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Appl. Environ. Microbiol. doi:10.1128/AEM.00428-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Sonoporation is an Efficient Tool for Intracellular Fluorescent Dextran Delivery and One-step Double-crossover Mutant Construction in Fusobacterium nucleatum

Department of Biological Sciences, School of Dental Medicine; Department of Pathology, School of Medicine, Department of Chemistry, School of Arts and Sciences; and Department of Biomedical Engineering, School of Engineering, Case Western Reserve University, Cleveland, Ohio 44106-4905

^* To whom correspondence should be addressed. Email: yiping.han{at}case.edu.

	Abstract

Studies of microorganisms are often hindered by the lack of effective genetic tools. One such example is Fusobacterium nucleatum, a Gram-negative anaerobe associated with various human infection including 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 was at least twice more efficiently by sonoporation than by electroporation, and sonoporation was non-bactericidal, unlike electroporation. The delivery efficiency was affected by the acoustic pressure amplitude, duty cycle, the quantity of microbubbles used to initiate cavitation, but not by 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 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 post-sonoporation repair and survival. Since sonoporation has consistently generated one-step double-crossover mutants in F. nucleatum using intact suicide plasmids, this technology may be developed into an efficient tool for streamlining mutant construction in bacteria.