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Characterization and field trials of a bioluminescent bacterial reporter of iron bioavailability

机译：铁生物利用度的生物发光细菌报告基因的表征和现场试验

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To better understand Fe cycling in marine and freshwater systems, we have developed a biomolecular tool to track the perceived bioavailability of Fe to heterotrophic bacteria. Bioluminescent reporters, constructed by fusing the fepA-fes promoter of Escherichia coli (an Enterobactin biosynthesis gene regulated by the ferric uptake regulatory [Fur] system) to a luxCDABE cassette, were integrated into the chromosome of a halotolerant Pseudomonas putida, which uses the Fur system to regulate high-affinity Fe uptake. The resultant P. putida bioreporter has been successfully tested both in lab and field studies. Laboratory cultures were maintained at a range of concentrations of total Fe (0-25 nM) or limited by the addition of concentrations of well-characterized siderophores (desferrioxamine B [DFB], ferrichrome, 2,2′-dipyridyl [DP] and Rhodotorulic acid [RA], 0-200 nM) and used to establish the dynamic range of this reporter system. Analysis of sample incubations after only 4 h suggest that both of the trihydroxamate-type siderophores DFB and ferrichrome efficiently reduced Fe availability, resulting respectively in a 1.77- and 1.88-fold increase in luminescence relative to Fe-replete conditions. In contrast, additions of the dihydroxamate-type siderophore RA and the synthetic chelator DP resulted in no response from the system, suggesting that cells could access Fe complexed to these compounds without activating high-affinity Fe transport systems. Field studies were performed in the central basin of Lake Erie, which has previously been shown to undergo sporadic Fe limitation during summer stratification. DFB concentrations were titrated across a range of 0-50 nM into unfiltered water to manipulate Fe availabilities. Bioreporters expressed Fe stress (ca. a 2-fold increase in luminescence) at concentrations of DFB equivalent to the total (dissolved + particulate) Fe in the system ( ≈ 30 nM), indicative of the concentration of bioavailable Fe. In a similar experiment with 0.2-μm pre-filtered water (2.25-5.24 nM Fe), a 6-fold increase in luminescence (relative to controls) was observed at the lowest (15 nM) concentration of chelators. The results of this study demonstrate the validity of bioreporters as a complimentary tool to measurements of total Fe. Moreover, these results suggest that a significant source of bioavailable Fe flux may be particulate in nature, and support the hypothesis that Fe recycling from microorganisms may play a key role in the regeneration of bioavailable forms of Fe.
机译：为了更好地了解海洋和淡水系统中的Fe循环，我们开发了一种生物分子工具来追踪Fe对异养细菌的生物利用度。通过将大肠杆菌的fepA-fes启动子（受铁摄取调节[Fur]系统调节的肠杆菌素生物合成基因）融合到luxCDABE盒中而构建的生物发光报告基因，被整合到使用假毛皮的卤代假单胞菌的染色体中系统来调节高亲和力的铁吸收。合成的恶臭假单胞菌生物报告物已在实验室和现场研究中成功测试。实验室培养物的总铁浓度保持在0-25 nM的范围内，或通过添加特征明确的铁载体（去铁胺X [B]，亚铁铬，2,2'-联吡啶[DP]和红杜鹃酸[RA]，0-200 nM），并用于建立此报告系统的动态范围。仅在4小时后进行的样品孵育分析表明，三氢肟酸酯型铁载体DFB和亚铁铬铁有效降低了铁的利用率，相对于富铁条件，发光率分别提高了1.77和1.88倍。相反，添加二氢肟酸酯型铁载体RA和合成螯合剂DP不会导致系统反应，这表明细胞可以进入与这些化合物复合的铁，而无需激活高亲和力的铁转运系统。在伊利湖中部盆地进行了实地研究，此前已证明该地区在夏季分层过程中会受到零星的铁限制。将DFB浓度在0-50 nM的范围内滴定到未过滤的水中，以控制Fe的利用率。生物报告者在DFB浓度等于系统中总Fe（溶解+颗粒）（≈30 nM）时表示Fe胁迫（发光强度增加2倍），表明生物可利用的Fe浓度。在使用0.2μm预过滤水（2.25-5.24 nM Fe）的类似实验中，在最低（15 nM）螯合剂浓度下，观察到的发光增加了6倍（相对于对照）。这项研究的结果证明了生物报告剂作为测量总铁的补充工具的有效性。此外，这些结果表明，生物利用铁通量的重要来源可能是自然界中的颗粒，并支持以下假设：从微生物中回收铁可能在生物利用铁的再生中起关键作用。

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