Control of Competence in Vibrio fischeri
Control of Competence in Vibrio fischeri
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DOI：
10.1128/aem.01962-20
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发表时间：
2021-03-01
影响因子：
4.4
通讯作者：
Visick, Karen L.
中科院分区：
文献类型：
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作者：
Cohen, Joshua J.;Eichinger, Steven J.;Visick, Karen L.
弧菌物种，包括鱿鱼共生体费氏弧菌，在特定条件下能够吸收DNA。例如，当在几丁质寡糖存在下生长或在感受态调节因子Tfox过量产生时，费氏弧菌变得感受态。虽然对控制费氏弧菌能力的调控途径知之甚少，但这种微生物编码控制霍乱弧菌能力的因子的同源物。为了进一步发展费氏弧菌作为一种遗传上易处理的生物体，我们评估了其中一些感受态同源物的作用。使用Tfox过量生产细胞，我们发现感受态依赖于LitR，霍乱弧菌主群体感应和感受态调节因子HapR的同源物，以及霍乱弧菌中促进DNA摄取的推定菌毛基因的同源物。破坏LitR上游的负调控基因，即LuxO蛋白和小RNA（sRNA）Qrr 1，导致转化频率增加。然而，与LitR控制的光产生不同，在tfoX过表达条件下，感受态不随细胞密度而变化。与霍乱弧菌的情况类似，对LitR的需求可以通过Dns核酸酶的缺失而被抑制。我们还发现了一个假定的能力调节CytR的作用。最后，我们确定转化频率根据TfoX编码质粒而变化，并且我们开发了一种新的双tfoX和litR过表达构建体，其显著增加了遗传上不太易处理的菌株的转化频率。这些发现将有助于快速发现参与生理相关过程的基因，如生物膜形成和宿主定植。重要意义细菌摄取DNA（感受态）和将外源DNA整合到其基因组中（转化）的能力使它们能够快速进化并获得新的性状和/或获得抗生素抗性。它还促进了对特定表型机制的实验室研究，例如参与宿主定植的机制。费氏弧菌长期以来一直是共生细菌-宿主相互作用以及其生理学的其他方面（如生物发光和生物膜形成）的模型。在感受态因子TfoX过表达时，可以容易地诱导费氏弧菌的感受态。尽管已知在霍乱弧菌能力中重要的因子的同源物存在，但对费氏弧菌能力途径的了解相对较少。通过探索控制费氏弧菌转化的假定能力因子的重要性，这项工作加深了我们对能力过程的理解，并提高了我们对这一重要模式生物进行遗传操作的能力。
Vibrio species, including the squid symbiont Vibrio fischeri, become competent to take up DNA under specific conditions. For example, V. fischeri becomes competent when grown in the presence of chitin oligosaccharides or upon overproduction of the competence regulatory factor TfoX. While little is known about the regulatory pathway(s) that controls V. fischeri competence, this microbe encodes homologs of factors that control competence in the well-studied V. cholerae. To further develop V. fischeri as a genetically tractable organism, we evaluated the roles of some of these competence homologs. Using TfoX-overproducing cells, we found that competence depends upon LitR, the homolog of V. cholerae master quorumsensing and competence regulator HapR, and upon homologs of putative pilus genes that in V. cholerae facilitate DNA uptake. Disruption of genes for negative regulators upstream of LitR, namely, the LuxO protein and the small RNA (sRNA) Qrr1, resulted in increased transformation frequencies. Unlike LitR-controlled light production, however, competence did not vary with cell density under tfoX overexpression conditions. Analogous to the case with V. cholerae, the requirement for LitR could be suppressed by loss of the Dns nuclease. We also found a role for the putative competence regulator CytR. Finally, we determined that transformation frequencies varied depending on the TfoX-encoding plasmid, and we developed a new dual tfoX and litR overexpression construct that substantially increased the transformation frequency of a less genetically tractable strain. By advancing the ease of genetic manipulation of V. fischeri, these findings will facilitate the rapid discovery of genes involved in physiologically relevant processes, such as biofilm formation and host colonization.IMPORTANCE The ability of bacteria to take up DNA (competence) and incorporate foreign DNA into their genomes (transformation) permits them to rapidly evolve and gain new traits and/or acquire antibiotic resistances. It also facilitates laboratory-based investigations into mechanisms of specific phenotypes, such as those involved in host colonization. Vibrio fischeri has long been a model for symbiotic bacterium-host interactions as well as for other aspects of its physiology, such as bioluminescence and biofilm formation. Competence of V. fischeri can be readily induced upon overexpression of the competence factor TfoX. Relatively little is known about the V. fischeri competence pathway, although homologs of factors known to be important in V. cholerae competence exist. By probing the importance of putative competence factors that control transformation of V. fischeri, this work deepens our understanding of the competence process and advances our ability to genetically manipulate this important model organism.