Specific Incorporation of Precursors into DNA by Feeding Labeled Bacteria to Paramecium aurelia

SYNOPSIS. Studies were carried out on the introduction of labeled precursors into the DNA of Paramecium aurelia (syngen 4, stock 51) by way of the bacteria that are used for food. A thymine-requiring strain of Escherichia coli (15 T⁻) was labeled by growth in either H³-methyl thymidine or 2-C¹⁴ bromouracil, washed free of the exogenous label, and fed to the paramecia. The tritium label from the bacteria was incorporated almost exclusively into the DNA of the paramecia, whereas it was much less specifically incorporated when introduced directly from the medium. The Cu label from bromouracil was also incorporated mainly into the DNA of the paramecia although a small amount appeared in RNA. The formation of labeled food vacuoles was followed. Food vacuoles were formed at a nearly constant rate, with the total number of vacuoles increasing throughout the cycle. The lifetime of the vacuoles was about 2.5 hours. Incorporation of the label into the DXA of the paramecia begins within a few minutes of the formation of the first labeled vacuole. DNA synthesis begins about 1.5 hr after the previous fission (total cell cycle about 5.8 hr) and progresses at a nearly constant rate throughout the remainder of the cycle.     

Catalase is the bacteria-derived detoxifying substance against paramecia-killing toxin in wheat grass powder infusion

Paramecium cells are usually cultured in a wheat grass powder infusion inoculated with Klebsiella pneumoniae. However, non-bacterized wheat grass powder infusion is toxic to paramecia, and bacteria-derived substance detoxifies the toxic substance. Here, the detoxifying substance from K. pneumoniae, which was found to be proteinaceous, was purified to homogeneity. The protein had an apparent molecular mass of about 200 kDa by gel filtration and 92 kDa by SDS-polyacrylamide gel electrophoresis. Although the amino acid sequence of the amino terminal region did not show a high sequence homology with any reported proteins, amino acid sequences of internal regions of the protein were nearly identical to catalase HPII from Escherichia coli. When the wheat grass powder infusion was treated at 25 degrees C for 1 h with commercially available catalase from bovine liver, the toxicity of the infusion against paramecia was completely abolished. The initial concentration of hydrogen peroxide in the wheat grass powder infusion was about 30 microM and was completely decomposed by the catalase treatment. Therefore, the toxic substance in the wheat grass powder infusion and the detoxifying substance from K. pneumoniae are considered as hydrogen peroxide and catalase, respectively.     

Competitive advantages of Caedibacter-infected Paramecia

Intracellular bacteria of the genus Caedibacter limit the reproduction of their host, the freshwater ciliate Paramecium. Reproduction rates of infected strains of paramecia were significantly lower than those of genetically identical strains that had lost their parasites after treatment with an antibiotic. Interference competition occurs when infected paramecia release a toxic form of the parasitic bacterium that kills uninfected paramecia. In mixed cultures of infected and uninfected strains of either P tetraurelia or of P novaurelia, the infected strains outcompeted the uninfected strains. Infection of new host paramecia seems to be rare. Infection of new hosts was not observed in either mixtures of infected with uninfected strains, or after incubation of paramecia with isolated parasites. The competitive advantages of the host paramecia, in combination with their vegetative reproduction, makes infection of new hosts by the bacterial parasites unnecessary, and could be responsible for the continued existence of "killer paramecia" in nature. Caedibacter parasites are not a defensive adaptation. Feeding rates and reproduction of the predators Didinium nasutum (Ciliophora) and Amoeba proteus (Amoebozoa, Gymnamoebia) were not influenced by whether or not their paramecia prey were infected. Infection of the predators frequently occurred when they preyed on infected paramecia. Caedibacter-infected predators may influence competition between Paramecium strains by release of toxic parasites into the environment that are harmful to uninfected strains.     

Sensitization to heat by X-rays

1. Paramecium caudatum is sensitized to heat by sublethal dosages of x-rays. Thus if paramecia are irradiated, then exposed to a sublethal dosage of heat they are killed, but if the same heat exposure precedes the same dosage of radiations, they are not. 2. Sensitivity to both heat and x-rays is much greater in paramecia from the log growth phase than in those from the stationary phase of a culture. 3. Recovery from heat sensitization in animals from the stationary phase of a culture is slow, requiring several days. 4. Division is readily retarded and even temporarily inhibited by sublethal dosage of x-rays. Recovery of the division rate is fairly slow requiring several days. 5. Paramecia can be killed by a dosage of 1,200,000 r (of which about one-half reach the animal) units of x-radiation alone. Smaller dosages are not lethal if the paramecia are transferred to fresh medium immediately upon completion of irradiation. 6. The possibility of utilization of heat sensitization in treatment of malignant growths is discussed.     

Analysis of the prestarvation response in growing cells of Dictyostelium discoideum

We have previously shown that growing cells of Dictyostelium discoideum (strains NC4 and AX3) produce a soluble substance that accumulates in the medium in proportion to cell density; this substance regulates the production of certain proteins previously thought to be induced by starvation [Clarke et al., 1987]. We suggest the name PSF (prestarvation factor) for this substance. During growth, Dictyostelium cells monitor the relative concentrations of PSF and food bacteria. When PSF reaches a sufficiently high level relative to the concentration of bacteria, synthesis of PSF-regulated proteins is induced. We propose the name prestarvation response for this induction, which takes place in exponentially growing cells several generations before the food bacteria are depleted. We have explored the mechanism by which the food bacteria inhibit the response of Dictyostelium cells to PSF. We find that the bacteria do not inactivate PSF or inhibit its production; instead, they affect the ability of NC4 cells to detect PSF, possibly by binding to the same cell surface receptor. In the absence of bacteria, as during axenic growth of AX3 cells, the prestarvation response occurs at much lower cell densities, probably accounting for the presence of certain developmentally regulated mRNAs and proteins in axenic cultures.     

Growth and disinfestation of 6 different bacteria in embryogenic suspension cultures of cotton

Summary Growth of 6 different common laboratory bacteria (Escherichia coli, Flavobacterium balustrum, Xanthomonas maltophilia, Enterobacter cloacae, Pseudomonas fluorescens, and Agrobacterium tumefaciens) in a bacterial medium, fresh plant medium, and spent plant media was initially measured. In all cases, bacteria grew best in the bacterial medium followed by the fresh plant medium. The spent plant medium did not support growth of the bacteria and apparently was actively toxic to bacterial cells. Proliferating, embryogenic suspension cultures of cotton (Gossypium hirsutum) were then inoculated with these 6 different bacteria. Two to three d following bacterial inoculation, embryogenic tissues were placed in various concentrations of bleach for various amounts of time, rinsed with sterile water, and placed on a bacterial culture medium. Clumps of embryogenic tissue which showed no visible bacterial growth after 3 d of culture were then transferred to an agar-solidified plant tissue culture medium to determine viability of bleachdisinfested tissues. Viable, single pieces of the disinfested embryogenic tissue were then used to reinitiate embryogenic suspension cultures. Treatment of contaminated tissue with a 1% bleach solution for 1–5 min resulted in the highest recovery of viable, disinfested tissues using 5 of the 6 bacteria. It was not possible to remove F. balustrum from clumps of embryogenic tissue without also killing the plant tissue.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid Salaries and research support were provided by an Ohio Academy of Sciences/National Science Foundation summer internship award to JAS and by State and Federal funds appropriated to OSU-OARDC. OARDC Journal Article No. 391-89     

Tracing the role of R-bodies in the killer trait: Absence of toxicity of R-body producing recombinant E. coli on paramecia

R-bodies are coiled proteinaceous ribbons produced by Paramecium endosymbionts belonging to the genus Caedibacter. These intracellular bacteria confer upon their hosts a phenomenon called the killer trait. It is the ability to kill symbiont-free competitors called sensitives. The R-body is the crucial element of this process, but despite many efforts, the actual role of R-bodies in killing sensitive paramecia is still not satisfactory clarified. The open question is whether the R-body acts as transmitter for a yet unidentified toxin or whether it directly kills sensitive paramecia having intrinsic cytotoxic effects. In the present study, this problem is addressed by heterologous expression of Caedibacter taeniospiralis R-body in Escherichia coli followed by a detailed analysis of its potential intrinsic toxic effect on feeding sensitive Paramecium tetraurelia. Using this approach, we can exclude any eventual effects of additional, unidentified factors produced by C. taeniospiralis and thus observe the impact of the recombinant R-body itself. No cytotoxic effects of recombinant R-bodies were detected following this approach, strengthening the hypothesis that R-bodies act as releasing system for an unidentified C. taeniospiralis toxin.     

The Toxic Symbiont Caedibacter caryophila in the Cytoplasm of Paramecium novaurelia

Endosymbiotic bacteria were observed to inhabit the cytoplasm of the freshwater ciliateParamecium novaurelia. Transmission electron microscopy and toxicity tests with sensitive paramecia showed that the endosymbionts belong to the genusCaedibacter. The bacteria conferred a killer trait to their host paramecia. The production of a proteinaceous inclusion body (“R-body”) in the bacterial cell makes them toxic to other paramecia after they become enclosed in food vacuoles. R-bodies ofCaedibacter sp were associated with phages, which are known in most otherCaedibacter species to code for the R-body proteins. The killer-effect ofP. novaurelia on sensitiveP. caudatum strains was of the “paralysis” type, which is a characteristic of the symbiont speciesCaedibacter caryophila. Until nowC. caryophila was known to inhabit the macronucleus ofParamecium caudatum only. Sequencing of the 16S rRNA-gene proved thatCaedibacter sp from the cytoplasm ofP. novaurelia belongs to the speciesC. caryophila as well. The rDNA-sequence of 1695 bp length differed in a total of only 1 bp from the corresponding gene inC. caryophila from the macronucleus ofP. caudatum. The results indicate that the infection of specific host cell compartments may depend on host genes, but not on different traits of the infecting symbiont species. The occurrence of killer and sensitive paramecia strains together in one pond is discussed with respect to the competitive advantage of the killer trait.     

Effect of phosphate-solubilizing bacteria and vesicular-arbuscular mycorrhizal fungi on the utilization of Bayovar rock phosphate by alfalfa plants using a sand-vermiculite medium

Phosphate-solubilizing bacteria (PSB) exhibited a high efficiency to improve plant growth and nutrition in the presence of Bayovar rock phosphate when sand-vermiculive was used as a culture medium. Treatments with dual inoculum (PSB plus mycorrhiza) significantly (P≤0.05) increased alfalfa growth. Bacteria-microbial fungi interactions resulted in a greater utilization of the rock phosphate added to the rooting medium. Although Bayovar rock phosphateper se can be considered an inert substrate because it did not stimulate plant growth, metabolites released by PSB were able to transform the rock into available forms which could be utilized by alfalfa plants.Glomus fasciculatum was the most efficient mycorrhizal endophyte under the experimental conditions employed.     

Growth limitation in hybridoma cell cultures: The role of inhibitory or toxic metabolites

Hybridoma cells usually grow to fairly low cell densities in batch cultures (1–3×10⁶ cells/ml). The reason for this is either that essential nutritional components of the medium are consumed, or that the cells produce some kind of inhibitory or toxic metabolite. This investigation presents evidence for the latter. Spent medium from cultures of hybridoma cells did not support growth of cells at lower cell densities (1–3×10⁵ cells/ml). The ability to support cell growth could not be restored by adding additional serum, energy sources (glucose, pyruvate) or L-glutamine. Furthermore, the consumption of amino acids could not account for this growth inhibition. On the contrary, the spent medium contained a substance that inhibited cell growth. This substance or metabolite was found in a fraction eluted from a gel filtration column when spent medium was applied to the column. This substance was found in the spent medium from all hybridoma and myeloma cell lines that were tested. The molecular weight of the substance was about 5 kD. Spent medium from two hybridoma cell lines also contained a substance that was eluted in the same fraction as albumin (67 kD). It is likely that this (or these) substance(s) is responsible for the growth limitation in hybridoma cell cultures.Abbreviations PBS phosphate buffered saline     

Rhizoremediation of lindane by root‐colonizing Sphingomonas

We used a two‐step enrichment approach to isolate root‐colonizing hexachlorocyclohexane (HCH)‐degrading microorganisms. The first step consists of the use of classical liquid enrichment to isolate γ‐HCH degraders. The γ‐HCH‐degrading microbes were attached in mass to corn seeds sown in soil with γ‐HCH, and after plant development we rescued bacteria growing on root tips. Bacteria were then subjected to a second enrichment round in which growth on liquid medium with γ‐HCH and inoculation of corn seeds were repeated. We then isolated bacteria on M9 minimal medium with γ‐HCH from root tips. We were able to isolate four Sphingomonas strains, all of which degraded α‐, β‐, γ‐ and δ‐HCH. Two of the strains were particularly good colonizers of corn roots, reaching high cell density in vegetated soil and partly removing γ‐HCH. In contrast, these bacteria performed poorly in unplanted soils. This study supports the hypothesis that the removal of persistent toxic chemicals can be accelerated by combinations of plants and bacteria, a process generally known as rhizoremediation.     

Toxic effects of toluene on the growth of activated sludge bacteria

Two bacteria were isolated from the activated sludge sample of a wastewater treatment plant in Dublin by enrichment culture technique with toluene as the sole source of carbon and energy. They were identified as Aeromonas caviae (To-4) and Pseudomonas putida (To-5). The growth of these bacteria depended on the manner in which toluene was supplied. In general, growth was better when toluene was supplied in the vapour phase. When toluene was added directly to the growth medium it was found to be toxic to the organisms but the toxic effect could be alleviated in the presence of other carbon sources and by the acclimation of the cells. The growth of To-4 on toluene has never been previously reported.     

The utilization of paramecia by the carnivorous plant Utricularia gibba

Summary A method has been devised to ensure capture of large numbers of live paramecia within a short period of time under controlled conditions by the bladders of Utricularia gibba. The method permits a direct evaluation of the role of entrapped animals in the nutrition of this carnivorous plant. Paramecia captured by the bladders of plants growing in a near optimal inorganic medium do not cause an increase in number or length of internodes. In contrast, feeding paramecia to plants grown in a poorly balanced or incomplete medium does result in an increase in both number and length of internodes produced. Feeding paramecia to Utricularia also results in an increase in number of bladders.This study was supported in part by an Undergraduate Research Participant stipend from Public Health Service, grant number 2-TIHE 5 303-09, to the first author and in part by Agricultural Research Service, U.S. Department of Agriculture, Grant No. 12-14-100-7981 (34) to the second author, administered by Crops Research Division, Beltsville, Maryland.     

阿根廷淡水大米草沼泽中野生豚鼠的种群统计和微生境的利用

本文研究了秋冬季节淡水大米草(Spartina densiflora)沼泽中野生豚鼠(Cavia a perea)的丰度、繁殖和微生境利用,以及其对当地植被和棉鼠类(Sigmodontine)啮齿动物的影响。野生豚鼠喜好S.densiflora覆盖度高的生境。繁殖个体(成体)主要利用矮草为主的斑块,幼体则主要利用禾本科植物为主的斑块。结果说明,野生豚鼠微生境的利用受捕食风险和食物种类的影响。在淡水沼泽中,野生豚鼠的丰度、繁殖、体重和微生境利用没有季节性变化,它们对植物的取食和活动跑道的建造对植被结构和同域的啮齿类动物没有负面影响。通过对具有中度季节性变化的淡水生境中的豚鼠种群和具有高度季节变化的草地和路边中的豚鼠种群进行比较,表明野生豚鼠的种群动态以及豚鼠种群对植被和与其共生的啮齿动物群落的影响都受到冬季植被盖度的限制。     

Isolation of toxic metal-tolerant bacteria from soil and examination of their bioaugmentation potentiality by pot studies in cadmium- and lead-contaminated soil

Heavy metals, also regarded as toxic metals, are the important environmental pollutants that affect all forms of life. Accumulation of toxic metals in plants results in various biochemical, physiological and structural disturbances, leading to inhibited growth and sometimes plant death. Toxic metal contamination disturbs the soil ecology as well as the agricultural productivity. Several indigenous microbes can withstand the effect of toxic metal and play a vital role in the revival of tarnished soil. In the present study, soil samples were collected from contaminated crop field of Cachar district of Assam, India. Segregation, enumeration and identification of bacteria from soil samples were performed. Among all the tested isolates, very few were able to withstand a high concentration of Cd and Pb in nutrient agar plates. Toxic metal-tolerant bacteria were identified as Pseudomonas aeruginosa and Bacillus cereus. The isolates having a higher tolerance for Cd and Pb were taken into consideration for pot studies. P. aeruginosa strain SN4 and strain SN5 showed significant results at Cd- and Pb-contaminated soil, evidenced by the healthy growth of Oryza sativa seedlings. However, B. cereus strain SN6 showed high tolerance towards Cd and Pb, but pot experimental studies showed adverse effects on seedling germination and shoot growth of O. sativa. P. aeruginosa strains were significantly able to reduce the negative impact of Cd and Pb in the soil, thus finding an alternative in removal, recovery and remediation of toxic metal-contaminated crop field.     

Cow dung extract: a medium for the growth of pseudomonads enhancing their efficiency as biofertilizer and biocontrol agent in rice

Some pseudomands are being utilized as biofertilizers and biopesticides because of their role in plant growth promotion and plant protection against root parasites, respectively. Two strains of Pseudomonas, P. jessenii LHRE62 and P. synxantha HHRE81, recovered from wheat rhizosphere, have shown their potential in field bioinoculation tests under rice-wheat and pulse-wheat rotation systems. Normally, pseudomonads are cultivated on synthetic media-like King’s B and used for inoculation on seeds/soil drench with talcum or charcoal as carrier material. Cow dung is being used for different purposes from the ancient time and has a significant role in crop growth because of the content in humic compounds and fertilizing bioelements available in it. Here, cow dung extract was tested as a growth medium for strains LHRE62 and HHRE81, in comparison with growth in King’s B medium. The log phase was delayed by 2 h as compared to growth in King’s B medium. The bacterial growth yield, lower in plain cow dung extract as compared to King’s B medium, was improved upon addition of different carbon substrates. Growth of rice var. Pant Dhan 4 in pot cultures was increased using liquid formulation of cow dung extract and bacteria as foliar spray, compared to their respective controls. Biocontrol efficacy of the bioagents was assessed by challenging rice crop with Rhizoctonia solani, a sheath blight pathogen. The growth promotion and biocontrol efficiencies were more pronounced in the case of mixed inocula of strains LHRE62 and HHRE81.     

新疆药用植物牛至内生细菌多样性与抗菌活性

【背景】植物内生菌作为微生物群落中一类非常重要的组成部分,在多个领域都有广泛的应用价值,如促进植物生长、抵抗病虫害、生物固氮、降解有毒有害化合物等。【目的】进一步丰富新疆野果林苹果腐烂病害的生防资源以及分析牛至内生细菌多样性特征。【方法】通过纯培养方法,对健康植物牛至组织进行内生细菌的分离纯化,并进行16S rRNA基因序列分析;再利用平板对峙法筛选具有抑制苹果腐烂病病原菌生长的内生细菌。【结果】共分离到168株内生细菌,最终确定为4门5纲8目12科17属,其中优势属为芽胞杆菌属(Bacillus)。由分离实验结果可知,M1号TSA培养基,M5号组氨酸-棉子糖培养基和M6号NA培养基是分离牛至内生细菌较为理想的培养基;采自新源县野果林的牛至内生细菌多样性较为丰富,在牛至根部内生细菌种类更多;通过拮抗实验共筛选出59株牛至内生细菌具有较好的抑菌效果。【结论】新疆药用植物牛至内生细菌的物种多样性较为丰富,而且富含一批有效抑制野苹果腐烂病病原菌生长的内生细菌菌株。因此,本研究在新疆野果林苹果腐烂病的生物防治和药用植物内生细菌种质资源的填补等方面具有重要意义。     

Toxic effects of antimalarial drugs in Paramecium : role of calcium channels

The antimalarial drugs, quinacrine, quinine and mefloquine, as well as the structurally-similar compound, W-7, inhibit calcium-dependent backward swimming and calcium currents in Paramecium calkinsi. These drugs are also toxic to paramecia at high concentrations. Therefore, one site of toxic action of the drugs may be the calcium channel. To test this hypothesis, the toxicity of the antimalarials and W-7 was compared in paramecia with and without calcium channels. Since calcium channels are located on the cilia, calcium channels were removed from the paramecia by deciliating the cells. Deciliated cells were found to be less susceptible to the lethal effects of the antimalarials and W-7 than their ciliated counterparts. Moreover, Pawns, mutants of P. tetraurelia that possess cilia but lack functional calcium channels, were also less susceptible to the antimalarials than wild-type cells. Thus, calcium channels may be one site of toxic action of the antimalarial drugs in paramecia and perhaps in other protists. Accepted: 27 December 1996     

Joint prediction of multiple quantitative traits using a Bayesian multivariate antedependence model

Predicting organismal phenotypes from genotype data is important for preventive and personalized medicine as well as plant and animal breeding. Although genome-wide association studies (GWAS) for complex traits have discovered a large number of trait- and disease-associated variants, phenotype prediction based on associated variants is usually in low accuracy even for a high-heritability trait because these variants can typically account for a limited fraction of total genetic variance. In comparison with GWAS, the whole-genome prediction (WGP) methods can increase prediction accuracy by making use of a huge number of variants simultaneously. Among various statistical methods for WGP, multiple-trait model and antedependence model show their respective advantages. To take advantage of both strategies within a unified framework, we proposed a novel multivariate antedependence-based method for joint prediction of multiple quantitative traits using a Bayesian algorithm via modeling a linear relationship of effect vector between each pair of adjacent markers. Through both simulation and real-data analyses, our studies demonstrated that the proposed antedependence-based multiple-trait WGP method is more accurate and robust than corresponding traditional counterparts (Bayes A and multi-trait Bayes A) under various scenarios. Our method can be readily extended to deal with missing phenotypes and resequence data with rare variants, offering a feasible way to jointly predict phenotypes for multiple complex traits in human genetic epidemiology as well as plant and livestock breeding.