Department of Physics, East Carolina University, Greenville, North Carolina 27858-4353, United States.
Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB3 0AS, U.K.
Anal Chem. 2021 Jan 26;93(3):1443-1450. doi: 10.1021/acs.analchem.0c03341. Epub 2020 Dec 28.
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 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 () between the addition of xylose and the start of cell burst was heterogeneous in cell populations; however, the period (Δ) 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 and Δ 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 time. The results of this study provide new insights into the heterogeneity and dynamics of lysis of individual cells derived from spores upon prophage induction.
一个原噬菌体包含整合到宿主细菌 DNA 中的噬菌体基因组，这通常允许细胞正常生长和分裂。然而，原噬菌体可以被各种应激诱导，或者诱导可以自发发生。原噬菌体诱导后，病毒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，原噬菌体诱导的个别细胞的裂解的异质性以及正在经历原噬菌体诱导的细胞的裂解动力学尚未得到充分描述。在这里，我们使用拉曼镊子和活细胞相差显微镜来描述单个细胞在携带 PBSX 原噬菌体的孢子的萌发、生长和随后的营养生长过程中裂解时发生的拉曼光谱和细胞长度变化。这项工作的主要发现如下：（i）加入木糖触发原噬菌体诱导后，诱导培养物中单细胞的核酸相关拉曼光谱带的强度逐渐降至零，而未诱导培养物中蛋白质带强度的变化要小得多，核酸带没有变化；（ii）单个诱导细胞的核酸带强度表现出快速下降，随后是一个很长的滞后期；（iii）加入富含木糖的营养培养基后，单个孢子经历了一个很长的时期（228 ± 41.4 min）进行萌发、生长和营养生长，然后在细胞长度为 8.2 ± 5.5 μm 时，在 1.5 ± 0.8 min 内经历一个短的细胞爆裂期；（iv）细胞群体中木糖添加和细胞爆裂开始之间的潜伏时间（）是异质的；然而，从潜伏时间到细胞裂解完成的时间（Δ）非常小；（v）在仅含有 L-丙氨酸的较差培养基中，加入木糖会导致原噬菌体诱导，随后是孢子的萌发，但潜伏期（）和Δ时间更长，且没有细胞伸长；（vi）在最低营养培养基中，无需添加木糖，即可观察到单个孢子的原噬菌体自发诱导和裂解，细胞裂解前的细胞长度约为 4.1 μm，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，在孢子萌发和生长后很久的时间添加木糖会显著缩短平均潜伏期（）时间。这项研究的结果为原噬菌体诱导时单个孢子衍生细胞裂解的异质性和动力学提供了新的见解。
