STRUCTURAL AND COMPOSITIONAL ANALYSIS OF BACTERIA IMPORTANT IN BIO-REMEDIATION

对生物修复很重要的细菌的结构和成分分析

• 批准号: 7721721
• 负责人: Patricia A Sobecky
• 金额: $ 2.22万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721721
• 关键词: Acid Phosphatase; Address; Aerobic; Bacillus (bacterium); Bacteria; Bacteriology; Biological; Bioremediations; Biotechnology; Calcium; Cell Survival; Cell surface; Cells; Charge; Chemistry; Class; Classification; Computer Retrieval of Information on Scientific Projects Database; Condition; Cytoplasm; Cytoplasmic Granules; DNA Sequence; Department of Energy; Development; Drug Metabolic Detoxication; Dyes; Ecology; Electron Microscopy; Electrons; Environment; Environmental Microbiology; Enzymes; Excision; Exhibits; Figs - dietary; Freeze Substitution; Freezing; Funding; Future; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Grant; Growth; Heavy Metals; Image; Image Analysis; Imagery; Immobilization; In Situ; Industrial Microbiology; Institutes; Institution; Journals; Knowledge; Laboratories; Liquid substance; Localized; Location; Mediating; Membrane; Membrane Lipids; Metals; Microbe; Minerals; Modeling; Nuclear Weapon; Nutrient; Organophosphates; Oxidants; Oxygen; Pattern; Peptidoglycan; Phenotype; Phosphoric Monoester Hydrolases; Phosphorus; Physiology; Polyphosphates; Precipitation; Preparation; Radioisotopes; Rahnella; Research; Research Personnel; Resolution; Resources; Respiration; Science; Site; Soil; Solid; Source; Staining method; Structure; System; Techniques; Technology; Tennessee; Toxic effect; USA Georgia; United States; United States National Institutes of Health; Uranium; War; Western Asia Georgia; Work; alpha-glycerophosphoric acid; base; cell envelope; cell fixing; depolymerization; extracellular; improved; inorganic phosphate; insight; microbial; mineralization; nanoscale; oxidation; periplasm; remediation; research study; sample fixation; spectroscopic imaging

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background Soil and groundwater systems contaminated with toxic heavy metals and radionuclides remain a legacy of Cold War nuclear weapons development. As a result, the United States Department of Energy (DOE) is charged with the remediation and long-term stewardship of such contaminated sites at many U.S. national laboratories. The focus of DOE funded bioremediation research aims at utilizing naturally occurring bacterial strains, obtained from contaminated soils, for the in situ immobilization of soluble uranium. Two distinct strategies for the removal of radionuclides such as uranium from contaminated soils and groundwater are known to be directly mediated by bacteria: 1. Bio-reduction: in the absence of oxygen, uranium is utilized as a terminal electron acceptor for respiration. Soluble U(VI) is reduced to the insoluble U(IV) oxidation state. 2. Bio-mineralization: in the presence of oxygen, uranium precipitation occurs when bacterially liberated phosphate yields an insoluble mineral phosphate (Fig.1). Fig.1. Three potential cell associated locations (i.e. cell surface, cytoplasm and periplasm) of bacterial phosphatase activity hypothesized to occur in heavy-metal and radionuclide contaminated soils and groundwater (A). The hypothesized mechanism of microbial phosphatase activity which yields a uranium phosphate precipitate (B). Current Research Our research focuses on the aerobic bio-mineralization strategy of uranium via phosphate-liberating bacterial strains that we have isolated from heavy metal and radionuclide contaminated soils from the Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee. We hypothesize that such phosphate liberation into the surrounding environment is a result of either: 1) Bacteria that constitutively express a sub-class of phosphatase enzymes known as non-specific acid phosphatases (NSAP) or 2) Bacterial strains that are able to secrete de-polymerized cytoplasmic polyphosphate granules. Our work has identified two ORNL bacterial strains belonging to the genera, Rahnella and Bacillus that we have used as model bio-mineralizing strains. Both strains exhibit a phosphate-liberating phenotype when grown on both solid and liquid media. The Rahnella and Bacillus strains demonstrate great promise as they represent naturally occurring bacteria that have been shown to liberate sufficient phosphate from organophosphate substrates such as glycerol-3-phosphate and subsequently precipitate uranium in the form of calcium autinite, Ca(UO2)2(PO4)2 (Martinez et al.; Beazley et al., in preparation). Our ORNL Rahnella and Bacillus strains are representatives of the two fundamental classifications of bacterial cell envelopes (i.e., gram-negative and gram-positive cell envelopes) (Fig 2). The identification of these cells was done through DNA sequencing rather than the traditional method of staining with the use basic dyes. The distinctions between these two cell envelopes are based upon the presence of a single peptidoglycan layer located between two concentric lipid membranes (i.e., periplasmic domain) for gram-negative bacteria. Gram-positive bacteria are characterized as having a single cytoplasmic lipid membrane and a large peptidoglycan layer. Until the recent application of energy filtering electron microscopy on frozen hydrated sections, it was believed that a periplasmic domain was absent in gram-positive bacteria. Recently, the Beveridge laboratory has demonstrated through the use of this technique as well as freeze substituted fixation that gram-positive bacteria do indeed have a periplasmic space similar to gram-negative bacteria (Matias et al. 2006). This finding suggests that, like gram-negative bacteria, gram-positive bacteria may have localized chemistry in this previously unseen periplasmic domain. The insight gained through the visualization of this new structure can further the current understanding of gram-positive physiology. Specifically, phosphate accumulation via the activity of NSAPs, which have been previously shown to be localized in the periplasm of gram-negative bacteria, may be functioning in a comparable fashion in our ORNL gram-positive bacterial strain. A second mechanism by which both gram-negative and gram-positive bacteria liberate phosphate and sequester heavy metals is through the depolymerization of cytoplasmic polyphosphate granules (Fig. 2A, 2B). The storage of polymerized phosphate acts as both a reservoir for growth under phosphate-limiting conditions and as a metal detoxification system. The depolymerization of polyphosphate has been shown to be involved in the extracellular precipitation of heavy metals (Boswell et al., 2001). Additionally, polyphosphate granules have also been shown to sequester radionuclides that have been transported into the cytoplasm of gram-positive bacteria (Suzuki and Banfield, 2004). Fig.2. Generalized composition of the two fundamental bacterial cell envelops (gram-negative and gram-positive) both containing polyphosphate storage granules (A). The three hypothesized uranium phosphate localization sites (i.e., outer membrane, periplasmic domain and polyphosphate granule sequestration) predicted in our model ORNL bacterial strains (B). Presently, our findings have demonstrated the capability of bacteria extant within the ORNL soils to bio-precipitate soluble uranium under similar in situ growth conditions. To understand the effect uranium phosphate precipitation has on ORNL Rahnella and Bacillus strains that have never been visualized on the nanometer scale, we would like to address the following questions: 1. Do the cell envelopes of ORNL Rahnella and Bacillus strains exposed to uranium differ from those unexposed to uranium? 2. For ORNL Rahnella and Bacillus strains exposed to uranium, is phosphorus and uranium localized on the outer membrane, periplasmic space or associated with polyphosphate granules within the cytoplasm? The visualization of the cell envelopes as well as the localization of both phosphorus and uranium in these strains will allow us to definitively determine the source of accumulating phosphate (i.e., NSAP activity within the periplasm or cytoplasmic depolymerization of polyphosphate granules). As bacterial viability is crucial for optimal bio-precipitation, the localization pattern of phosphorus and uranium is important. Cytoplasmic polyphosphate sequestration of uranium would be less detrimental to cell viability than a periplasmic accumulation. Due to the inhibition of electron and nutrient transport, periplasmic mineralization of uranium would contribute to a loss in cell viability. Goals at the RVBC The microanalysis and ultra-structure imaging will be critical for our understanding of heavy-metal and radionuclide toxicity. Knowledge gained by such analysis will allow optimal utilization of bacterial strains for future bioremediation experiments. Therefore, we feel the imaging and spectroscopic capabilities of the Resource for the Visualization of Biological Complexity (RVBC) will further our understanding of microbes capable of metal and radionuclide precipitation. The proven expertise of the RVBC staff will allow us to conduct imaging analysis of dehydrated cells, freeze substitution fixed cells and frozen hydrated cells. Additionally, the energy filtering capabilities of the RVBC allow for EELS elemental microanalysis as well as improved resolution at the nanometer scale. Conclusion The microanalysis and ultra-structure imaging of the ORNL Rahnella and Bacillus strains has never been conducted. The knowledge gained from the microanalysis and ultra-structure imaging will yield new insights applicable to future in situ bioremediation strategies. Such strategies will aim to stimulate soil microbes capable of bio-precipitating metals found in contaminated soils and groundwater systems. This research is conducted at the Georgia Institute of Technology in the laboratory of Dr. Patricia A. Sobecky and supported by the Office of Science (BER), U.S. Department of Energy Grant No. DE-FG02-04ER63906. References Beazley, M., Martinez, R.J., Webb, S.M., Sobecky, P.A., Taillefert, M. Environmental Science and Technology (in preparation). Boswell, C. D., Dick, R. E.,Eccles, H. 2001. Journal of Industrial Microbiology & Biotechnology. 26, 333-340. Martinez, R.J., Beazley, M., Taillefert, M., Arakaki, A.K., Skolnick, J. and Sobecky, P.A. Environmental Microbiology (in preparation). Matias, V. R. F., Beveridge, T. J. 2006. Journal of Bacteriology. 3, 1011-1021. Suzuki, Y., Banfield, J. F. 2004. Geomicrobiology Journal. 21, 113-121.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 背景 被有毒重金属和放射性核素污染的土壤和地下水系统仍然是冷战核武器发展的遗产。 因此，美国能源部 (DOE) 负责对许多美国国家实验室的此类污染场地进行修复和长期管理。 美国能源部资助的生物修复研究的重点是利用从受污染土壤中获得的天然细菌菌株来原位固定可溶性铀。 众所周知，从受污染的土壤和地下水中去除放射性核素（例如铀）的两种不同策略是由细菌直接介导的： 1.生物还原：在没有氧气的情况下，铀被用作呼吸的末端电子受体。可溶性 U(VI) 被还原为不溶性 U(IV) 氧化态。 2.生物矿化：在氧气存在的情况下，当细菌释放的磷酸盐产生不溶性矿物磷酸盐时，就会发生铀沉淀（图1）。 图1.假设发生在重金属和放射性核素污染的土壤和地下水中的细菌磷酸酶活性的三个潜在细胞相关位置（即细胞表面、细胞质和周质）（A）。 产生磷酸铀沉淀的微生物磷酸酶活性的假设机制 (B)。 目前的研究 我们的研究重点是通过从田纳西州橡树岭国家实验室 (ORNL) 受重金属和放射性核素污染的土壤中分离出磷酸盐释放细菌菌株来进行铀的需氧生物矿化策略。 我们假设这种磷酸盐释放到周围环境中的原因是：1）组成型表达磷酸酶亚类（称为非特异性酸性磷酸酶（NSAP））的细菌或2）能够分泌解聚细胞质多磷酸盐颗粒的细菌菌株。 我们的工作已经鉴定出属于 Rahnella 和 Bacillus 属的两种 ORNL 细菌菌株，我们将其用作模型生物矿化菌株。 当在固体和液体培养基上生长时，两种菌株均表现出释放磷酸盐的表型。 Rahnella 和 Bacillus 菌株表现出巨大的前景，因为它们代表天然存在的细菌，已被证明可以从有机磷酸盐底物（如 3-磷酸甘油）中释放出足够的磷酸盐，并随后以钙奥氏体 Ca(UO2)2(PO4)2 的形式沉淀铀（Martinez 等人；Beazley 等人，正在准备中）。 我们的 ORNL Rahnella 和 Bacillus 菌株是细菌细胞包膜的两个基本分类（即革兰氏阴性和革兰氏阳性细胞包膜）的代表（图 2）。 这些细胞的鉴定是通过 DNA 测序完成的，而不是使用碱性染料染色的传统方法。 这两个细胞包膜之间的区别是基于位于革兰氏阴性细菌的两个同心脂质膜（即周质结构域）之间的单个肽聚糖层的存在。 革兰氏阳性细菌的特征是具有单一的细胞质脂质膜和大的肽聚糖层。 直到最近在冷冻水合切片上应用能量过滤电子显微镜之前，人们认为革兰氏阳性细菌中不存在周质结构域。 最近，Beveridge 实验室通过使用该技术以及冷冻替代固定证明，革兰氏阳性细菌确实具有与革兰氏阴性细菌相似的周质空间（Matias 等人，2006）。 这一发现表明，与革兰氏阴性细菌一样，革兰氏阳性细菌可能在这个以前看不见的周质域中具有局部化学作用。 通过这种新结构的可视化获得的见解可以进一步加深目前对革兰氏阳性生理学的理解。 具体来说，通过 NSAP 的活性进行磷酸盐积累，先前已证明 NSAP 位于革兰氏阴性细菌的周质中，可能在我们的 ORNL 革兰氏阳性细菌菌株中以类似的方式发挥作用。 革兰氏阴性和革兰氏阳性细菌释放磷酸盐和螯合重金属的第二种机制是通过细胞质多磷酸盐颗粒的解聚（图2A、2B）。 聚合磷酸盐的储存既充当磷酸盐限制条件下生长的储存库，又充当金属解毒系统。 多磷酸盐的解聚已被证明与重金属的细胞外沉淀有关（Boswell 等，2001）。 此外，多磷酸盐颗粒还被证明可以隔离已转运到革兰氏阳性细菌细胞质中的放射性核素（Suzuki 和 Banfield，2004）。 图2.两种基本细菌细胞包膜（革兰氏阴性和革兰氏阳性）的一般组成均含有多磷酸盐储存颗粒 (A)。 在我们的模型 ORNL 细菌菌株中预测了三个假设的磷酸铀定位位点（即外膜、周质结构域和多磷酸盐颗粒隔离）（B）。 目前，我们的研究结果证明了橡树岭国家实验室土壤中现存的细菌在类似的原位生长条件下生物沉淀可溶性铀的能力。 为了了解磷酸铀沉淀对从未在纳米尺度上观察到的 ORNL Rahnella 和 Bacillus 菌株的影响，我们想解决以下问题： 1. 暴露于铀的 ORNL Rahnella 和 Bacillus 菌株的细胞包膜与未暴露于铀的菌株有何不同？ 2. 对于暴露于铀的 ORNL Rahnella 和 Bacillus 菌株，磷和铀是否位于外膜、周质空间或与细胞质内的聚磷酸盐颗粒相关？ 细胞包膜的可视化以及这些菌株中磷和铀的定位将使我们能够明确确定磷酸盐积累的来源（即周质内的 NSAP 活性或多磷酸盐颗粒的细胞质解聚）。 由于细菌活力对于最佳生物沉淀至关重要，因此磷和铀的定位模式也很重要。 铀的细胞质多磷酸盐封存对细胞活力的损害小于周质积累。 由于电子和营养物质运输的抑制，铀的周质矿化将导致细胞活力的丧失。 RVBC 的目标 微量分析和超微结构成像对于我们了解重金属和放射性核素毒性至关重要。 通过此类分析获得的知识将使细菌菌株能够在未来的生物修复实验中得到最佳利用。因此，我们认为生物复杂性可视化资源 (RVBC) 的成像和光谱功能将进一步加深我们对能够沉淀金属和放射性核素的微生物的理解。 RVBC 工作人员经过验证的专业知识将使我们能够对脱水细胞、冷冻替代固定细胞和冷冻水合细胞进行成像分析。 此外，RVBC 的能量过滤功能可实现 EELS 元素微量分析并提高纳米级分辨率。 结论 从未对 ORNL Rahnella 和 Bacillus 菌株进行过微量分析和超微结构成像。 从微量分析和超微结构成像中获得的知识将产生适用于未来原位生物修复策略的新见解。这些策略的目的是刺激能够生物沉淀受污染土壤和地下水系统中发现的金属的土壤微生物。 这项研究是在佐治亚理工学院 Patricia A. Sobecky 博士的实验室中进行的，并得到了美国能源部科学办公室 (BER) 的支持，资助号为 DE-FG02-04ER63906。 参考 Beazley, M.、Martinez, R.J.、Webb, S.M.、Sobecky, P.A.、Taillefert, M. 环境科学与技术（准备中）。 Boswell, C. D.、Dick, R. E.、Eccles, H. 2001。工业微生物学与生物技术杂志。 26、333-340。 Martinez, R.J.、Beazley, M.、Taillefert, M.、Arakaki, A.K.、Skolnick, J. 和 Sobecky, P.A.环境微生物学（准备中）。 Matias, V. R. F., Beveridge, T. J. 2006。细菌学杂志。 3、1011-1021。 Suzuki, Y., Banfield, J. F. 2004。地球微生物学杂志。 21、113-121。