Pellicle formation of MR-1. Shewanella oneidensis MR-1

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. In recent years, biofilm development of S. oneidensis has been extensively studied because it is essential to reduce solid metals. As a special form of biofilm, however, pellicles are largely overlooked. The goal of this work was to understand requirements of S. oneidensis pellicle formation and the molecular basis of pellicle formation. We demonstrated that successful pellicle formation and survival was likely to require the threshold level of cell density and higher concentration of oxygen. Proteinase K and EDTA were potent pellicle disrupter. DNA microarray experiments were used to study the gene expression profile of young air–liquid interface pellicle relative to planktonic cells, which indicated that the air–liquid interface pellicle was more metabolically active than the planktonic cells. Most notably, consistently up-regulation of iron or heme uptake and transportation proteins was observed in the S. oneidensis MR-1 pellicle. However, neither the hmuT nor hugA heme transport mutant was defective in pellicle formation. An examination of the influence of several metal cations on the anti-pellicle activity of EDTA showed that Ca (II), Mn(II), Cu(II), and Zn(II) fully protected S. oneidensis MR-1 pellicle against EDTA treatment while additional of iron enabled the initiation of pellicle formation but maturation was significantly impaired. Collectively, iron was less important than other metals with respect to pellicle formation in S. oneidensis. Overall design: A fresh colony grown overnight on a LB plate was used to inoculate 50 ml LB and incubated in a shaker (200 rpm) to an OD600 of 0.8 at the room temperature. This culture was then diluted 500-fold with fresh LB, resulting in the starting cultures. Aliquots of 30ml starting cultures were transferred to 50-ml Pyrex beakers and allowed to develop pellicles at the room temperature. When a complete but thin (young pellicle) at the interface were formed (about 30h hours), planktonic culture and pellicle were separated and applied to centrifugation at 8000 rpm for 3 min at room temperature. 3 parallel starting cultures were used and 3 samlpes of pellicle cells or planktonic cells were collected at 30h. RNA from the pellicle cells was fluorescently labeled with Cy3, and that from the planktonic was labeled with Cy5.

奥奈达希瓦氏菌（Shewanella oneidensis）因具备多样的呼吸能力，是生物修复研究中的重要模式生物。近年来，由于其对固态金属的还原不可或缺，奥奈达希瓦氏菌的生物被膜（biofilm）发育得到了广泛研究。然而，作为生物被膜的一种特殊形式，菌膜（pellicle）在很大程度上被忽视了。本研究的目标是阐明奥奈达希瓦氏菌菌膜形成的必要条件，以及菌膜形成的分子基础。我们证实，成功形成菌膜并存活可能需要达到阈值的细胞密度以及较高的氧气浓度。蛋白酶K（Proteinase K）与乙二胺四乙酸（EDTA）均为有效的菌膜破坏剂。本研究采用基因芯片（DNA microarray）技术，对比分析了气液界面幼龄菌膜与浮游态细胞的基因表达谱，结果显示气液界面菌膜的代谢活性高于浮游态细胞。最为显著的是，在奥奈达希瓦氏菌MR-1菌株的菌膜中，铁或血红素的摄取与转运蛋白呈现持续上调表达。然而，hmuT与hugA血红素转运突变株的菌膜形成均未出现缺陷。对多种金属阳离子在EDTA抗菌膜活性中的影响进行检测后发现，Ca(II)、Mn(II)、Cu(II)及Zn(II)可完全保护奥奈达希瓦氏菌MR-1菌株的菌膜免受EDTA处理的破坏；而添加铁虽可启动菌膜形成，但会显著损害其成熟过程。综上，相较于其他金属，铁在奥奈达希瓦氏菌菌膜形成过程中的重要性相对较低。总体实验设计：将在LB平板上过夜培养的新鲜菌落接种至50 mL LB培养基中，于室温下以200 rpm摇床培养至OD600为0.8。随后将该培养液以500倍稀释于新鲜LB培养基中，获得初始培养液。取30 mL初始培养液转移至50 mL Pyrex烧杯中，于室温下培养以形成菌膜。当气液界面形成完整但较薄的幼龄菌膜（约30小时）后，将浮游态培养液与菌膜分离，并于室温下以8000 rpm离心3分钟进行收集。本研究设置3组平行初始培养液，于30小时时分别收集3份菌膜细胞样本与3份浮游态细胞样本。将菌膜细胞的RNA用Cy3进行荧光标记，浮游态细胞的RNA则用Cy5进行荧光标记。