Breuer Rebecca J, Bandyopadhyay Arpan, O'Brien Sofie A, Barnes Aaron M T, Hunter Ryan C, Hu Wei-Shou, Dunny Gary M
Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, United States of America.
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, United States of America.
PLoS Genet. 2017 Jul 3;13(7):e1006878. doi: 10.1371/journal.pgen.1006878. eCollection 2017 Jul.
In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.
在粪肠球菌中，性信息素介导的抗生素抗性质粒转移可在不利条件下发生，例如，当诱导性信息素浓度低而抑制性信息素浓度高时。为了更好地理解这一矛盾现象，我们采用荧光原位杂交链式反应（HCR）方法在单细胞水平上同时定量多种粪肠球菌转录本。我们提供了直接证据，证明单个细胞对特定诱导条件的最短周期、最大反应水平和反应持续时间存在变异性。使用荧光报告基因对单个细胞的诱导模式进行时间跟踪支持了HCR的研究结果。它还揭示了快速反应者的亚群，即使在低诱导性信息素浓度下，整个群体的总体反应较慢时也是如此。在低信息素浓度下培养的细胞中少数细胞的强烈、快速诱导与肠球菌信息素反应随机模型的预测一致。先前记录的控制信息素反应的复杂调节回路可能导致该系统中的随机变化。除了增加我们对由细胞间信号传导调节的水平基因转移系统生物学的基本理解外，信息素反应随机性的证明也会影响未来开发针对该系统的治疗药物的任何努力。使用HCR进行定量单细胞分析也有很大潜力阐明以前无法在单细胞水平上进行研究的重要细菌调节机制，并加快功能基因组学研究的步伐。
