Sonoporation is an efficient tool for intracellular fluorescent dextran delivery and one-step double-crossover mutant construction in Fusobacterium nucleatum - PubMed (original) (raw)

Comparative Study

. 2007 Jun;73(11):3677-83.

doi: 10.1128/AEM.00428-07. Epub 2007 Apr 20.

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Comparative Study

Sonoporation is an efficient tool for intracellular fluorescent dextran delivery and one-step double-crossover mutant construction in Fusobacterium nucleatum

Yiping W Han et al. Appl Environ Microbiol. 2007 Jun.

Abstract

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.

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Figures

FIG. 1.

FIG. 1.

Delivery of Texas Red-conjugated dextran into F. nucleatum 12230 by sonoporation and electroporation. (a) i, fluorescent dextran uptake by F. nucleatum 12230 following sonoporation in the presence of Definity; ii, fluorescent dextran uptake by F. nucleatum 12230 without US treatment; and iii, fluorescent dextran uptake by F. nucleatum 12230 following electroporation. (b) Quantization of fluorescent dextran delivery into F. nucleatum 12230 following sonoporation in the presence of Definity, sonoporation in the absence of Definity, no US treatment, and electroporation (black bars, scales on the left). The bacterial survival rate following each treatment is shown with a gray bar (scales on the right). The results shown here are the averages for four individual experiments. The standard deviations are shown above the bars.

FIG. 2.

FIG. 2.

Effects of different sonoporation parameters, i.e., acoustic pressure amplitude (a), PRF and duty cycle (DC) (b), sonoporation duration (c), and quantity of Definity (d), on delivery of Texas Red-conjugated dextran into F. nucleatum 12230. The percentages of fluorescent F. nucleatum cells were calculated as follows: percent fluorescent cells = (number of fluorescent cells/total number of cells) × 100. Values are expressed as means and standard deviations (error bars) for three separate experiments performed in triplicate.

FIG. 3.

FIG. 3.

Nucleotide and deduced amino acid sequences of the recA gene of F. nucleatum 12230. The putative Shine-Dalgarno sequence (SD) is underlined. The consensus sequence of RecA is listed directly below the amino acid sequence. The 11 residues in F. nucleatum 12230 RecA different from those in the consensus sequence are underlined. The P-loop motif (GXXXXGKT) is boxed.

FIG. 4.

FIG. 4.

Construction of the _recA_-defective mutant US1610 of F. nucleatum 12230. (a) Schematic representation of the construction of the recA::ermF-ermAM mutant. The ermF-ermAM cassette was inserted at the HincII site in recA, conferring clindamycin resistance. (b) PCR analysis of F. nucleatum 12230 and US1610 chromosomes using primers _recA_F and _recA_2R. Lane 1, F. nucleatum 12230; lane 2, US1610. M, molecular size makers as indicated on the right. (c) Effects of different sonoporation conditions on the integrity of pYH1380. Lane 1, linearized pYH1380 following EcoRV digestion; lane 2, untreated intact pYH1380; lane 3, untreated intact pYH1380 in the presence of Definity; lanes 4, 5, 8, and 9, pYH1380 treated with sonoporation in the absence of Definity; lanes 6, 7, 10, and 11, pYH1380 treated with sonoporation in the presence of Definity. M, DNA molecular size markers. The sonoporation durations and the presence (+) or absence (−) of Definity are indicated below the gel. Each condition was tested in duplicate.

FIG. 5.

FIG. 5.

Susceptibilities of F. nucleatum 12230 and US1610 to UV (a) and sonoporation (b). Percent bacterial viability was expressed as the number of CFU following treatment over the total number of CFU prior to treatment. The sonoporation conditions used were 0.5 MPa, 1-Hz PRF, 90-s duration, 50% duty cycle, and 33% Definity (vol/vol). The standard deviations are indicated above and below the squares (a) or above the bars (b).

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