Salvadori Gabriela, Junges Roger, Morrison Donald A, Petersen Fernanda C
Department of Oral Biology, Faculty of Dentistry, University of Oslo Oslo, Norway.
Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois at Chicago Chicago, IL, USA.
Front Microbiol. 2016 Jul 5;7:1009. doi: 10.3389/fmicb.2016.01009. eCollection 2016.
Streptococcus mitis is a predominant oral colonizer, but difficulties in genetic manipulation of this species have hampered our understanding of the mechanisms it uses for colonization of oral surfaces. The aim of this study was to reveal optimal conditions for natural genetic transformation in S. mitis and illustrate its application in direct genome editing.
Luciferase reporter assays were used to assess gene expression of the alternative sigma factor (σ(X)) in combination with natural transformation experiments to evaluate the efficiency by which S. mitis activates the competence system and incorporates exogenous DNA. Optimal amounts and sources of donor DNA (chromosomal, amplicon, or replicative plasmid), concentrations of synthetic competence-stimulating peptide, and transformation media were assessed.
A semi-defined medium showed much improved results for response to the competence stimulating peptide when compared to rich media. The use of a donor amplicon with large homology flanking regions also provided higher transformation rates. Overall, an increase of transformation efficiencies from 0.001% or less to over 30% was achieved with the developed protocol. We further describe the construction of a markerless mutant based on this high efficiency strategy.
We optimized competence development in S. mitis, by use of semi-defined medium and appropriate concentrations of synthetic competence factor. Combined with the use of a large amplicon of donor DNA, this method allowed easy and direct editing of the S. mitis genome, broadening the spectrum of possible downstream applications of natural transformation in this species.
缓症链球菌是口腔中的主要定植菌，但该菌种基因操作的困难阻碍了我们对其口腔表面定植机制的理解。本研究的目的是揭示缓症链球菌自然遗传转化的最佳条件，并说明其在直接基因组编辑中的应用。
使用荧光素酶报告基因检测来评估替代sigma因子（σ(X)）的基因表达，并结合自然转化实验来评估缓症链球菌激活感受态系统并整合外源DNA的效率。评估了供体DNA（染色体、扩增子或复制质粒）的最佳量和来源、合成感受态刺激肽的浓度以及转化培养基。
与丰富培养基相比，半限定培养基对感受态刺激肽的反应结果有显著改善。使用具有大同源侧翼区域的供体扩增子也能提供更高的转化率。总体而言，通过所开发的方案，转化效率从0.001%或更低提高到了30%以上。我们进一步描述了基于这种高效策略构建无标记突变体的方法。
我们通过使用半限定培养基和适当浓度的合成感受态因子优化了缓症链球菌的感受态发育。结合使用大的供体DNA扩增子，该方法允许对缓症链球菌基因组进行简单直接的编辑，拓宽了该菌种自然转化可能的下游应用范围。
