Development of a modified gentamicin resistance cassette for genetic manipulation of the oral spirochete Treponema denticola

Abstract

Herein, we report that a modified gentamicin cassette and a PCR-based method can be used for targeted mutagenesis of the oral spirochete Treponema denticola. This approach minimizes polar effects and spontaneous antibiotic resistance. Therefore, it can serve as a reliable tool for genetic manipulation of T. denticola.

Fig 1

The mutated aacC_m cassette is resistant to the cleavage of T. denticola. PCR amplified 534 bp of the wild-type aacC1 cassette, and mutated aacC_m fragments were treated with the crude cell extract of Td35405. The samples obtained were then analyzed by 2% agarose gel electrophoresis and visualized with ethidium bromide staining. Lane M contains DNA markers. The two white arrows point toward the cleaved products.

Fig 2

Schematic illustration of constructing the PrcA::aacCm vector for the deletion of prcA gene. The vector was constructed via three PCR amplification steps. Arrows indicate the relative positions of PCR primers for constructing the vector. The sequences of these primers are listed in Table 1.

Fig 3

Isolation and characterization of ΔprcA mutant. (a) Illustration of the prcA locus in Td35405 (wild type) and the aacC_m cassette in the ΔprcA mutant. (b) PCR analysis showing that the prcA gene was deleted and replaced by aacC_m in the ΔprcA mutant. The primers used for PCR analysis are labeled in panel a. (c) Western blot analysis of the ΔprcA mutant. Similar amounts of whole-cell lysates from the wild type and the mutant were analyzed by SDS-PAGE and then probed with a specific PrcA antibody (αPrcA, anti-PrcA antibody). Immunoblots were developed using horseradish peroxidase secondary antibody with an enhanced chemiluminescence (ECL) luminol assay as previously described (2, 3).

Fig 4

Western immunoblots of Td35405, ΔprcA mutant, and two previously constructed prcA mutants (PNE and CF547) (13, 16). The blots were probed with antibodies against T. denticola PrcA, PrtP, or FlaA. FlaA expression was used as a loading control (3, 13).