Disruption of a type II endonuclease (TDE0911) enables Treponema denticola ATCC 35405 to accept an unmethylated shuttle vector

Abstract

The oral spirochete Treponema denticola is associated with human periodontal disease. T. denticola ATCC 35405 and ATCC 33520 are two routinely used laboratory strains. Compared to T. denticola ATCC 33520, ATCC 35405 is more virulent but less accessible to genetic manipulations. For instance, the shuttle vectors of ATCC 33520 cannot be transformed into strain ATCC 35405. The lack of a shuttle vector has been a barrier to study the biology and virulence of T. denticola ATCC 35405. In this report, we hypothesize that T. denticola ATCC 35405 may have a unique DNA restriction-modification (R-M) system that prevents it from accepting the shuttle vectors of ATCC 33520 (e.g., the shuttle plasmid pBFC). To test this hypothesis, DNA restriction digestion, PCR, and Southern blot analyses were conducted to identify the differences between the R-M systems of these two strains. DNA restriction digestion analysis of these strains showed that only the cell extract from ATCC 35405 was able to digest pBFC. Consistently, PCR and Southern blot analyses revealed that the genome of T. denticola ATCC 35405 encodes three type II endonucleases that are absent in ATCC 33520. Among these three endonucleases, TDE0911 was predicted to cleave unmethylated double-stranded DNA and to be most likely responsible for the cleavage of unmethylated pBFC. In agreement with this prediction, the mutant of TDE0911 failed to cleave unmethylated pBFC plasmid, and it could accept the unmethylated shuttle vector. The study described here provides us with a new tool and strategy to genetically manipulate T. denticola, in particular ATCC 35405, and other strains that may carry similar endonucleases.

Fig. 1.

Schematic of construction of TDE0911::ermB for targeted mutagenesis of TDE0911. The method illustrated here was used to delete the entire reading frame of TDE0911 and to replace it with the ermB cassette. Arrows indicate the approximate positions of the primers used for multiple-step PCR amplifications. The sequences of these primers are listed in Table 1. Thin black and gray rectangles represent the engineered adaptors for the fusion of individual amplicons.

Fig. 2.

pBFC digested by the crude cell extracts of ATCC 35405 and ATCC 33520. pBFC was first linearized by SpeI and then coincubated with the crude cell extracts of strain ATCC 35405 or ATCC 33520, as described below. Lane 1, pBFC (methylated) treated with boiled crude cell extract of ATCC 35405; lanes 2 and 4, pBFC (methylated) treated with ATCC 35405 extract (lane 2) or ATCC 33520 extract (lane 4); lanes 3 and 5, pBFC (unmethylated) treated with extracts of ATCC 35405 (lane 3) and ATCC 33520 (lane 5). After the digestions, the samples were analyzed by 1% agarose gel electrophoresis and visualized with ethidium bromide staining (a); results were further confirmed by Southern blotting with DIG-labeled pBFC (b). M, DNA marker.

Fig. 3.

Detection of TDE0228, TDE0911, and TDE1268 by Southern blot analysis. The purified chromosomal DNAs of ATCC 33520 and ATCC 35405 were digested with ClaI and HindIII and then probed with three different DIG-labeled DNA fragments: TDE0228 (a), TDE0911 (b), and TDE1268 (c). Lanes 1 and 2, genomic DNA of ATCC 33520 digested with ClaI and HindIII; lanes 3 and 4, genomic DNA of ATCC 35405 digested with ClaI and HindIII. The numbers to the right of the gels present the sizes of detected genes.

Fig. 4.

Characterization of the TdΔ911 mutant. The purified chromosomal DNAs of ATCC 35405 and the TdΔ911 mutant were treated with ClaI and HindIII and then detected by Southern blotting with probes for TDE0911 (a) and ermB (b). Lanes 1 and 2, genomic DNA of ATCC 35405 treated with ClaI (lane 1) or HindIII (lane 2); lanes 3 and 4, genomic DNA of TdΔ911 mutant treated with ClaI (lane 3) or HindIII (lane 4). The numbers to the right of the gels represent the sizes of the detected DNA fragments. (c) PCR analysis of the TdΔ911 mutant with two pairs of primers, P₃/P₁₉ and P₃/P₄. The PCR samples were loaded in the following order: ATCC 35405 amplified with P₃/P₁₉ (lane 1); TdΔ911 amplified with P₃/P₁₉ (lane 2); ATCC 35405 amplified with P₃/P₄ (lane 3); TdΔ911amplified with P₃/P₄ (lane 4). The sequences and descriptions of these three primers are listed in Table 1.

Fig. 5.

Identification of the recognition and cleavage site of TDE0911 (TdeIII). PCR-amplified 534 bp of the wild-type (G₉₇GCCC) and mutated aacC1 (A₉₇GCCC) fragments were treated with the crude cell extract of ATCC 35405 and TdΔ911. The obtained samples were then analyzed by 2% agarose gel electrophoresis and visualized with ethidium bromide staining. The samples were loaded in the following order: the mutated aacC1 (lane 1) and the wild-type aacC1 (lane 2) treated with ATCC 35405 extract, and the mutated aacC1 (lane 3) and the wild-type aacC1 (lane 4) treated with TdΔ911 extract. M, DNA marker. The cleaved fragments in lane 2 were cloned and sequenced to determine the cleavage site.

Fig. 6.

The TdΔ911 mutant failed to cleave the unmethylated pBFC. The SpeI-treated methylated or unmethylated pBFC was coincubated with the crude cell extract of ATCC 35405 or TdΔ911 and then analyzed on a 1.0% agarose gel (a), followed by Southern blot analysis (b). The samples are in the following lane order: methylated pBFC treated with boiled crude cell extract of ATCC 35405 (lane 1); methylated (lane 2) and unmethylated (lane 3) pBFC treated with ATCC 35405 extract; methylated (lane 4) and unmethylated (lane 5) pBFC treated with TdΔ911 extract. M, DNA marker.

Fig. 7.

Transformation of pBFC into different T. denticola strains. Ten micrograms of methylated (mpBFC, top panels) or unmethylated pBFC plasmid (umpBFC, bottom panels) was electroporated into ATCC 35405, ATCC 33520, and the TdΔ911 mutant. Images were taken 7 days after plating. A total of 20 antibiotic-resistant colonies were picked from the positive transformants (c and f), and the presence of pBFC in these colonies was identified by extracting plasmid followed by DNA restriction digestion. All examined colonies contained the plasmid.