| Analysis of Pseudomonas aeruginosa c-di-GMP High and Low Subpopulations Using Flow-assisted Cell Sorting (FACS) and Quantitative Reverse Transcriptase PCR (qRT-PCR)
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Author:
Date:
2021-01-20
[Abstract] Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger signaling molecule that drives the transition from planktonic to the biofilm mode of growth in many bacterial species. Pseudomonas aeruginosa has at least two surface sensing systems that produce c-di-GMP in response to surface attachment, the Wsp and Pil-Chp systems. We recently used a plasmid-based c-di-GMP reporter (pPcdrA::gfp) to describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells during early biofilm formation. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the ...
[摘要] [摘要]环状双鸟苷酸单磷酸酯（c-di-GMP）是第二个信使信号分子，它驱动许多细菌物种从浮游生物向生物膜生长的转变。铜绿假单胞菌具有至少两个响应于表面附着而产生c-di-GMP的表面传感系统，即Wsp和Pil-Chp系统。我们最近使用了基于质粒的c-di-GMP报告基因（pP cdrA :: gfp ）描述Wsp系统如何在表面感应中产生异质性，从而在早期生物膜形成过程中导致两个生理上不同的细胞亚群。一个亚群的c-di-GMP升高并产生生物膜基质，从而成为最初的微殖民地的奠基人。另一个亚群的c-di-GMP较低，并且具有表面运动性，可以探索表面。在这里，我们描述了一项关键实验的协议，以确认我们在表面传感过程中对c-di-GMP异质性的初步观察：使用流辅助细胞分选（FACS）来分离具有高和低c- di- G的细胞亚群GMP报告基因活性，然后是已知响应于细胞c-di-GMP水平（pelA和pslA ）被转录调控的基因的定量逆转录酶PCR（qRT-PCR ）。该方案可以被其他人修改以分离高c-di-GMP铜绿假单胞菌细胞的亚群，这些亚群在基因上是相同的，但在表型上是不同的，以便将来像我们一样检查特定的mRNA转录本，或者大概用于其他应用如RNAseq，蛋白质组学或TNseq。
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[背景]第二信使信号分子环状单鸟苷酸单磷酸酯（c-di-GMP）可使细菌响应环境条件而迅速修饰其细胞表面。双鸟苷酸环化酶（DGC）是含有从两个GTP分子合成c-di-GMP的GGDEF氨基酸序列基序的蛋白质，磷酸二酯酶（PDE）是具有EAL或HD-GYP基序的蛋白，可将c-di-GMP水解为线性pGpG或GMP ...
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| Rapid Isolation and Purification of Secreted Bacteriocins from Streptococcus mutans and Other Lactic Acid Bacteria
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Author:
Date:
2020-11-20
[Abstract] Bacteriocins are small ribosomally synthesized antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB), a group of Gram-positive bacteria (cocci, rods) expressing high tolerance for low pH. Bacteriocins kill bacteria rapidly and are biologically active at very low concentrations. Bacteriocins produced by LAB are primarily active against closely related bacterial species. Many bacteriocins have been investigated with respect to their potential use in promoting human, plant, and animal health, and as food biopreservatives. Bacteriocins produced by LAB are particularly interesting since several LAB have been granted GRAS (Generally Recognized as Safe) status. Because it is not always possible to extract active bacteriocins secreted from cells grown in ...
[摘要] [摘要]细菌素是由一些核糖体合成的抗菌肽微生物，包括乳酸菌（LAB），一组革兰氏阳性菌（球菌，棒）表现出对低pH的高耐受性。细菌素可迅速杀死细菌，并在极高的温度下具有生物活性。低浓度。LAB生产的细菌素主要对紧密相关的细菌具有活性种类。已经研究了许多细菌素在促进细菌生长方面的潜在用途。对人类，植物和动物健康，以及作为食品生物防腐剂。LAB生产的细菌素是特别有趣的是，由于一些实验室已获得GRAS（通常被认为是安全的）状态。因为并非总是可能提取液体中生长的细胞分泌的活性细菌素介质，我们开发了一种使用半固体的简单而廉价的肽提取程序营养丰富的琼脂培养基。我们在此提出一种详细的程序，以快速提取出从口腔物种分泌的生物活性细菌素肽变异链球菌，多产的菌种的生产及其在其他实验室提取细菌素的潜在应用（例如，链球菌，乳球菌，肠球菌）。我们还提出了一种简单的检测方法纯化的细胞外肽提取物的细菌素活性测定
[背景]自然界中的大多数细菌并不是独立存在，而是存在于复杂的多物种中生物膜群落（López等，2010）。两者之间存在大量的身体和营养相互作用细菌有助于生物膜的生长和存活。细菌素的产生和分泌细胞外空间为生产者与其他竞争者赋予了独特的生态优势存在于同一生态位中的细菌（Donia and ...
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