxidative stress and cell death within unsaturated Pseudomonas putida biofilms.

Here we examined how water limitation (matric stress) and high osmolarity (solute stress) influence the extent of endogenous oxidative stress and cell death patterns within Pseudomonas putida biofilms. The temporal dynamics and spatial organization of reactive oxygen species (ROS) accumulation and dead cells in biofilms developed under water-replete and solute stress conditions were similar to each other. Arrays of dead cells, typically one cell width in diameter, were distributed throughout the biofilm and occasionally they spanned the entire depth of the biofilm. These arrays of dead cells were not observed under water-limiting conditions, although the extent of ROS accumulation and cell death was substantially greater. Despite the greater death rate under water-limiting conditions, culturable population sizes were transiently maintained at levels comparable to those under water-replete and solute stress conditions. There was greater spatial stratification of dead cells under water-limiting than water-replete conditions with viable cells primarily located at the air interface, which could facilitate cell dispersal following a wetting event. Under water-limiting conditions, ROS accumulation is greater in an DeltaalgD mutant compared with the wild type, suggesting that the exopolysaccharide alginate attenuates the extent of dehydration-mediated oxidative stress. We conclude that endogenous ROS accumulation is correlated with cell death within P. putida biofilms, although mechanisms contributing to their accumulation may differ under water-replete and water-limiting conditions.

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.

Whole-genome transcriptional profiling of Bradyrhizobium japonicum during chemoautotrophic growth.

Bradyrhizobium japonicum is a facultative chemoautotroph capable of utilizing hydrogen gas as an electron donor in a respiratory chain terminated by oxygen to provide energy for cellular processes and carbon dioxide assimilation via a reductive pentose phosphate pathway. A transcriptomic analysis of B. japonicum cultured chemoautotrophically identified 1,485 transcripts, representing 17.5% of the genome, as differentially expressed when compared to heterotrophic cultures. Genetic determinants required for hydrogen utilization and carbon fixation, including the uptake hydrogenase system and components of the Calvin-Benson-Bassham cycle, were strongly induced in chemoautotrophically cultured cells. A putative isocitrate lyase (aceA; blr2455) was among the most strongly upregulated genes, suggesting a role for the glyoxylate cycle during chemoautotrophic growth. Addition of arabinose to chemoautotrophic cultures of B. japonicum did not significantly alter transcript profiles. Furthermore, a subset of nitrogen fixation genes was moderately induced during chemoautotrophic growth. In order to specifically address the role of isocitrate lyase and nitrogenase in chemoautotrophic growth, we cultured aceA, nifD, and nifH mutants under chemoautotrophic conditions. Growth of each mutant was similar to that of the wild type, indicating that the glyoxylate bypass and nitrogenase activity are not essential components of chemoautotrophy in B. japonicum.