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Characterization of Heterogeneity and Dynamics of Lysis of Single Bacillus subtilis Cells upon Prophage Induction During Spore Germination, Outgrowth, and Vegetative Growth Using Raman Tweezers and Live-Cell Phase-Contrast Microscopy
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-12-28 , DOI: 10.1021/acs.analchem.0c03341 Mei-yan Wu 1 , Wen-wei Li , Graham Christie 2 , Peter Setlow 3 , Yong-qing Li 1, 4
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-12-28 , DOI: 10.1021/acs.analchem.0c03341 Mei-yan Wu 1 , Wen-wei Li , Graham Christie 2 , Peter Setlow 3 , Yong-qing Li 1, 4
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A prophage comprises a bacteriophage genome that has integrated into a host bacterium’s DNA, which generally permits the cell to grow and divide normally. However, the prophage can be induced by various stresses, or induction can occur spontaneously. After prophage induction, viral replication and production of endolysins begin until the cell lyses and phage particles are released. However, the heterogeneity of prophage induction and lysis of individual cells in a population and the dynamics of a cell undergoing lysis by prophage induction have not been fully characterized. Here, we used Raman tweezers and live-cell phase-contrast microscopy to characterize the Raman spectral and cell length changes that occur during the lysis of individual Bacillus subtilis cells from spores that carry PBSX prophage during spores’ germination, outgrowth, and then vegetative growth. Major findings of this work are as follows: (i) After addition of xylose to trigger prophage induction, the intensities of Raman spectral bands associated with nucleic acids of single cells in induced cultures gradually fell to zero, in contrast to the much smaller changes in protein band intensities and no changes in nucleic acid bands in uninduced cultures; (ii) the nucleic acid band intensities from an individual induced cell exhibited a rapid decrease, following a long lag period; (iii) after the addition of nutrient-rich medium with xylose, single spores underwent a long period (228 ± 41.4 min) for germination, outgrowth, and vegetative growth, followed by a short period of cell burst in 1.5 ± 0.8 min at a cell length of 8.2 ± 5.5 μm; (iv) the latent time (Tlatent) between the addition of xylose and the start of cell burst was heterogeneous in cell populations; however, the period (ΔTburst) from the latent time to the completion of cell lysis was quite small; (v) in a poor medium with l-alanine alone, addition of xylose caused prophage induction following spore germination but with longer Tlatent and ΔTburst times and without cell elongation; (vi) spontaneous prophage induction and lysis of individual cells from spores in a minimal nutrient medium were observed without xylose addition, and cell length prior to cell lysis was ∼4.1 μm, but spontaneous prophage induction was not observed in a rich medium; (vii) in a rich medium, addition of xylose at a time well after spore germination and outgrowth significantly shortened the average Tlatent time. The results of this study provide new insights into the heterogeneity and dynamics of lysis of individual B. subtilis cells derived from spores upon prophage induction.
中文翻译：
使用拉曼镊子和活细胞相差显微镜在孢子萌发，生长和营养生长期间，通过噬菌体诱导的单个枯草芽孢杆菌细胞的异质性和溶解动力学
噬菌体包含噬菌体基因组，该噬菌体基因组已整合到宿主细菌的DNA中，通常可使细胞正常生长和分裂。但是，可以通过各种压力来诱发预言，或者可以自发发生诱发。在前体诱导之后，病毒的复制和内溶素的产生开始，直到细胞裂解并释放噬菌体颗粒。然而，尚未充分表征群体中单个细胞的前噬诱导和裂解的异质性以及通过前噬诱导进行裂解的细胞的动力学。在这里，我们使用了拉曼镊子和活细胞相差显微镜来表征单个枯草芽孢杆菌裂解过程中发生的拉曼光谱和细胞长度变化来自孢子的细胞，在孢子萌发，生长和营养生长期间携带PBSX噬菌体。这项工作的主要发现如下：（i）在添加木糖以触发前噬菌体诱导之后，与诱导培养物中单个细胞的核酸相关的拉曼光谱带的强度逐渐降至零，与之相比，变化很小。蛋白带强度，未诱导培养物中核酸带无变化；（ii）在长时间的滞后之后，来自单个诱导细胞的核酸条带强度迅速下降；（iii）在添加了富含木糖的营养丰富的培养基后，单个孢子经历了较长时间（228±41.4分钟）的萌发，生长和营养生长，然后在1.5±0.8分钟内短时间细胞破裂。单元长度为8.2±5.5μm;Ť潜）加入木糖和细胞突发的开始之间在细胞群异质; 然而，在期间（Δ Ť突发）从潜时间细胞裂解的完成是相当小; （v）在仅含1-丙氨酸的贫乏培养基中，添加木糖会引起孢子萌发后诱发腐肉，但T潜伏期较长且ΔT爆发次且无细胞伸长；（vi）在没有添加木糖的条件下，观察到自发的原噬菌体诱导和裂解的单个细胞在没有添加木糖的情况下发生，细胞裂解前的细胞长度为〜4.1μm，但在丰富的培养基中未观察到自发的原噬菌体诱导; （vii）在丰富的培养基中，在孢子萌发和长出后很好的时间添加木糖显着缩短了平均T潜伏时间。这项研究的结果提供了新的见解，对诱导腐肉后衍生自孢子的单个枯草芽孢杆菌细胞裂解的异质性和动力学。
更新日期：2021-01-26
中文翻译：
使用拉曼镊子和活细胞相差显微镜在孢子萌发，生长和营养生长期间，通过噬菌体诱导的单个枯草芽孢杆菌细胞的异质性和溶解动力学
噬菌体包含噬菌体基因组，该噬菌体基因组已整合到宿主细菌的DNA中，通常可使细胞正常生长和分裂。但是，可以通过各种压力来诱发预言，或者可以自发发生诱发。在前体诱导之后，病毒的复制和内溶素的产生开始，直到细胞裂解并释放噬菌体颗粒。然而，尚未充分表征群体中单个细胞的前噬诱导和裂解的异质性以及通过前噬诱导进行裂解的细胞的动力学。在这里，我们使用了拉曼镊子和活细胞相差显微镜来表征单个枯草芽孢杆菌裂解过程中发生的拉曼光谱和细胞长度变化来自孢子的细胞，在孢子萌发，生长和营养生长期间携带PBSX噬菌体。这项工作的主要发现如下：（i）在添加木糖以触发前噬菌体诱导之后，与诱导培养物中单个细胞的核酸相关的拉曼光谱带的强度逐渐降至零，与之相比，变化很小。蛋白带强度，未诱导培养物中核酸带无变化；（ii）在长时间的滞后之后，来自单个诱导细胞的核酸条带强度迅速下降；（iii）在添加了富含木糖的营养丰富的培养基后，单个孢子经历了较长时间（228±41.4分钟）的萌发，生长和营养生长，然后在1.5±0.8分钟内短时间细胞破裂。单元长度为8.2±5.5μm;Ť潜）加入木糖和细胞突发的开始之间在细胞群异质; 然而，在期间（Δ Ť突发）从潜时间细胞裂解的完成是相当小; （v）在仅含1-丙氨酸的贫乏培养基中，添加木糖会引起孢子萌发后诱发腐肉，但T潜伏期较长且ΔT爆发次且无细胞伸长；（vi）在没有添加木糖的条件下，观察到自发的原噬菌体诱导和裂解的单个细胞在没有添加木糖的情况下发生，细胞裂解前的细胞长度为〜4.1μm，但在丰富的培养基中未观察到自发的原噬菌体诱导; （vii）在丰富的培养基中，在孢子萌发和长出后很好的时间添加木糖显着缩短了平均T潜伏时间。这项研究的结果提供了新的见解，对诱导腐肉后衍生自孢子的单个枯草芽孢杆菌细胞裂解的异质性和动力学。
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