In Vivo Characterization of Bacteria-mediated Extracellular Reduction of Chromium

描述(由申请人提供)： 六价铬[Cr(VI)]存在于超级基金的地点，在那里它对人类造成了重大的健康问题。细菌介导的水溶性和毒性的铬(VI)原位转化为难溶/毒性的铬(III)是一种很有前途的控制和处理铬(VI)的方法。然而，细菌还原铬(VI)的分子机制仍不清楚。异化铁(Fe)还原细菌希瓦氏杆菌对铬(VI)的还原导致细胞表面形成纳米尺寸的铬(III)颗粒。我们推测，暴露在细胞表面的细胞色素MtrC和OMCA通过直接作为铬(VI)末端还原酶和间接作为Fe(III)氧化物末端还原酶参与了铬(III)的形成，从而产生Fe(II)，而Fe(II)又反过来还原了Cr(VI)。我们计划使用我们开发的纳米级AFM相关SERS成像显微镜(AFM-CSIM)以及其他方法来验证我们的假设。AFM-CSIM可以在纳米分辨率上同时测定细胞色素和纳米金属颗粒的位置以及它们在细菌表面的氧化状态。利用AFM-CSIM将MtrC和OMCA与纳米级的Cr(III)和Fe(III)粒子共定位，将为我们的假设提供直接的证据。其他方法(如扫描电子显微镜的免疫金标记法和纯化的MtrC和OMCA的铬(VI)还原酶活性的测量)将用于验证AFM-CSIM结果。该项目的成功完成不仅将有助于实现我们了解细菌还原铬(VI)的分子机制的长期目标，而且还将开发基于科学的解决方案，以减轻超级基金地点的铬(VI)的毒性影响。

DESCRIPTION (provided by the applicant): Hexavalent chromium[Cr(VI)]exists at the Superfund sites where it poses major health concerns to humans. Bacteria-mediated in situ transformation of water soluble and toxic Cr(VI) to less soluble/toxic Cr(III) represents a promising method to contain and treat Cr(VI). However, the molecular mechanisms by which bacteria reduce Cr(VI) remain undetermined. Reduction of Cr(VI) by dissimilatory iron (Fe) reducing bacterium Shewanella oneidensis results in formation of nano-sized Cr(III) particles on the cell surfaces. We hypothesize that cell surface-exposed cytochromes MtrC and OmcA of S. oneidensis are involved in the formation of Cr(III) by reducing Cr(VI) directly as Cr(VI) terminal reductases and indirectly as Fe(III) oxide terminal reductases to produce Fe(II) that in turn reduces Cr(VI). We plan to use nanoscale AFM correlated SERS imaging microscopy (AFM-CSIM) developed by us along with other methods to test our hypothesis. AFM-CSIM can simultaneously determine the locations of cytochromes and nano-sized metal particles as well as their oxidation states on the bacterial surfaces at nanometer-resolution. Co-localization of MtrC and OmcA with nano-sized Cr(III) and Fe(III) particles by AFM-CSIM will provide direct evidences to support our hypothesis. Other approaches (e.g. immuno-gold localization with scanning electron microscopy and the measurements of Cr(VI) reductase activity of purified MtrC and OmcA) will be used to validate the AFM-CSIM results. Successful completion of this project will help not only achieve our long-term goal of understanding the molecular mechanisms by which bacteria reduce Cr(VI), but also develop science-based solutions to mitigate the toxic effects of Cr(VI) at the Superfund sites.