Streptomyces caniscabiei sp. nov., which causes potato common scab and is distributed across the world.

Fourteen strains of Streptomyces isolated from scab lesions on potato are described as members of a novel species based on genetic distance, morphological observation and biochemical analyses. Morphological and biochemical characteristics of these strains are distinct from other described phytopathogenic species. Strain NE06-02DT has white aerial mycelium and grey, cylindrical, smooth spores on rectus-flexibilis spore chains. Members of this species group can utilize most of the International Streptomyces Project sugars, utilize melibiose and trehalose, produce melanin, grow on 6-7 % NaCl and pH 5-5.5 media, and are susceptible to oleandomycin (100 µg ml-1), streptomycin (20 µg ml-1) and penicillin G (30 µg ml-1). Though the 16S rRNA gene sequences from several members of this novel species are identical to the Streptomyces bottropensis 16S rRNA gene sequence, whole-genome average nucleotide identity and multi-locus sequence analysis confirm that the strains are members of a novel species. Strains belonging to this novel species have been isolated from the United States, Egypt and China with the earliest known members being isolated in 1961 from common scab lesions of potato in both California, USA, and Maine, USA. The name Streptomyces caniscabiei sp. nov. is proposed for strain NE06-02DT (=DSM111602T=ATCC TSD-236T) and the other members of this novel species group.

Description of Streptomyces griseiscabiei sp. nov. and reassignment of Streptomyces sp. strain NRRL B-16521 to Streptomyces acidiscabies.

Streptomyces strain NRRL B-2795T (DSM 112329T=NRRL B-2795T) is described as the type strain of Streptomyces griseiscabiei sp. nov. using whole-genome average nucleotide identity and multilocus sequence analyses in addition to phenotypic characterization of carbon source utilization, spore chain morphology, melanin production, salt tolerance, pH tolerance, plant pathogenicity and antibiotic resistance. This strain was previously classified as Streptomyces scabiei but suggested as a potential novel species. A second Streptomyces strain, NRRL B-16521, previously named Streptomyces scabiei, and also previously suggested as a potential novel species, is assigned to Streptomyces acidiscabies based on whole-genome average nucleotide identity. Morphological and biochemical characterizations also support this designation for NRRL B-16521. Both Streptomyces sp. strain NRRL B-2795T and NRRL B-16521 cause common scab on multiple cultivars of potato.

Global-level population genomics reveals differential effects of geography and phylogeny on horizontal gene transfer in soil bacteria.

Although microorganisms are known to dominate Earth's biospheres and drive biogeochemical cycling, little is known about the geographic distributions of microbial populations or the environmental factors that pattern those distributions. We used a global-level hierarchical sampling scheme to comprehensively characterize the evolutionary relationships and distributional limitations of the nitrogen-fixing bacterial symbionts of the crop chickpea, generating 1,027 draft whole-genome sequences at the level of bacterial populations, including 14 high-quality PacBio genomes from a phylogenetically representative subset. We find that diverse Mesorhizobium taxa perform symbiosis with chickpea and have largely overlapping global distributions. However, sampled locations cluster based on the phylogenetic diversity of Mesorhizobium populations, and diversity clusters correspond to edaphic and environmental factors, primarily soil type and latitude. Despite long-standing evolutionary divergence and geographic isolation, the diverse taxa observed to nodulate chickpea share a set of integrative conjugative elements (ICEs) that encode the major functions of the symbiosis. This symbiosis ICE takes 2 forms in the bacterial chromosome-tripartite and monopartite-with tripartite ICEs confined to a broadly distributed superspecies clade. The pairwise evolutionary relatedness of these elements is controlled as much by geographic distance as by the evolutionary relatedness of the background genome. In contrast, diversity in the broader gene content of Mesorhizobium genomes follows a tight linear relationship with core genome phylogenetic distance, with little detectable effect of geography. These results illustrate how geography and demography can operate differentially on the evolution of bacterial genomes and offer useful insights for the development of improved technologies for sustainable agriculture.

Determination of nicotine and its metabolites accumulated in fish tissue using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

The determination of nicotine and its major metabolites (cotinine and anabasine) in fish tissue was performed using liquid chromatography and tandem mass spectrometry. Marine and freshwater fish were purchased from local grocery stores and were prepared based on a quick, easy, cheap, effective, rugged, and safe sample preparation protocol. To determine the highly polar compounds, hydrophilic interaction liquid chromatography was also used. There were modest suppressions on measured nicotine signals (10%) due to the matrix effects from marine fish but no obvious effects on freshwater fish signals. Method validation was incorporated with internal standards and carried out with matrix-matched calibration. The detection limits for nicotine, cotinine, and anabasine were 9.4, 3.0, and 1.5 ng/g in fish, respectively. Precision was quite acceptable returning less than 8% RSD at low, medium, and high concentrations. Acceptable and reproducible extraction recoveries (70-120%) of all three compounds were achieved, except for anabasine at low concentration (61%). The method was then applied to define nicotine bioaccumulation in a fathead minnow model, which resulted in rapid uptake with steady state internal tissue levels, reached within 12 h. This developed method offers a fast, easy, and sensitive way to evaluate nicotine and its metabolite residues in fish tissues.

NopP2 effector of Bradyrhizobium elkanii USDA61 is a determinant of nodulation in Vigna radiata cultivars.

The symbiotic relationship between legumes and rhizobia is known to be influenced by specific rhizobial type III effectors (T3Es) in certain cases. In this study, we present evidence that the symbiosis between Vigna radiata and Bradyrhizobium elkanii USDA61 is controlled by a T3E called NopP2, and this interaction is highly dependent on the genetic makeup of the host plant. NopP2 plays a crucial role in promoting nodulation in various V. radiata varieties. Additionally, NopP2 is essential for early infection and the formation of nodules in compatible plants. Through evolutionary analysis, we discovered that bradyrhizobial NopPs can be categorized into two distinct clusters: NopP1 and NopP2. Furthermore, both types of bradyrhizobial NopPs were conserved within their respective groups. Our findings suggest that NopP2 serves as a mechanism for optimizing the symbiotic relationship between V. radiata and B. elkanii USDA61 by interacting with the pathogenesis related-10 (PR10) protein and reducing effector-triggered immunity (ETI) responses.

Hydrophilic interaction liquid chromatography coupled with MS/MS to detect and quantify dicarboxyethyl glutathione, a metabolic biomarker of the fumarate hydratase deficient cancer cell.

The identification of a glutathione (GSH) fumarate conjugate, dicarboxyethyl glutathione, formed during the nonenzymatic succination of GSH by fumarate was confirmed in fumarate hydratase deficient cells using a product ion scan approach followed by hydrophilic interaction liquid chromatography coupled with MS/MS. GSH and its conjugates, including dicarboxyethyl glutathione and glutathione disulfide, were successfully separated on a zwitterionic stationary phase and detected by MS/MS operated under negative ESI mode. The relative quantitation of the analytes in cell extracts was carried out and a correction model was established to determine correction factors under matrix effects and the response mismatch between the analytes. These factors were calculated and iteratively used to measure all analytes in cell extracts, based on calibration curves constructed in neat solution. The model was a closed-loop calculation, consisting of two sides with each side of the loop presenting a calculation pathway. Deviation of the correction factors obtained from these pathways manifested the model accuracy. The model was evaluated and there was no significant difference between the two pathways.

Evaluation of matrix effects in analysis of estrogen using liquid chromatography-tandem mass spectrometry.

Matrix effects of different biological samples, including phosphate-buffered saline-bovine serum albumin (PBS-BSA), gelded horse serum, mouse serum, and mouse brain, were investigated for the determination of 17α- and ß-estradiol using derivatization with dansyl chloride prior to LC-MS/MS. Matrix effects were evaluated based on the slopes of regression lines plotted from results obtained in biological matrices versus pure standard solutions. Such plots indicate the enhancement or suppression of signal based on the presence of a particular biological fluid for a particular method. The matrix effects from PBS-BSA were similar to those of mouse serum. In contrast, analyses performed from horse serum and mouse brain yielded significant ion suppression, especially for 17ß-estradiol. Precipitation during derivatization was observed when pre-concentrated samples were processed with ethyl acetate as an extraction solvent. This was overcome with the use of methyl tert-butyl ether; however, matrix effects from this preparation were still present, evidenced by signal suppression and poor linearity in the standard curve. This work affirms that caution should be taken in the transfer of methods for use with different biological matrices, especially in the case where surrogate matrices are necessary for calibration purposes.

The Bradyrhizobium diazoefficiens type III effector NopE modulates the regulation of plant hormones towards nodulation in Vigna radiata.

Host-specific legume-rhizobium symbiosis is strictly controlled by rhizobial type III effectors (T3Es) in some cases. Here, we demonstrated that the symbiosis of Vigna radiata (mung bean) with Bradyrhizobium diazoefficiens USDA110 is determined by NopE, and this symbiosis is highly dependent on host genotype. NopE specifically triggered incompatibility with V. radiata cv. KPS2, but it promoted nodulation in other varieties of V. radiata, including KPS1. Interestingly, NopE1 and its paralogue NopE2, which exhibits calcium-dependent autocleavage, yield similar results in modulating KPS1 nodulation. Furthermore, NopE is required for early infection and nodule organogenesis in compatible plants. Evolutionary analysis revealed that NopE is highly conserved among bradyrhizobia and plant-associated endophytic and pathogenic bacteria. Our findings suggest that V. radiata and B. diazoefficiens USDA110 may use NopE to optimize their symbiotic interactions by reducing phytohormone-mediated ETI-type (PmETI) responses via salicylic acid (SA) biosynthesis suppression.

Rhizobia use a pathogenic-like effector to hijack leguminous nodulation signalling.

Legume plants form a root-nodule symbiosis with rhizobia. This symbiosis establishment generally relies on rhizobium-produced Nod factors (NFs) and their perception by leguminous receptors (NFRs) that trigger nodulation. However, certain rhizobia hijack leguminous nodulation signalling via their type III secretion system, which functions in pathogenic bacteria to deliver effector proteins into host cells. Here, we report that rhizobia use pathogenic-like effectors to hijack legume nodulation signalling. The rhizobial effector Bel2-5 resembles the XopD effector of the plant pathogen Xanthomonas campestris and could induce nitrogen-fixing nodules on soybean nfr mutant. The soybean root transcriptome revealed that Bel2-5 induces expression of cytokinin-related genes, which are important for nodule organogenesis and represses ethylene- and defense-related genes that are deleterious to nodulation. Remarkably, Bel2-5 introduction into a strain unable to nodulate soybean mutant affected in NF perception conferred nodulation ability. Our findings show that rhizobia employ and have customized pathogenic effectors to promote leguminous nodulation signalling.

Retention behavior of estrogen metabolites on hydrophilic interaction chromatography stationary phases.

Estrogens and estrogen metabolites are important biological mediators of the endocrine system. They have also been implicated in detrimental carcinogenesis and beneficial neuroprotective processes. The retention behavior of estrogen metabolites was investigated on five polar stationary phases, used for hydrophilic interaction chromatography, and coupled with ESI-MS. Data were fit to partitioning and surface adsorption models. Retention of the compounds, especially estrogen glucuronides, on the amide- and diol-bonded stationary phases, could be best described by the surface adsorption model; however, mixed modes of retention were observed on most stationary phases. Retention time increased while the peak efficiency decreased proportional to the number of hydroxyl groups in the analytes. The effects of salt concentration and salt type were also investigated. The presence of solvated salt ions, which interact with the stationary phase and the analyte, enhanced retention of the analytes. This was believed to be due to two effects. The increased ionic strength reduced the contribution of secondary electrostatic interactions (mixed-mode effects). It also enhanced hydrogen-bonding and partitioning (hydrophilic interaction) between the analyte and the stationary phase, likely facilitated by the associated solvated salt ions.

Novel rhizobia exhibit superior nodulation and biological nitrogen fixation even under high nitrate concentrations.

Legume-rhizobium symbiosis leads to the formation of nitrogen-fixing root nodules. However, externally applied chemical nitrogen fertilizers (nitrate and ammonia) strongly inhibit nodule formation and nitrogen fixation. Here, we isolated several rhizobial strains exhibiting a superior nodulation and nitrogen fixation with soybean at high nitrate concentrations. The nodulation of soybean symbiont Bradyrhizobium diazoefficiens USDA110 was significantly inhibited at 12.5 mM nitrate; however, three isolates (NKS4, NKM2 and NKTG2) were capable of forming nitrogen-fixing nodules, even at 20 mM nitrate. These isolates exhibited higher nodulation competitiveness and induced larger nodules with higher nitrogen-fixation activity than USDA110 at 5 mM nitrate. Furthermore, these isolates induced more nodules than USDA110 even in nitrate-free conditions. These isolates had a distant lineage within the Bradyrhizobium genus; though they were relatively phylogenetically close to Bradyrhizobium japonicum, their morphological and growth characteristics were significantly different. Notably, in the presence of nitrate, expression of the soybean symbiosis-related genes (GmENOD40 and GmNIN) was significantly higher and expression of GmNIC1 that is involved in nitrate-dependent nodulation inhibition was lower in the roots inoculated with these isolates in contrast with inoculation of USDA110. These novel rhizobia serve as promising inoculants for soybeans cultivated in diverse agroecosystems, particularly on nitrate-applied soils.

Identification of Bradyrhizobium elkanii USDA61 Type III Effectors Determining Symbiosis with Vigna mungo.

Bradyrhizobium elkanii USDA61 possesses a functional type III secretion system (T3SS) that controls host-specific symbioses with legumes. Here, we demonstrated that B. elkanii T3SS is essential for the nodulation of several southern Asiatic Vigna mungo cultivars. Strikingly, inactivation of either Nod factor synthesis or T3SS in B. elkanii abolished nodulation of the V. mungo plants. Among the effectors, NopL was identified as a key determinant for T3SS-dependent symbiosis. Mutations of other effector genes, such as innB, nopP2, and bel2-5, also impacted symbiotic effectiveness, depending on host genotypes. The nopL deletion mutant formed no nodules on V. mungo, but infection thread formation was still maintained, thereby suggesting its pivotal role in nodule organogenesis. Phylogenetic analyses revealed that NopL was exclusively conserved among Bradyrhizobium and Sinorhizobium (Ensifer) species and showed a different phylogenetic lineage from T3SS. These findings suggest that V. mungo evolved a unique symbiotic signaling cascade that requires both NFs and T3Es (NopL).

The advantages of ESI-MS detection in conjunction with HILIC mode separations: Fundamentals and applications.

The analysis of highly hydrophilic, ionic, and polar compounds has been performed by HILIC-ESI-MS for the last few years. The use of low aqueous/high polar organic solvent content in HILIC separation mobile phase is almost ideal for ESI-MS detection in many cases, resulting in increased sensitivity. Although the addition of modifiers such as acids or salts is necessary in some circumstances for a good separation, the optimum concentrations used are still highly amenable for ESI-MS analysis, showing few deleterious effects. In this review, the mechanism of HILIC separation and ESI ion generation will be briefly discussed, followed by a summary of method development and applications in several fields of research including pharmaceutical, biomolecular, food, metabolic, and environmental analysis.

InnB, a Novel Type III Effector of Bradyrhizobium elkanii USDA61, Controls Symbiosis With Vigna Species.

Bradyrhizobium elkanii USDA61 is incompatible with mung bean (Vigna radiata cv. KPS1) and soybean (Glycine max cv. BARC2) and unable to nodulate either plant. This incompatibility is due to the presence of a functional type III secretion system (T3SS) that translocates effector protein into host cells. We previously identified five genes in B. elkanii that are responsible for its incompatibility with KPS1 plants. Among them, a novel gene designated as innB exhibited some characteristics associated with the T3SS and was found to be responsible for the restriction of nodulation on KPS1. In the present study, we further characterized innB by analysis of gene expression, protein secretion, and symbiotic phenotypes. The innB gene was found to encode a hypothetical protein that is highly conserved among T3SS-harboring rhizobia. Similar to other rhizobial T3SS-associated genes, the expression of innB was dependent on plant flavonoids and a transcriptional regulator TtsI. The InnB protein was secreted via the T3SS and was not essential for secretion of other nodulation outer proteins. In addition, T3SS-dependent translocation of InnB into nodule cells was confirmed by an adenylate cyclase assay. According to inoculation tests using several Vigna species, InnB promoted nodulation of at least one V. mungo cultivar. These results indicate that innB encodes a novel type III effector controlling symbiosis with Vigna species.

Identification of Bradyrhizobium elkanii Genes Involved in Incompatibility with Vigna radiata.

The establishment of a root nodule symbiosis between a leguminous plant and a rhizobium requires complex molecular interactions between the two partners. Compatible interactions lead to the formation of nitrogen-fixing nodules, however, some legumes exhibit incompatibility with specific rhizobial strains and restrict nodulation by the strains. Bradyrhizobium elkanii USDA61 is incompatible with mung bean (Vigna radiata cv. KPS1) and soybean cultivars carrying the Rj4 allele. Here, we explored genetic loci in USDA61 that determine incompatibility with V. radiata KPS1. We identified five novel B. elkanii genes that contribute to this incompatibility. Four of these genes also control incompatibility with soybean cultivars carrying the Rj4 allele, suggesting that a common mechanism underlies nodulation restriction in both legumes. The fifth gene encodes a hypothetical protein that contains a tts box in its promoter region. The tts box is conserved in genes encoding the type III secretion system (T3SS), which is known for its delivery of virulence effectors by pathogenic bacteria. These findings revealed both common and unique genes that are involved in the incompatibility of B. elkanii with mung bean and soybean. Of particular interest is the novel T3SS-related gene, which causes incompatibility specifically with mung bean cv. KPS1.

Peripartum neuroactive steroid and γ-aminobutyric acid profiles in women at-risk for postpartum depression.

Neuroactive steroids (NAS) are allosteric modulators of the γ-aminobutyric acid (GABA) system. NAS and GABA are implicated in depression. The peripartum period involves physiologic changes in NAS which may be associated with peripartum depression and anxiety. We measured peripartum plasma NAS and GABA in healthy comparison subjects (HCS) and those at-risk for postpartum depression (AR-PPD) due to current mild depressive or anxiety symptoms or a history of depression. We evaluated 56 peripartum medication-free subjects. We measured symptoms with the Hamilton Depression Rating Scale (HAM-D17), Hamilton Anxiety Rating Scale (HAM-A) and Spielberger State-Trait Anxiety Inventory-State (STAI-S). Plasma NAS and GABA were quantified by liquid chromatography-mass spectrometry. We examined the associations between longitudinal changes in NAS, GABA and depressive and anxiety symptoms using generalized estimating equation methods. Peripartum GABA concentration was 1.9±0.7ng/mL (p=0.004) lower and progesterone and pregnanolone were 15.8±7.5 (p=0.04) and 1.5±0.7ng/mL (p=0.03) higher in AR-PPD versus HCS, respectively. HAM-D17 was negatively associated with GABA (ß=-0.14±0.05, p=0.01) and positively associated with pregnanolone (ß=0.16±0.06, p=0.01). STAI-S was positively associated with pregnanolone (ß=0.11±0.04, p=0.004), allopregnanolone (ß=0.13±0.05, p=0.006) and pregnenolone (ß=0.02±0.01, p=0.04). HAM-A was negatively associated with GABA (ß=-0.12±0.04, p=0.004) and positively associated with pregnanolone (ß=0.11±0.05, p=0.05). Altered peripartum NAS and GABA profiles in AR-PPD women suggest that their interaction may play an important role in the pathophysiology of peripartum depression and anxiety.

The Oxidative Stress Network of Mycobacterium tuberculosis Reveals Coordination between Radical Detoxification Systems.

M. tuberculosis (Mtb) survives a hostile environment within the host that is shaped in part by oxidative stress. The mechanisms used by Mtb to resist these stresses remain ill-defined because the complex combination of oxidants generated by host immunity is difficult to accurately recapitulate in vitro. We performed a genome-wide genetic interaction screen to comprehensively delineate oxidative stress resistance pathways necessary for Mtb to resist oxidation during infection. Our analysis predicted functional relationships between the superoxide-detoxifying enzyme (SodA), an integral membrane protein (DoxX), and a predicted thiol-oxidoreductase (SseA). Consistent with that, SodA, DoxX, and SseA form a membrane-associated oxidoreductase complex (MRC) that physically links radical detoxification with cytosolic thiol homeostasis. Loss of any MRC component correlated with defective recycling of mycothiol and accumulation of cellular oxidative damage. This previously uncharacterized coordination between oxygen radical detoxification and thiol homeostasis is required to overcome the oxidative environment Mtb encounters in the host.

Differentiating isobaric steroid hormone metabolites using multi-stage tandem mass spectrometry.

Steroid hormones and their metabolites are currently undergoing clinical trials as potential therapeutics for traumatic brain injury (TBI). To support this work, it is necessary to develop improved procedures for differentiating isobaric species in this compound class. Equilin sulfate (E-S), estrone sulfate (E1-S), 17α-dihydroequilin sulfate (ADHE-S), and 17ß-dihydroequilin sulfate (BDHE-S) are primary constituents in hormone replacement therapies, such as Premarin, which are among pharmaceuticals being investigated for TBI treatment. The latter three compounds are isomers and can be difficult to differentiate in trace analytical determinations. In this work, a systematic study of the fragmentation of ADHE-S, BDHE-S, E1-S, and E-S under different stages of higher order tandem mass spectrometry (MS(n)) and variation of collision energy, allowed optimization of conditions for distinguishing the isomeric structures. For epimeric variants (e.g., ADHE-S versus BDHE-S; α- versus ß-stereoisomerization in the C-17 position), differentiation was achieved at MS(4) and fragmentation was demonstrated through MS(5). Computational analysis was performed to further explore differences in the fragmentation pathways due to changes in stereochemistry.

Investigation of the temperature stability of premarin intravenous using liquid chromatography-mass spectrometry.

Stability of Premarin(®)Intravenous was investigated in dry and reconstituted forms by monitoring major components in samples for a period of six months, using liquid chromatography-mass spectrometry. The components, largely comprising a series of estrogen and steroid hormone sulfates, were considered to be fairly stable (variation≤10%) for dry samples stored at room temperature and at 38°C (100°F) during the experimental time frame. However, significant variation, especially after 2 months of storage, was observed in reconstituted solutions. This variation was significantly larger for samples stored at elevated vs. room temperature. It was interesting to note that the concentration of equilenin sulfate increased over time, whereas that of other major components were seen to fluctuate and decrease. This phenomenon was partially explained by the conversion of equilin compounds into their corresponding equilenin forms, a phenomenon which was further investigated through a storage study with pure standard solutions and by tandem mass spectrometry.

Matrix-assisted laser desorption/ionization-mass spectrometry of cuticular lipid profiles can differentiate sex, age, and mating status of Anopheles gambiae mosquitoes.

Malaria is a devastating mosquito-borne disease, which affects hundreds of millions of people each year. It is transmitted predominantly by Anopheles gambiae, whose females must be >10 days old to become infective. In this study, cuticular lipids from a laboratory strain of this mosquito species were analyzed using a mass spectrometry method to evaluate their utility for age, sex and mating status differentiation. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), in conjunction with an acenaphthene/silver nitrate matrix preparation, was shown to be 100% effective in classifying A. gambiae females into 1, 7-10, and 14 days of age. MALDI-MS analysis, supported by multivariate statistical methods, was also effective in detecting cuticular lipid differences between the sexes and between virgin and mated females. The technique requires further testing, but the obtained results suggest that MALDI-MS cuticular lipid spectra could be used for age grading of A. gambiae females with precision greater than with other available methods.