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

生长在生物膜中的白色9701018细菌形成了一个相对稳定的群落，这可能会极大地影响原位生物修复的速度，可能比本体阶段的细菌更多。然而，生物被膜细菌的微生物活性以及金属污染物的生物利用度仍然是难以研究的领域。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，以及生物膜群落在空间和时间上对金属离子的组成。我们将通过构建这两种金属的生物转运子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可生物利用的，细菌正在积极生产基因产品来解毒。我们已经生产出在汞存在下生物发光的生物报告菌株。我们进一步对该菌株进行了改造，编码了绿色荧光蛋白(GFP)的基因，这是一种非常明亮的荧光分子。使用我们灵敏的光检测设备，可以找到表达这种蛋白质的单个细胞。我们将在流动细胞生物膜形成装置中使用这种菌株，跟随细菌的荧光和生物发光检测器。生物膜群落将在实验室条件下利用污染场地的地下水建立。我们将整合生物报告细菌，并使用它们的荧光来跟踪它们的建立。这些细胞的持续存在及其数量将使用荧光生物报告程序来确定。同样的技术也将应用于降低铬含量的细菌。汞还原(MER)基因是高度保守的，我们将利用TIS的优势来检测生物膜种群中的MER基因。敏感的分子技术，如检测汞还原酶的原位聚合酶链式反应和原位逆转录酶聚合酶链式反应，将被用来与光测量的结果进行比较。这些实验的结果将对现场条件下的生物膜生物修复速率做出更明确的评估。

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.