Heterogeneity in flagella-mediated motility

许多细菌通过鞭毛运动，这提供了一种有效但代谢昂贵的运动，粘附和环境感知手段。许多物种，其中包括希瓦氏菌属的几个，具有两个不同的鞭毛系统。人们对这种二次系统是如何管理的知之甚少。在以前的研究中，我们已经证明了S。腐败菌CN-32具有一个功能齐全的次级系统，在诱导时，除了初级极性外，还形成额外的侧丝。在复杂的无鞭毛培养物中，群体就鞭毛表达而言分成几个亚群：不表达任何鞭毛的细胞，表达两个鞭毛系统之一的细胞，以及产生两个鞭毛系统的细胞。这意味着CN-32在运动性方面采用了赌注对冲策略。在上一个资助期内，我们已讨论了中学教育制度的表现形式所带来的好处。我们可以证明，具有一个极丝和一个侧丝的细胞亚群比仅具有单个鞭毛系统的细胞亚群在铺展方面显著更有效。我们证明了次级细丝的存在导致平均转向角的减小，从而增加了游泳的方向持久性。使用实验获得的轨迹参数的互补建模方法证实了更有效的传播。因此，我们发现了一种全新的提高细菌有效游动能力的方法，在后续研究中，我们将进一步探讨次级系统发挥作用的机制。为此，我们将使用荧光标记的鞭毛丝，使游泳细胞的活成像。为了完善和验证理论模型，我们还将研究在趋化梯度中游泳。为了进一步表征次级系统的潜在有益作用，我们将确定该系统在细胞-表面-和细胞-细胞-粘附中的作用。在一个互补的方法，我们将探讨二级鞭毛系统的调节和异质性是如何建立的。为此，我们已经构建了荧光和发光报告系统。通过流式细胞术分析，我们将确定环境信号后群体异质性的变化。此外，我们将专门筛选二次系统的潜在调节因素。为此，我们将直接筛选潜在的DNA结合蛋白，并采用两种不同的转座子诱变筛选。最后，我们将次级鞭毛系统转移到密切相关的物种S。oneidensis MR-1来确定希瓦氏菌之间是否保持功能和调节。这两种方法有望从根本上揭示鞭毛功能和调节的新方面。

Many bacteria are motile by means of flagella, which provide an effective but metabolically expensive means of locomotion, adhesion, and environmental sensing. Numerous species, among which are several of the genus Shewanella, possess two distinct flagellar systems. Little is known about how such secondary systems are regulated. In previous studies we have demonstrated that S. putrefaciens CN-32 possesses a fully functional secondary system which, upon induction, results in formation of additional lateral filaments in addition to a primary polar one. In complex planktonic cultures, the population splits into several subpopulations with respect to flagellar expression: cells that do not express any flagella, cells that express one of the two flagellar system, and cells in which both flagellar systems are produced. This implicates that CN-32 employs a bet-hedging strategy with respect to motility. In the previous funding period, we have addressed the question of the benefit the expression of the secondary system entails. We could show that the subpopulation of cell with both one polar and one lateral filament was significantly more efficient in spreading than their counterparts with a single flagellar system only. We demonstrated that the presence of the secondary filament leads to a decrease in the average turning angle, thereby increasing the directional persistence of swimming. A complementary modeling approach using the trajectory parameters obtained experimentally confirmed more efficient spreading. We have therefore found a fundamentally novel means of improving effective swimming used by bacteria.In the follow-up studies proposed here we will further address the mechanism by which the secondary system exerts its function. To this end, we will use fluorescently labeled flagellar filaments to enable live imaging of swimming cells. To refine and validate the theoretical model, we will also study swimming in chemotactic gradients. To further characterize potential benficial role of the secondary system, we will determine the role of the system in cell-surface- and cell-cell-adhesion. In a complementary approach we will explore the regulation of the secondary flagellar system and how heterogeneity is established. To this end, we have already constructed fluorescence and luminescence reporter systems. By flow cytometry analysis we will determine shifts in the population heterogeneity upon environmental signals. In addition, we will specifically screen for potential regulating factors of the secondary system. For this, we will directly screen for potential DNA-binding proteins and employ two different transposon mutagenesis screens. Finally, we will transfer the secondary flagellar system into the closely related species S. oneidensis MR-1 to determine whether function and regulation are maintained among Shewanella sp. Both approaches are expected to reveal fundamentally novel aspects of flagella function und regulation.