Han Yiping W, Ikegami Akihiko, Chung Peter, Zhang Lei, Deng Cheri X
Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44106-4905, USA.
Appl Environ Microbiol. 2007 Jun;73(11):3677-83. doi: 10.1128/AEM.00428-07. Epub 2007 Apr 20.
Studies of microorganisms are often hindered by a lack of effective genetic tools. One such example is Fusobacterium nucleatum, a gram-negative anaerobe associated with various human infections, including those causing periodontal disease and preterm birth. The first double-crossover allelic-exchange mutant in F. nucleatum was recently constructed using sonoporation, a novel ultrasound-mediated intracellular delivery method, demonstrating potential for bacterial gene transfection. To better unveil its mechanism, the current study examines the factors affecting the outcome of sonoporation. Delivery of Texas Red-conjugated dextran into F. nucleatum by sonoporation was at least twice as efficient as that by electroporation, and sonoporation was nonbactericidal, unlike electroporation. The delivery efficiency was affected by the acoustic pressure amplitude, the duty cycle, and the quantity of microbubbles used to initiate cavitation but not by the pulse repetition frequency of ultrasound application. To examine the involvement of homologous recombination in sonoporation-mediated mutant construction, the highly conserved recA gene, which carried most of the consensus residues, including the P loop, was identified in F. nucleatum, and a double-crossover recA mutant of F. nucleatum 12230, US1610, was constructed by sonoporation. The mutant exhibited increased sensitivity to UV exposure compared with that of the wild type, indicating that the RecA function in F. nucleatum was conserved. Interestingly, US1610 was also sensitive to ultrasound treatment, suggesting the likely involvement of RecA in postsonoporation repair and survival. Since sonoporation has consistently generated one-step double-crossover mutants in F. nucleatum by use of intact suicide plasmids, this technology may be developed into an efficient tool for streamlining mutant construction in bacteria.
对微生物的研究常常因缺乏有效的遗传工具而受阻。一个这样的例子是具核梭杆菌，一种革兰氏阴性厌氧菌，与各种人类感染有关，包括那些导致牙周病和早产的感染。具核梭杆菌中的首个双交叉等位基因交换突变体最近利用声穿孔法构建而成，声穿孔法是一种新型的超声介导的细胞内递送方法，证明了细菌基因转染的潜力。为了更好地揭示其机制，当前研究考察了影响声穿孔结果的因素。通过声穿孔将 Texas Red 偶联的葡聚糖递送至具核梭杆菌的效率至少是电穿孔法的两倍，而且与电穿孔不同，声穿孔不具有杀菌作用。递送效率受声压幅度、占空比以及用于引发空化的微泡数量的影响，但不受超声应用的脉冲重复频率的影响。为了考察同源重组在声穿孔介导的突变体构建中的作用，在具核梭杆菌中鉴定出了高度保守的 recA 基因，该基因带有包括 P 环在内的大多数共有残基，并且通过声穿孔构建了具核梭杆菌 12230 的双交叉 recA 突变体 US1610。与野生型相比，该突变体对紫外线照射表现出更高的敏感性，表明具核梭杆菌中的 RecA 功能是保守的。有趣的是，US1610 对超声处理也敏感，这表明 RecA 可能参与了声穿孔后的修复和存活过程。由于声穿孔通过使用完整的自杀质粒在具核梭杆菌中一直能产生一步双交叉突变体，这项技术可能会发展成为一种用于简化细菌突变体构建的高效工具。
