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首页> 外文学位 >Development of a Reproducible Conjugative Gene Transfer System for Bifidobacteria and its Use for Investigating the Stress Response of Bifidobacterium longum DJO10A In Situ in a Yogurt Fermentation.
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Development of a Reproducible Conjugative Gene Transfer System for Bifidobacteria and its Use for Investigating the Stress Response of Bifidobacterium longum DJO10A In Situ in a Yogurt Fermentation.

机译：双歧杆菌可复制结合基因转移系统的开发及其在酸奶发酵中用于调查长双歧杆菌DJO10A应激反应的用途。

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摘要

Bifidobacteria are considered a beneficial inhabitant of the human gastrointestinal tract and are widely used as a probiotic in the food industry. However, our understanding of how these bacteria interact with the host and with the stresses encountered during food processing is limited. One of the main research limitations is the lack of molecular tools for these bacteria, particularly gene transfer techniques that currently depend on strainspecific electroporation protocols. Therefore, it was necessary to develop an efficient and reproducible gene transfer system for bifidobacteria. This was achieved by employing a conjugative-base DNA transfer system able to transfer a mobilizable vector from E. coli to bifidobacteria. The developed system was dependent on the donor (E. coli) to recipient (bifidobacteria) ratio, such that higher efficiencies were observed with higher donor ratios. In addition, the system was successful in all the bifidobacteria strains tested. This tool coupled with a genome-wide transcriptional analysis, RT-qPCR and the identification of ncRNAs, allowed the identification of molecular players during a yogurt fermentation with B. longum DJO10A. These molecular players included dnaK that appeared to play an important role at all times during growth, groEL that seemed particularly important for heat stress, and ibpA, a small heat-shock protein, together with the novel bifidobacteria-specific gene, bspA, that were added to the chaperone network as the acid accumulated in the fermentation. In addition, three ncRNAs, termed Bl20, Bl30, and Bl39, were shown to affect the stress response. The constitutive expression of Bl20 reduced the mRNA levels from all the tested stress genes, while the expression of dnaK was specifically targeted by Bl39 and ibpA expression was considerably reduced by the constitutive expression of Bl30. In conclusion, B. longum DJO10A responds to stress conditions during the yogurt fermentation by coordinating the expression of stress genes that are influenced by the expression of ncRNAs. Furthermore, this research revealed for the first time in bifidobacteria the involvement of ibpA in acid stress, the expression in response to stress of the novel gene bspA, the presence and expression of ncRNAs and a reproducible, efficient and strain-independent means of mobilizing DNA into this bacterial group.
机译：双歧杆菌被认为是人类胃肠道的有益菌，并且在食品工业中被广泛用作益生菌。但是，我们对这些细菌如何与宿主相互作用以及在食品加工过程中遇到的压力的理解是有限的。主要研究限制之一是缺乏用于这些细菌的分子工具，尤其是目前依赖于菌株特异性电穿孔方案的基因转移技术。因此，有必要为双歧杆菌开发有效且可再现的基因转移系统。这是通过使用共轭碱基的DNA转移系统实现的，该系统能够将可动员的载体从大肠杆菌转移到双歧杆菌。开发的系统取决于供体（大肠杆菌）与受体（双歧杆菌）的比率，因此，以较高的供体比率观察到更高的效率。此外，该系统在所有测试的双歧杆菌菌株中均成功。该工具与全基因组转录分析，RT-qPCR和ncRNA鉴定相结合，可以鉴定长双歧杆菌DJO10A在酸奶发酵过程中的分子分子。这些分子参与方包括dnaK似乎在生长过程中始终发挥着重要作用，groEL似乎对热应激特别重要，以及小小的热休克蛋白ibpA以及新颖的双歧杆菌特异性基因bspA都在其中。由于发酵中积累的酸而被添加到伴侣网络中。另外，显示了三个ncRNA，称为Bl20，Bl30和Bl39，它们影响应激反应。 Bl20的组成型表达降低了所有测试的应激基因的mRNA水平，而Bl39特异性靶向dnaK的表达，而Bl30的组成型表达大大降低了ibpA表达。总之，长双歧杆菌DJO10A通过协调受ncRNA表达影响的应激基因的表达来响应酸奶发酵过程中的应激条件。此外，这项研究首次揭示了双歧杆菌中ibpA参与酸胁迫，新基因bspA响应胁迫的表达，ncRNA的存在和表达以及动员DNA的可再现，有效且不依赖菌株的方法。进入这个细菌群。

著录项

  • 作者

    Dominguez, Wilfredo.;

  • 作者单位

    University of Minnesota.;

  • 授予单位 University of Minnesota.;
  • 学科 Biology Molecular.;Biology Microbiology.;Agriculture Food Science and Technology.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 272 p.
  • 总页数 272
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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