Bioreporters of Genetic Expression Demonstrate Bioavailability of Metals During Biofilm Growth and Development

白色9701018在生物膜中生长的细菌形成了一个相对稳定的群落，可能会极大地影响原位生物修复速率，可能比本体相中的细菌更大。 然而，生物膜细菌的微生物活性以及金属污染物的生物可利用性仍然是研究的难点。 生物膜群落是许多物种的复杂混合物，包括可培养的和不可培养的，并且生物膜群落的组成在空间和时间上与金属离子有关。 我们将通过构建这两种金属的生物转运菌株来研究生物膜群落对金属离子（汞和铬）的响应。 生物报告基因的表达表明金属是生物可利用的，并且细菌正在积极地产生基因产物以解毒。 我们已经生产出在汞存在下生物发光的生物报告菌株。 我们进一步修饰了该菌株，使其编码绿色荧光蛋白（GFP）基因，GFP是一种非常明亮的荧光分子。 表达这种蛋白质的单个细胞可以使用我们灵敏的光检测设备找到。 我们将在流动池生物膜形成装置中使用该菌株，用荧光和生物发光检测器跟踪细菌。 生物膜群落将在实验室条件下使用污染场地的地下水建立。 我们将结合生物报告细菌，并使用它们的荧光跟踪它们的建立。 将使用荧光生物报告器确定细胞的持续存在及其数量。同样的技术将应用于减少铬的细菌。 汞还原（mer）基因是高度保守的，我们将利用这一优势来检测生物膜群体中的mer基因。 敏感的分子技术，如用于检测汞还原剂的原位PCR和用于检测mer还原剂活性的原位逆转录酶PCR，将用于与光测量结果进行比较。 这些实验的结果将在原位条件下给出生物膜生物修复速率的更明确的评估。

White 9701018 Bacteria that are growing in biofilms form a relatively stable community that may greatly affect bioremediation rate in situ, perhaps more than bacteria in the bulk phase. However, microbial activity of biofilm bacteria, as well as the bioavaliability o metal contaminatants, remain difficult areas to study. Biofilm communities are complex mixtures of many species, both culturable and non-culturable, and the composition of the biofilm communities to metal ions spatially and temporally. We will study the response of biofilm communities to metal ions (mercury and chromium) by constructing bioeporter strains for these two metals. Expression of the bioreporter genes indicates that the metal is bioavailable, and that the bacteria are actively producing gene products to detoxify. We have produced bioreporter strains that bioluminesce in the presence of mercury. We have further modified this strain to encode a gene for Green Fluorescent Protein (GFP), which is an extremely bright fluorescent molecule. Individual cells expressing this protein can be found using our sensitive photodetection equipment. We will use this strain in a flow cell biofilm formation apparatus, following the bacteria with both fluorescence and bioluminescence detectors. The biofilm community will be established under laboratory conditions using groundwater from a contaminated site. We will incorporate the bioreporter bacteria and track their establishment using their fluorescence. The continued presence of the cells, and their number, will be established using the fluorescence bioreporter. The same technique will be applied to bacteria that reduce chromium. The mercury reduction (mer) genes are highly conserved, and we will use tis advantage to detect mer genes in the biofilm population. Sensitive molecular techniques, such as in situ PCR for detecting mercury reducers, and in situ Reverse Transcriptase PCR for detecting activity from mer reducers, will be employed for comparison to results form light measurements. The results of these experiments will give a more definite assessment of biofilm bioremediationrates under in situ conditions.