Non-invasive in situ monitoring and quantification of TOL plasmid segregational loss within Pseudomonas putida biofilms.
Non-invasive in situ monitoring and quantification of TOL plasmid segregational loss within Pseudomonas putida biofilms.
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DOI：
10.1002/bit.24953
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发表时间：
2013-11
影响因子：
3.8
通讯作者：
Bryers JD
中科院分区：
文献类型：
--
作者：
Ma H;Katzenmeyer KN;Bryers JD
检测自然环境中质粒丢失的方法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、低估特定细胞群以及缺乏对非均质环境有洞察力的数据等局限性。我们开发了一种非侵入性显微分析方法来量化正在发育的生物膜内细菌群体的局部质粒分离损失。在不施加任何选择压力的情况下，直接原位测定携带 TOL 质粒 (pWWO::gfpmut3b) 的恶臭假单胞菌的浮游和生物膜培养物中质粒分离损失的概率。与悬浮液体培养相比，我们报告生物膜生长模式增强了质粒分离损失。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) 显着大于浮游培养物中测定的质粒丢失概率 (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损失概率从基质表面的 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质粒分离损失的可能性较高。原位估计正在发育的生物膜中质粒分离损失。 A. 黄色荧光宿主细胞（带有 TOL 质粒 pWWO::gfpmut3b）和红色荧光分离子（恶臭假单胞菌 TUM-PP12 (miniTn5Putdsred)）的空间分布。在连续生物膜形成的第 4 天收集堆叠法师。箭头表示嵌入微菌落中的红色荧光白鹭。棒尺寸：20μm。 B. 携带质粒 () 和无质粒 () 细胞在不同生物膜深度的分布。
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. In situ estimation of plasmid segregational loss in a developing biofilm. A. Spatial distribution of yellow luorescent host cells (P. putida TUM-PP12 (mini-Tn5Putdsred) harboring the TOL plasmid pWWO::gfpmut3b)) and red fluorescent segregants (P. putida TUM-PP12 (miniTn5Putdsred)). The stack mage was collected on Day 4 of continuous biofilm formation. Arrows indicate the red fluorescent egregants embedded in microcolonies. Bar size: 20 μm. B. Distribution of both plasmid-bearing () and lasmid-free () cells at the different biofilm depths.
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影响因子：
3.8
作者：
HUANG, CT;PERETTI, SW;BRYERS, JD
通讯作者：
BRYERS, JD
影响因子：
5
作者：
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