Induction of Natural Competence in Genetically-modified Lactococcus lactis
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Author:
Date:
2018-07-05
[Abstract] Natural competence can be activated in Lactoccocus lactis subsp lactis and cremoris upon overexpression of ComX, a master regulator of bacterial competence. Herein, we demonstrate a method to activate bacterial competence by regulating the expression of the comX gene by using a nisin-inducible promoter in an L. lactis strain harboring either a chromosomal or plasmid-encoded copy of nisRK. Addition of moderate concentrations of the inducer nisin resulted in concomitant moderate levels of ComX, which led to an optimal transformation rate (1.0 x 10-6 transformants/total cell number/g plasmid DNA). Here, a detailed description of the optimized protocol for competence induction is presented.
[摘要] 在过度表达细菌能力的主要调节因子ComX后,天然能力可以在乳酸乳球菌亚种乳酸和 cremoris 中激活。 在本文中,我们展示了通过在 L中使用乳链菌肽诱导型启动子调节 comX 基因的表达来激活细菌能力的方法。 含有 nisRK 的染色体或质粒编码拷贝的lactis 菌株。 加入中等浓度的诱导剂乳链菌肽导致伴随的中等水平的ComX,其导致最佳转化率(1.0×10 2 sup / -6>转化子/总细胞数/ g质粒DNA)。 在此,提出了用于能力归纳的优化协议的详细描述。
【背景】自然能力是细菌通过专门的摄取机制获得外源DNA的过程,之后内化的DNA整合到其基因组中或作为质粒DNA维持。一些细菌在特定的环境触发因素如基因毒性应激或饥饿时进入能力状态(Seitz和Blokesch,2013; Blokesch,2016)。群体感应系统,如 comCDE 或 comRS ,控制着革兰氏阳性菌的自然能力的激活(Håvarstein et al。,1995; Pestova et al。,1996; Kleerebezem et al。,1997b; Fontaine et al。,2015)。更具体地说, comC 和 comS 编码信息素,而 comD 编码组氨酸激酶和 comE 和 comR 编码响应调节器(Håvarstein et al。,1995; ...
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Bacterial Competition Assay Based on Extracellular D-amino Acid Production
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Author:
Date:
2018-04-05
[Abstract] Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in fitness/survival of bacterial subpopulations within a community. Co-cultivation methods usually involve the growth of the competing bacteria in the same container. Therefore, within such mixed cultures the effect on growth caused by extracellular metabolites cannot be distinguished from direct physical interactions between species (e.g., T6SS effectors). However, this problem can be easily solved by using a filtration unit that allows free ...
[摘要] 在激烈的竞争中,细菌生活在多种微生物群落中。为了坚持特定的生态位,许多物种会产生有毒的细胞外效应物作为干扰附近微生物生长的策略。这种效应子之一是非规范的D-氨基酸。在这里我们描述一种方法来测试D-氨基酸生产对社区内细菌亚群的适应/存活的影响。共培养方法通常涉及相同容器中竞争细菌的生长。因此,在这种混合培养物中,细胞外代谢物对生长的影响不能与物种间的直接物理相互作用区分开(例如T6SS效应物)。然而,通过使用允许小分解代谢物(例如L-和D-氨基酸)自由扩散的过滤单元可以容易地解决这个问题,同时将不同亚群保持在独立区室中。
通过这种方法,我们已经证明D-精氨酸是由霍乱弧菌产生的杀菌剂效应物,其强烈影响不同微生物亚群的存活。此外,D-精氨酸可作为混合菌群中的一种协同工具,用于保护非生产成员免受竞争细菌的侵害。
【背景】细菌生活在多种多样的物种共存并争夺现有资源的多种微生物群落中。细菌设计为在特定生态位持续存在的许多策略之一是产生有毒的细胞外代谢物作为干扰其他微生物生长和/或生存力的策略。已知D-氨基酸长时间在细菌培养物中具有细胞形状和活力的强大作用(Bopp,1965; Fox等人,1944; Kobayashi等人, 1948年; Yaw和Kakavas,1952年; Lark和Lark,1959年; Grula,1960年; ...
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A Bioassay Protocol for Quorum Sensing Studies Using Vibrio campbellii
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Author:
Date:
2016-07-20
[Abstract] Quorum Sensing (QS), or bacterial cell-to-cell communication, is a finely-tuned mechanism that regulates gene expression on a population density-dependent manner through the production, secretion and reception of extracellular signaling molecules termed autoinducers (AIs). Given that QS plays an important role in bacterial biofilm formation and virulence factor production in many pathogenic strains, QS disruptors have become a hot topic in current antimicrobial research. There are several reporter strains exhibiting QS-regulated phenotypes that have been engineered for the identification of QS inhibitors, including, for example, pigment production (González and Keshavan, 2006; Steindler and Venturi, 2007), gfp, lacZ or lux reporter gene fusions (González and ...
[摘要] Quorum Sensing(QS)或细菌细胞与细胞间的通信是一种微调的机制,通过称为自体诱导物(AI)的细胞外信号分子的产生,分泌和接受,以群体密度依赖性方式调节基因表达。鉴于QS在许多致病菌株中的细菌生物膜形成和毒力因子产生中起重要作用,QS干扰物已经成为当前抗微生物研究的热门话题。存在几种报道菌株,其表现出QS-调节的表型,其已被工程化用于鉴定QS抑制剂,包括例如颜料产生(González和Keshavan,2006; Steindler和Venturi,2007),gfp < (gonzález和keshavan,2006;="" steindler和venturi,2007)或致死性基因融合下游qs控制的启动子(weiland-bräuer=""> et al 。,2015)。使用三个平行的QS电路,生物发光海洋细菌(Vibrio campbellii)(以前称为harveyi,Lin等人,2010)构成复杂的革兰氏阴性模型,存在广泛的知识,包括一系列突变生物传感器。在中。 campbellii ...
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