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

在生物膜中生长的细菌形成一个相对稳定的群落，可能会极大地影响原位生物修复率，可能比体相的细菌影响更大。然而，生物膜细菌的微生物活性以及对金属污染物的生物利用度仍然是研究的难点。生物膜群落是多种生物的复杂混合物，既有可培养的，也有不可培养的，生物膜群落的组成在空间和时间上都与金属离子有关。我们将通过构建金属离子（汞和铬）的生物报告菌株来研究生物膜群落对金属离子（汞和铬）的反应。生物报告基因的表达表明金属是生物可利用的，并且细菌正在积极产生基因产物来解毒。我们已经生产了生物报告菌株，在汞存在下生物发光。我们进一步修改了这种菌株，使其编码绿色荧光蛋白（GFP）的基因，这是一种非常明亮的荧光分子。使用我们灵敏的光探测设备可以发现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该菌株，用荧光和生物发光检测器跟踪细菌。利用污染场地的地下水，在实验室条件下建立生物膜群落。我们将纳入生物报告细菌，并利用它们的荧光追踪它们的建立。细胞的持续存在及其数量将使用荧光生物报告仪来确定。同样的技术将应用于减少铬的细菌。汞还原（mer）基因高度保守，我们将利用这一优势在生物膜群体中检测mer基因。灵敏的分子技术，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，将被用于与光测量结果进行比较。这些实验结果将对原位条件下的生物膜生物修复率给出更明确的评估。

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.