Department of Bioengineering, University of Washington, Seattle, Washington, 98195.
Biotechnol Bioeng. 2013 Nov;110(11):2949-58. doi: 10.1002/bit.24953. Epub 2013 May 23.
Methods for the detection of plasmid loss in natural environments have typically relied on replica plating, selective markers and PCR. However, these traditional methods have the limitations of low sensitivity, underestimation of specific cell populations, and lack of insightful data for non-homogeneous environments. We have developed a non-invasive microscopic analytical method to quantify local plasmid segregational loss from a bacterial population within a developing biofilm. The probability of plasmid segregational loss in planktonic and biofilm cultures of Pseudomonas putida carrying the TOL plasmid (pWWO::gfpmut3b) was determined directly in situ, in the absence of any applied selection pressure. Compared to suspended liquid culture, we report that the biofilm mode of growth enhances plasmid segregational loss. Results based on a biofilm-averaged analysis reveal that the probability of plasmid loss in biofilm cultures (0.016 ± 0.004) was significantly greater than that determined in planktonic cultures (0.0052 ± 0.0011). Non-invasive assessments showed that probabilities of plasmid segregational loss at different locations in a biofilm increased dramatically from 0.1% at the substratum surface to 8% at outside layers of biofilm. Results suggest that higher nutrient concentrations and subsequentially higher growth rates resulted in higher probability of plasmid segregational loss at the outer layers of the biofilm.
方法检测质粒丢失在自然环境中通常依赖于复制平板、选择性标记和 PCR。然而，这些传统方法存在灵敏度低、低估特定细胞群体、以及缺乏对非均匀环境的深入数据等局限性。我们开发了一种非侵入性的微观分析方法，用于量化在形成生物膜的细菌群体中局部质粒分离丢失的概率。在不存在任何应用选择压力的情况下，直接原位确定携带 TOL 质粒 (pWWO::gfpmut3b) 的假单胞菌浮游生物和生物膜培养物中质粒分离丢失的概率。与悬浮液培养相比，我们报告说生物膜生长模式增强了质粒分离丢失。基于生物膜平均分析的结果表明，生物膜培养物中质粒丢失的概率（0.016 ± 0.004）明显大于浮游培养物中确定的概率（0.0052 ± 0.0011）。非侵入性评估表明，生物膜中不同位置的质粒分离丢失的概率从基质表面的 0.1%急剧增加到生物膜外层的 8%。结果表明，较高的营养浓度和随后较高的生长速率导致生物膜外层的质粒分离丢失的概率更高。
