Identification and manipulation of soil properties to improve the biological control performance of phenazine-producing Pseudomonas fluorescens

Pseudomonas fluorescens 2-79RN(10) protects wheat against take-all disease caused by Gaeumannomyces graminis var. tritici; however, the level of protection in the field varies from site to site. Identification of soil factors that exert the greatest influence on disease suppression is essential to improving biocontrol. In order to assess the relative importance of 28 soil properties on take-all suppression, seeds were treated with strain 2-79RN(10) (which produces phenazine-1-carboxylate [PCA(+)]) or a series of mutants with PCA(+) and PCA(-) phenotypes. Bacterized seeds were planted in 10 soils, representative of the wheat-growing region in the Pacific Northwest. Sixteen soil properties were correlated with disease suppression. Biocontrol activity of PCA(+) strains was positively correlated with ammonium-nitrogen, percent sand, soil pH, sodium (extractable and soluble), sulfate-sulfur, and zinc. In contrast, biocontrol was negatively correlated with cation-exchange capacity (CEC), exchangeable acidity, iron, manganese, percent clay, percent organic matter (OM), percent silt, total carbon, and total nitrogen. Principal component factor analysis of the 16 soil properties identified a three-component solution that accounted for 87 percent of the variance in disease rating (biocontrol). A model was identified with step-wise regression analysis (R(2) = 0.96; Cp statistic = 6.17) that included six key soil properties: ammonium-nitrogen, CEC, iron, percent silt, soil pH, and zinc. As predicted by our regression model, the biocontrol activity of 2-79RN(10) was improved by amending a soil low in Zn with 50 micro g of zinc-EDTA/g of soil. We then investigated the negative correlation of OM with disease suppression and found that addition of OM (as wheat straw) at rates typical of high-OM soils significantly reduced biocontrol activity of 2-79RN(10).     

Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA⁺ (toluene o-monooxygenase) genes from Burkholderia cepacia PR1₂₃(TOM_23C). A transposon integration vector was used to insert tomA⁺ into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf. B. cepacia G4 PR1₂₃(TOM_23C), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h⁻¹) and colonized wheat (3 × 10⁶ CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h⁻¹; level of colonization, 4 × 10⁶ CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil.     

Changes in Populations of Rhizosphere Bacteria Associated with Take-All Disease of Wheat

Take-all, caused by Gaeumannomyces graminis var. tritici, is one of the most important fungal diseases of wheat worldwide. Knowing that microbe-based suppression of the disease occurs in monoculture wheat fields following severe outbreaks of take-all, we analyzed the changes in rhizosphere bacterial communities following infection by the take-all pathogen. Several bacterial populations were more abundant on diseased plants than on healthy plants, as indicated by higher counts on a Pseudomonas-selective medium and a higher fluorescence signal in terminal restriction fragment length polymorphism analyses of amplified 16S ribosomal DNA (rDNA). Amplified rDNA restriction analysis (ARDRA) of the most abundant cultured populations showed a shift in dominance from Pseudomonas to Chryseobacterium species in the rhizosphere of diseased plants. Fluorescence-tagged ARDRA of uncultured rhizosphere washes revealed an increase in ribotypes corresponding to several bacterial genera, including those subsequently identified by partial 16S sequencing as belonging to species of alpha-, beta-, and gamma-proteobacteria, sphingobacteria, and flavobacteria. The functional significance of some of these populations was investigated in vitro. Of those isolated, only a small subset of the most abundant Pseudomonas spp. and a phlD⁺ Pseudomonas sp. showed any significant ability to inhibit G.graminis var. tritici directly. When cultured strains were mixed with the inhibitory phlD⁺ Pseudomonas strain, the Chryseobacterium isolates showed the least capacity to inhibit this antagonist of the pathogen, indicating that increases in Chryseobacterium populations may facilitate the suppression of take-all by 2,4-diacetylphloroglucinol-producing phlD⁺ pseudomonads.     

Environmental Factors Modulating Antibiotic and Siderophore Biosynthesis by Pseudomonas fluorescens Biocontrol Strains

Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of Pseudomonas fluorescens is an essential step toward improving the level and reliability of their biocontrol activity. We used liquid culture assays to identify several minerals and carbon sources which had a differential influence on the production of the antibiotics 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), and pyrrolnitrin and the siderophores salicylic acid and pyochelin by the model strain CHA0, which was isolated from a natural disease-suppressive soil in Switzerland. Production of PHL was stimulated by Zn²⁺, NH₄Mo²⁺, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated by the same factors as PHL. Production of PLT was stimulated by Zn²⁺, Co²⁺, and glycerol but was repressed by glucose. Pyrrolnitrin production was increased by fructose, mannitol, and a mixture of Zn²⁺ and NH₄Mo²⁺. Pyochelin production was increased by Co²⁺, fructose, mannitol, and glucose. Interestingly, production of its precursor salicylic acid was increased by different factors, i.e., NH₄Mo²⁺, glycerol, and glucose. The mixture of Zn²⁺ and NH₄Mo²⁺ with fructose, mannitol, or glycerol further enhanced the production of PHL and PLT compared with either the minerals or the carbon sources used alone, but it did not improve siderophore production. Extending fermentation time from 2 to 5 days increased the accumulation of PLT, pyrrolnitrin, and pyochelin but not of PHL. When findings with CHA0 were extended to an ecologically and genetically diverse collection of 41 P. fluorescens biocontrol strains, the effect of certain factors was strain dependent, while others had a general effect. Stimulation of PHL by Zn²⁺ and glucose was strain dependent, whereas PLT production by all strains that can produce this compound was stimulated by Zn²⁺ and transiently repressed by glucose. Inorganic phosphate reduced PHL production by CHA0 and seven other strains tested but to various degrees. Production of PLT but not pyrrolnitrin by CHA0 was also reduced by 100 mM phosphate. The use of 1/10-strength nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract, amended with glucose and/or glycerol resulted in dramatically increased accumulations of PHL (but not PLT), pyochelin, and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. The results of this study (i) provide insight into the biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for intensive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial inoculants.     

Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR

A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD⁺) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD⁺ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C_Ts) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD⁺ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD⁺ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r² = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.     

Low-Concentration Kinetics of Atmospheric CH4 Oxidation in Soil and Mechanism of NH4+ Inhibition

NH₄⁺ inhibition kinetics for CH₄ oxidation were examined at near-atmospheric CH₄ concentrations in three upland forest soils. Whether NH₄⁺-independent salt effects could be neutralized by adding nonammoniacal salts to control samples in lieu of deionized water was also investigated. Because the levels of exchangeable endogenous NH₄⁺ were very low in the three soils, desorption of endogenous NH₄⁺ was not a significant factor in this study. The K_m(app) values for water-treated controls were 9.8, 22, and 57 nM for temperate pine, temperate hardwood, and birch taiga soils, respectively. At CH₄ concentrations of ≤15 μl liter⁻¹, oxidation followed first-order kinetics in the fine-textured taiga soil, whereas the coarse-textured temperate soils exhibited Michaelis-Menten kinetics. Compared to water controls, the K_m(app) values in the temperate soils increased in the presence of NH₄⁺ salts, whereas the V_max(app) values decreased substantially, indicating that there was a mixture of competitive and noncompetitive inhibition mechanisms for whole NH₄⁺ salts. Compared to the corresponding K⁺ salt controls, the K_m(app) values for NH₄⁺ salts increased substantially, whereas the V_max(app) values remained virtually unchanged, indicating that NH₄⁺ acted by competitive inhibition. Nonammoniacal salts caused inhibition to increase with increasing CH₄ concentrations in all three soils. In the birch taiga soil, this trend occurred with both NH₄⁺ and K⁺ salts, and the slope of the increase was not affected by the addition of NH₄⁺. Hence, the increase in inhibition resulted from an NH₄⁺-independent mechanism. These results show that NH₄⁺ inhibition of atmospheric CH₄ oxidation resulted from enzymatic substrate competition and that additional inhibition that was not competitive resulted from a general salt effect that was independent of NH₄⁺.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.     

The external K+ concentration and mutations in the outer pore mouth affect the inhibition of kv1.5 current by Ni2+

By examining the consequences both of changes of [K⁺]_o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni²⁺ is similar to that for proton block. Ni²⁺ block is inhibited by increasing [K⁺]_o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni²⁺ had no effect on gating currents. We propose that, as with H_o⁺, Ni²⁺ binding to H463 facilitates an outer pore inactivation process that is antagonized by K_o⁺ and that requires R487. However, whereas H_o⁺ substantially accelerates inactivation of residual currents, Ni²⁺ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co²⁺ and Mn²⁺, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0 mM K_o⁺ is Zn²⁺ (K_D ~ 0.07 mM) ≥ Ni²⁺ (K_D ~ 0.15 mM) > Co²⁺ (K_D ~ 1.4 mM) > Mn²⁺ (K_D > 10 mM).     

Exploiting Genotypic Diversity of 2,4-Diacetylphloroglucinol-Producing Pseudomonas spp.: Characterization of Superior Root-Colonizing P. fluorescens Strain Q8r1-96

The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by P. fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed⁻¹ or soil⁻¹) to establish high rhizosphere population densities (10⁷ CFU g of root⁻¹) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P. fluorescens strains. Q8r1-96 maintained a rhizosphere population density of approximately 10⁵ CFU g of root⁻¹ after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.     

Rare Earth Element Transfer from Soil to Navel Orange Pulp (Citrus sinensis Osbeck cv. Newhall) and the Effects on Internal Fruit Quality

The effects of soil rare earth element (REE) on navel orange quality and safety in rare earth ore areas have gained great attention. This study investigated the transfer characteristics of REE from soil to navel orange pulp (Citrus sinensis Osbeck cv. Newhall) and examined the effects of soil REE on internal fruit quality in Xinfeng County, Jiangxi province, China. Path analysis showed that soil REE, pH, cation exchange capacity (CEC), and Fe oxide (Fe_ox) significantly affected pulp REE concentrations. A Freundlich-type prediction model for pulp REE was established: log[REE_pulp] = -1.036 + 0.272 log[REE_soil] - 0.056 pH - 0.360 log[CEC] + 0.370 log[Fe_ox] (n = 114, R² = 0.60). From the prediction model, it was inferred that even when soil REE and Fe_ox were as high as 1038 mg kg^-1 and 96.4 g kg^-1, respectively, and pH and CEC were as low as 3.75 and 5.08 cmol kg^-1, respectively, pulp REE concentrations were much lower than the food limit standard. Additionally, soil REE levels were significantly correlated with selected fruit quality indicators, including titratable acidity (r = 0.52, P < 0.01), total soluble solids (r = 0.48, P < 0.01) and vitamin C (r = 0.56, P < 0.01). Generally, under routine methods of water and fertilization management, the cultivation of navel oranges in rare earth ore areas of south China with soil REE ranging from 38.6 to 546 mg kg^-1 had improved in internal fruit quality.     

Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause characterized by expansion of autoreactive lymphocytes. Regulatory T cells (T_regs) are a component of the normal immune system and contribute to the maintenance of peripheral tolerance. T_reg abnormalities have been associated with several autoimmune diseases and there is interest in the role of T_regs in SLE. We previously demonstrated that transfer of expanded CD4⁺CD25⁺CD62L^HI T_regs slows the development of lupus in (NZBxNZW)F₁ (B/W) mice. However in the absence of T_reg specific surface antigens, cell purification remains a compromise between the breadth and purity of the population isolated. Importantly, purified populations always contain Foxp3⁻ effector T cells (T_effs) that theoretically could exacerbate autoimmunity in the recipient. Here we explore the impact of transferring the more comprehensive, but less pure T_reg subset defined by CD4⁺CD25⁺ expression on development of murine lupus. All cells were FACS sorted and expanded prior to adoptive transfer. Development of proteinuria and survival were measured. We found that exogenous expansion of CD4⁺CD25⁺ cells produced a population containing 70–85% CD4⁺Foxp3⁺T_regs. Expanded T_regs had higher CTLA-4 and Foxp3 expression, increased in vitro suppression capacity, and prolonged in vivo survival as compared to freshly isolated cells. Adoptive transfer of expanded CD4⁺CD25⁺ T_regs inhibited the onset of glomerulonephritis and prolonged survival in mice. Importantly the population of T_eff contained within the adoptively transferred cells had reduced survival and proliferation capacity as compared to either co-transferred T_regs or transferred T_effs expanded in the absence of T_regs. These studies demonstrate that adoptive transfer of expanded CD4⁺CD25⁺Foxp3⁺T_regs has the capacity to inhibit the onset of murine lupus and that this capacity is significant despite transfer of co-cultured T_eff cells. These data indicate that when co-expanded with regulatory T cells, exogenously activated T_effs from autoimmune patients may not pose a significant risk of promoting disease.     

Genome-Wide Association Study to Identify the Genetic Determinants of Otitis Media Susceptibility in Childhood

Background

Otitis media (OM) is a common childhood disease characterised by middle ear inflammation and effusion. Susceptibility to recurrent acute OM (rAOM; ≥3 episodes of AOM in 6 months) and chronic OM with effusion (COME; MEE ≥3 months) is 40–70% heritable. Few underlying genes have been identified to date, and no genome-wide association study (GWAS) of OM has been reported.

Methods and Findings

Data for 2,524,817 single nucleotide polymorphisms (SNPs; 535,544 quality-controlled SNPs genotyped by Illumina 660W-Quad; 1,989,273 by imputation) were analysed for association with OM in 416 cases and 1,075 controls from the Western Australian Pregnancy Cohort (Raine) Study. Logistic regression analyses under an additive model undertaken in GenABEL/ProbABEL adjusting for population substructure using principal components identified SNPs at CAPN14 (rs6755194: OR = 1.90; 95%CI 1.47–2.45; P_adj-PCA = 8.3×10⁻⁷) on chromosome 2p23.1 as the top hit, with independent effects (rs1862981: OR = 1.60; 95%CI 1.29–1.99; P_adj-PCA = 2.2×10⁻⁵) observed at the adjacent GALNT14 gene. In a gene-based analysis in VEGAS, BPIFA3 (P_Gene = 2×10⁻⁵) and BPIFA1 (P_Gene = 1.07×10⁻⁴) in the BPIFA gene cluster on chromosome 20q11.21 were the top hits. In all, 32 genomic regions show evidence of association (P_adj-PCA<10⁻⁵) in this GWAS, with pathway analysis showing a connection between top candidates and the TGFβ pathway. However, top and tag-SNP analysis for seven selected candidate genes in this pathway did not replicate in 645 families (793 affected individuals) from the Western Australian Family Study of Otitis Media (WAFSOM). Lack of replication may be explained by sample size, difference in OM disease severity between primary and replication cohorts or due to type I error in the primary GWAS.

Conclusions

This first discovery GWAS for an OM phenotype has identified CAPN14 and GALNT14 on chromosome 2p23.1 and the BPIFA gene cluster on chromosome 20q11.21 as novel candidate genes which warrant further analysis in cohorts matched more precisely for clinical phenotypes.     

Purification and Characterization of Monovalent Cation-Activated Levodione Reductase from Corynebacterium aquaticum M-13

(6R)-2,2,6-Trimethyl-1,4-cyclohexanedione (levodione) reductase was isolated from a cell extract of the soil isolate Corynebacterium aquaticum M-13. This enzyme catalyzed regio- and stereoselective reduction of levodione to (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (actinol). The relative molecular mass of the enzyme was estimated to be 142,000 Da by high-performance gel permeation chromatography and 36,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required NAD⁺ or NADH as a cofactor, and it catalyzed reversible oxidoreduction between actinol and levodione. The enzyme was highly activated by monovalent cations, such as K⁺, Na⁺, and NH₄⁺. The NH₂-terminal and partial amino acid sequences of the enzyme showed that it belongs to the short-chain alcohol dehydrogenase/reductase family. This is the first report of levodione reductase.     

Asymptomatic human CD4+ cytotoxic T-cell epitopes identified from herpes simplex virus glycoprotein B

The identification of “asymptomatic” (i.e., protective) epitopes recognized by T cells from herpes simplex virus (HSV)-seropositive healthy individuals is a prerequisite for an effective vaccine. Using the PepScan epitope mapping strategy, a library of 179 potential peptide epitopes (15-mers overlapping by 10 amino acids) was identified from HSV type 1 (HSV-1) glycoprotein B (gB), an antigen that induces protective immunity in both animal models and humans. Eighteen groups (G1 to G18) of 10 adjacent peptides each were first screened for T-cell antigenicity in 38 HSV-1-seropositive but HSV-2-seronegative individuals. Individual peptides within the two immunodominant groups (i.e., G4 and G14) were further screened with T cells from HLA-DR-genotyped and clinically defined symptomatic (n = 10) and asymptomatic (n = 10) HSV-1-seropositive healthy individuals. Peptides gB_161-175 and gB_166-180 within G4 and gB_661-675 within G14 recalled the strongest HLA-DR-dependent CD4⁺ T-cell proliferation and gamma interferon production. gB_166-180, gB_661-675, and gB_666-680 elicited ex vivo CD4⁺ cytotoxic T cells (CTLs) that lysed autologous HSV-1- and vaccinia virus (expressing gB)-infected lymphoblastoid cell lines. Interestingly, gB_166-180 and gB_666-680 peptide epitopes were strongly recognized by CD4⁺ T cells from 10 of 10 asymptomatic patients but not by CD4⁺ T cells from 10 of 10 symptomatic patients (P < 0.0001; analysis of variance posttest). Inversely, CD4⁺ T cells from symptomatic patients preferentially recognized gB_661-675 (P < 0.0001). Thus, we identified three previously unrecognized CD4⁺ CTL peptide epitopes in HSV-1 gB. Among these, gB_166-180 and gB_666-680 appear to be “asymptomatic” peptide epitopes and therefore should be considered in the design of future herpes vaccines.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

Platinum cross-linking of adenines and guanines on the quadruplex structures of the AG3(T2AG3)3 and (T2AG3)4 human telomere sequences in Na+ and K+ solutions

The quadruplex structures of the human telomere sequences AG₃(T₂AG₃)₃ I and (T₂AG₃)₄ II were investigated in the presence of Na⁺ and K⁺ ions, through the cross-linking of adenines and guanines by the cis- and trans-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ complexes 1 and 2. The bases involved in chelation of the cis- and trans-Pt(NH₃)₂ moieties were identified by chemical and 3′-exonuclease digestions of the products isolated after denaturing gel electrophoresis. These are the four adenines of each sequence and four out of the 12 guanines. Two largely different structures have been reported for I: A from NMR data in Na⁺ solution and B from X-ray data of a K⁺-containing crystal. Structure A alone agrees with our conclusions about the formation of the A1–G10, A13–G22, A1–A13 platinum chelates at the top of the quadruplex and A7–A19, G4–A19 and A7–G20 at the bottom, whether the Na⁺ or K⁺ ion is present. At variance with a recent proposal that structures A and B could be the major species in Na⁺ and K⁺ solutions, respectively, our results suggest that structure A exists predominantly in the presence of both ions. They also suggest that covalent platinum cross-linking of a human telomere sequence could be used to inhibit telomerase.     

Characterization of Cadmium Uptake in Lactobacillus plantarum and Isolation of Cadmium and Manganese Uptake Mutants

Two different Cd²⁺ uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn²⁺ uptake system which also takes up Cd²⁺ and is induced by Mn²⁺ starvation. The calculated K_m and V_max are 0.26 μM and 3.6 μmol g of dry cell⁻¹ min⁻¹, respectively. Unlike Mn²⁺ uptake, which is facilitated by citrate and related tricarboxylic acids, Cd²⁺ uptake is weakly inhibited by citrate. Cd²⁺ and Mn²⁺ are competitive inhibitors of each other, and the affinity of the system for Cd²⁺ is higher than that for Mn²⁺. The other Cd²⁺ uptake system is expressed in Mn²⁺-sufficient cells, and no K_m can be calculated for it because uptake is nonsaturable. Mn²⁺ does not compete for transport through this system, nor does any other tested cation, i.e., Zn²⁺, Cu²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺, or Ni²⁺. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn²⁺-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn²⁺ for growth as the parental strain. Mn²⁺ starvation-induced Cd²⁺ uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn²⁺ or Cd²⁺ accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn²⁺ and Cd²⁺ uptake system.     

Regulation of the Fast Vacuolar Channel by Cytosolic and Vacuolar Potassium

At resting cytosolic Ca²⁺, passive K⁺ conductance of a higher plant tonoplast is likely dominated by fast vacuolar (FV) channels. This patch-clamp study describes K⁺-sensing behavior of FV channels in Beta vulgaris taproot vacuoles. Variation of K⁺ between 10 and 400 mM had little effect on the FV channel conductance, but a pronounced one on the open probability. Shift of the voltage dependence by cytosolic K⁺ could be explained by screening of the negative surface charge with a density σ = 0.25 e⁻/nm². Vacuolar K⁺ had a specific effect on the FV channel gating at negative potentials without significant effect on closed-open transitions at positive ones. Due to K⁺ effects at either membrane side, the potential at which the FV channel has minimal activity was always situated at ~50 mV below the potassium equilibrium potential, E_K⁺. At tonoplast potentials below or equal to E_K⁺, the FV channel open probability was almost independent on the cytosolic K⁺ but varied in a proportion to the vacuolar K⁺. Therefore, the release of K⁺ from the vacuole via FV channels could be controlled by the vacuolar K⁺ in a feedback manner; the more K⁺ is lost the lower will be the transport rate.     

Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil

Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 × 10⁵ cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH₄)₂SO₄ application. AOB populations in amended treatments increased from an initial density of approximately 4 × 10⁶ cells/g of dry soil to peak values (day 7) of 35 × 10⁶ and 66 × 10⁶ cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 × 10⁹ and 2.2 × 10⁹ cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 × 10⁶ to 38.0 × 10⁶ cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH₄⁺ h⁻¹ cell⁻¹ and decreased with time in both microcosms and the field. Growth yields were 5.6 × 10⁶, 17.5 × 10⁶, and 1.7 × 10⁶ cells/mol of NH₄⁺ in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.     

Effects of Hypoxia on 13NH4+ Fluxes in Rice Roots : Kinetics and Compartmental Analysis

Techniques of compartmental (efflux) and kinetic influx analyses with the radiotracer ¹³NH₄⁺ were used to examine the adaptation to hypoxia (15, 35, and 50% O₂ saturation) of root N uptake and metabolism in 3-week-old hydroponically grown rice (Oryza sativa L., cv IR72) seedlings. A time-dependence study of NH₄⁺ influx into rice roots after onset of hypoxia (15% O₂) revealed an initial increase in the first 1 to 2.5 h after treatment imposition, followed by a decline to less than 50% of influx in control plants by 4 d. Efflux analyses conducted 0, 1, 3, and 5 d after the treatment confirmed this adaptation pattern of NH₄⁺ uptake. Half-lives for NH₄⁺ exchange with subcellular compartments, cytoplasmic NH₄⁺ concentrations, and efflux (as percentage of influx) were unaffected by hypoxia. However, significant differences were observed in the relative amounts of N allocated to NH₄⁺ assimilation and the vacuole versus translocation to the shoot. Kinetic experiments conducted at 100, 50, 35, and 15% O₂ saturation showed no significant change in the K_m value for NH₄⁺ uptake with varying O₂ supply. However, V_max was 42% higher than controls at 50% O₂ saturation, unchanged at 35%, and 10% lower than controls at 15% O₂. The significance of these flux adaptations is discussed.