Bacillus sp.ZJS3基因组测序及砷胁迫下相关基因表达分析

【背景】环境中高毒性As³⁺的微生物氧化在砷的生物地球化学循环中起重要作用,具有潜在的应用价值。【目的】Bacillus sp.ZJS3菌株是本实验室前期分离鉴定的一株As³⁺耐受菌株,而且对多种重金属具有耐受性,期望进一步明确该菌株在As³⁺胁迫下菌体形态变化及应对砷胁迫的遗传基础,为As³⁺耐受细菌的研究提供基础数据。【方法】使用单分子实时测序（single-molecule real-time sequencing,SMRT）及Illumina测序技术对Bacillus sp.ZJS3菌株进行全基因组测序,对其基因进行功能注释和生物信息学分析,并结合绝对定量PCR技术对砷抗性及砷代谢相关基因进行分析。【结果】Bacillus sp.ZJS3菌株基因组大小为5.82 Mb,GC含量为35.9%,包含染色体1个、质粒3个、CDS数量为5 981个、tRNA 104个、sRNA 136个、rRNA 42个、串联重复序列173个、基因岛13个、转运蛋白1 023个、跨膜蛋白1 717个和双组分调控基因160个。NR、Swiss-Prot、Pfam、COG、GO和KEGG数据库分别可注释Bacillus sp.ZJS3菌株基因组中97.66%、69.30%、78.52%、65.49%、67.65%和43.87%的基因。绝对定量PCR结果表明,arsC基因在砷处理条件下显著高于对照组,而arsB基因在砷处理条件下显著低于对照组。【结论】Bacillus sp.ZJS3菌株在As³⁺胁迫下可能导致细胞分裂无法正常进行,进而影响细胞形态。基因组中aqpZ、arsA、arsB、arsC等基因的存在表明该菌株具有As³⁺外排和还原As⁵⁺的能力,phoUpstBACS的存在表明菌株可以吸收As⁵⁺,但菌株受到外界环境As³⁺胁迫时arsB表达水平降低。     

A hydrogen-peroxide digestion system for tissue trace-metal analysis

Tissue digestion prior to analysis for trace metals is usually carried out with strong acids. Nitric acid, alone or in combination with perchloric acid, is most commonly used. In addition to the laborious acid washing of all glassware prior to use, the digestion necessitates exposure to potential environmental contamination. Use of perchloric acid mandates a specially constructed hood with facilities for washing to remove acid deposits. A simple digestion procedure using 30% hydrogen peroxide in polyethylene vials in an oven at approximately 75°C has been previously described for the measurement of zinc in tissues using flame or flameless atomic absorption spectrophotometry and of selenium in liver by flameless atomic absorption. Readings for reagent blanks were insignificant. The technique has been further developed with a reduction in digestion time using 50% H₂O₂. Analysis of liver has been extended to include copper, manganese, and arsenic. Although the level of arsenic present was too low to be detected, 50 and 100 ng of this element added to the liver powder was completely recovered. The digest obtained when dissolved in appropriate solvent is suitable for analysis for multiple trace metals. Author to whom all correspondence and reprint requests should be addressed.     

The influence of chemical form and concentration of arsenic on rice growth and tissue arsenic concentration

Arsenic absorption by rice (Oryza sativa, L.) in relation to the chemical form and concentration of arsenic added in nutrient solution was examined. A 4 × 3 × 2 factorial experiment was conducted with treatments consisting of four arsenic chemical forms [arsenite, As(III); arsenate, As(V); monomethyl arsenic acid, MMAA; and dimethyl arsenic acid, DMAA], three arsenic concentrations [0.05, 0.2, and 0.8 mg As L^-1], and two cultivars [Lemont and Mercury] with a different degree of susceptibility to straighthead, a physiological disease attributed to arsenic toxicity. Two controls, one for each cultivar, were also included. Arsenic phytoavailability and phytotoxicity are determined primarily by the arsenic chemical form present. Application of DMAA increased total dry matter production. While application of As(V) did not affect plant growth, both As(III) and MMAA were phytotoxic to rice. Availability of arsenic to rice followed the trend: DMAA<As(V)<MMAA<As(III). Upon absorption, DMAA was readily translocated to the shoot. Arsenic(III), As(V), and MMAA accumulated in the roots. With increased arsenic application rates the arsenic shoot/root concentration decreased for the As(III) and As(V) treatments. Monomethyl arsenic acid (MMAA), however, was translocated to the shoot upon increased application. The observed differential absorption and translocation of arsenic chemical forms by rice is possibly responsible for the straighthead disorder attributed to arsenic.     

土壤溶液中的砷及其与水稻生长效应的关系

通过盆栽试验和土壤中砷的吸附试验,探讨了砷在土壤溶液中的氧化－还原行为和溶解行为,并初步揭示了不同价态砷对水稻毒性差异的机理,以及土壤ｐＨ对砷的吸附作用和毒害作用的影响。     

Oxidative stress from environmental pollutants

Recently progress has been made on O₂ toxicity and pathology related to numerous environmental contaminants in insects. The pro-oxidants studied included: dioxin, paraquat, and an assorted array of quinones, 8-methoxypsorlen, arsenic, and mercury. The responses to these oxidants are diverse, but they arise from the reactive oxygen species. These pro-oxidants in insects cause lipid peroxidation, protein and enzyme oxidation, and GSH depletion. Potentially, they may also cause DNA oxidation, and form DNA adducts. Oxidative challenge is alleviated by antioxidant compounds, but more importantly by the induction of antioxidant enzymes, which are crucial for the termination of O₂ radical cascade and lipid peroxidation chain reaction. Insects exhibit a wasting syndrome under sub-acute stress. In acute toxicity vital physiological processes impaired are hemolymph melanization and diuresis. Thus, insects resemble vertebrates in both the response to oxidative stress and its pathological consequences. These results raise the prospect that insects may serve as non-mammalian model species for monitoring the oxidative-stress component of environmental toxicity. © 1995 Wiley-Liss, Inc.     

An insect model for assessing oxidative stress related to arsenic toxicity

The potential usefulness of an insect model to evaluate oxidative stress induced by environmental pollutants was examined with trivalent arsenic (As³⁺, NaAsO₂) and pentavalent arsenic (As⁵⁺, Na₂HAsO₄) in adult female house flies, Musca domestica, and fourth-instar cabbage loopers, Trichoplusia ni. M. domestica was highly susceptible to both forms of arsenic following 48 h exposure in the drinking water with LC₅₀s of 0.008 and 0.011% w/v for As³⁺ and As⁵⁺, respectively. T. ni larvae were susceptible to dietary As³⁺ with an LC₅₀ of 0.032% w/w but seem to tolerate As⁵⁺ well with an LC₅₀ of 0.794% concentration after 48 h exposure. The minimally acute LC₅ dose of both As³⁺ and As⁵⁺ varied considerably but averaged 0.005% for both insects. The potential of both valencies of arsenic for inducing oxidative stress in the insects exposed ad libitum to approximately LC₅ levels was assessed. The parameters examined were the alterations of the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), the peroxidase activity of glutathione transferase (GSTPX), and glutathione reductase (GR), and increases in lipid peroxidation and protein oxidation. SOD (1.3-fold), GST (1.6-fold), and GR (1.5-fold) were induced by As³⁺ in M. domestica but CAT and GSTPX were not affected. As⁵⁺ had no effect on M. domestica. In T. ni, the antioxidant enzyme activities were not affected by As³⁺ except for SOD which was suppressed by 29.4% and GST which was induced by 1.4-fold. As⁵⁺ had no effect except the suppression of SOD by 41.2%. Lipid peroxidation and protein oxidation, which represent stronger indices of oxidative stress, were elevated in both insects by up to 2.9-fold. However, based on the antioxidant enzyme response to the arsenic anions, the mode of action of arsenic induced oxidative stress may differ between the two insects. Until this aspect is further clarified, evidence at this time favors the prospect of As³⁺ as a pro-oxidant, especially for M. domestica. © 1995 Wiley-Liss, Inc.     

Cloning of genes that have environmental and clinical importance from rhodococci and related bacteria

Generalised and specialised transduction systems were developed for Rhodococcus by means of bacteriophage Q4. The latter was used in conjunction with DNA from an unstable genetic element of R. rhodochrous to construct resistance plasmids which replicate in strains of R. equi, R. erythropolis and R. rhodochrous. One of the plasmids, pDA21, was joined with Erythropolis coli suicide vector pEcoR251 to obtain shuttle plasmids maintained in both rhodococci and E. coli. Conjugation between these rhodococcal strains demonstrated all were interfertile with each other and that some of the determinants for this were located on the unstable genetic element. Plasmids derived from this element, such as pDA21, carried the conjugative and self- incompatibility capacities; deletion analysis revealed that DNA necessary for self-incompatibility overlapped with that for arsenic resistance.Rifampicin is one of the principal chemotherapeutic agents used to treat infections by rhodococci and related organisms. The genes responsible for two types of inactivation have been cloned. The sequence of the R. equi DNA responsible for decomposition of the antibiotic strongly resembled those of monooxygenases acting upon phenolic compounds, consistent with the presence of a naphthalenyl moiety in the rifampicin molecule. Antibiotic resistance conferred by the gene was surprisingly specific to the semisynthetic compounds rifampicin (150-fold increase) and rifapentine (70-fold). Similar specificity was observed with the other inactivation gene cloned, which ribosylates rifampicin at the 23-hydroxyl position. A 60-bp sequence upstream of the monooxygenase and ribosylation genes is strikingly similar suggesting a shared pattern of regulation.Rhodococcal arsenic resistance and azo dye degradation genes have been cloned and characterised.     

Assessing Risk of Inorganic Arsenic in Drinking Water in the United States

Limitations of the current EPA risk assessment for inorganic arsenic in drinking water in the U.S. are discussed. An empirical approach is suggested that would sample survey the populations in regions with the highest arsenic levels in drinking water for signs of arsenicism, which has been much more prevalent and appeared much earlier in exposed populations than cancer (e.g., of the skin). Biomarkers of exposure, such as arsenic content in urine, nails, hair, and skin scales, may provide even earlier indications of subpopulations with excessive arsenic exposure and identify individuals at risk. Further study is needed to evaluate fully the potential for use of biomarkers, focusing on the accuracy and reliability of analytical methods, the utility of biomarkers as indicators of short-term and long-term exposure and as precursors to clinical signs of arsenicism, and the use of “normal” ranges of biomarkers for interpretation of field observations.     

Arsenic and antimony release from sediments in a Mediterranean estuary

An ancient realgar mine is responsible for the contamination of a riverine system in central Corsica. Nearby the mine, high As and Sb concentrations are found (up to 2–3 mg l^-1 and 2–3 g l^-1 respectively). A significant increase of the As and Sb concentrations in water is found in the water of the mixing zone of river and sea (Bravona estuary; chlorinity 7000–7800). In the same area, a decrease of As and Sb concentrations in sediments is observed. Fe and Mn exhibit the same behaviour. The possible involvement of redox processes is discussed. However, owing to insufficient reducing capacity and insignificant variation of redox potential values along the various estuarine sites (from 320 down to 280 mV), this scenario is rather doubtful. Desorption from FeOOH particles is the most likely process in the Bravona estuary. The impact of these contaminated waters on the marine environment is discussed. Owing to decrease by dilution, as well as the reduced water flow of the estuary, the net supply of As and Sb to the Tyrrhenian Sea is approximately 5 and 1.5 t y^-1 for As and Sb respectively. The possible harmful influence of the Bravona river should be of local concern only, and the emphasis of this study is, therefore, on a better knowledge of in situ processes.