Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva - PubMed (original) (raw)

Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva

D K Mercer et al. Appl Environ Microbiol. 1999 Jan.

Abstract

Competitive PCR was used to monitor the survival of a 520-bp DNA target sequence from a recombinant plasmid, pVACMC1, after admixture of the plasmid with freshly sampled human saliva. The fraction of the target remaining amplifiable ranged from 40 to 65% after 10 min of exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been exposed to degradation by fresh saliva was capable of transforming naturally competent Streptococcus gordonii DL1 to erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s. S. gordonii DL1 transformants were obtained in the presence of filter-sterilized saliva and a 1-microg/ml final concentration of pVACMC1 DNA. Addition of filter-sterilized saliva instead of heat-inactivated horse serum to S. gordonii DL1 cells induced competence, although with slightly lower efficiency. These findings indicate that DNA released from bacteria or food sources within the mouth has the potential to transform naturally competent oral bacteria. However, further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.

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Figures

FIG. 1

FIG. 1

Construction of the competitor plasmid to pVACMC1 and its use in competitive PCR. (A) Plasmid pVACMC1 and its competitor, pVACMCcomp, obtained by insertion of a 100-bp _Sal_I fragment (see text). (B) Trial competitive PCR experiment in which different ratios of competitor plasmid and pVACMC1 were subjected to PCR amplification with the forward PCR primer CMCP2 and the reverse primer pVACrev. The R. flavefaciens cellulase gene, endA, encodes CMCase activity.

FIG. 2

FIG. 2

Persistence of a 520-bp fragment of pVACMC1 DNA after incubation with fresh, whole saliva for 9 min. PCR amplification of degraded DNA was carried out as described in Materials and Methods, and products were analyzed on a 1.4% (wt/vol) agarose gel. A 1-kb DNA ladder (Gibco BRL), 0.5 to 12.2 kbp, was used.

FIG. 3

FIG. 3

(A) Survival of pVACMC1 DNA in human saliva analyzed on a 0.8% (wt/vol) agarose gel. A 1-kb DNA ladder (Gibco BRL), 0.5 to 12.2 kbp, was used. OC, open circular; L, linear. A faint band of covalently closed circular DNA was visible in lane 0). (B) Transformation of S. gordonii DL1 with pVACMC1 DNA that had been previously exposed to fresh, whole saliva for the times indicated.

FIG. 4

FIG. 4

Transformation of S. gordonii DL1 with pVACMC1 in the presence and absence of filter-sterilized saliva. Competent cells were exposed to DNA and saliva or sterile dH2O for the times shown before plating on selective medium to identify transformants. Maximum transformation frequencies for the saliva of a second volunteer were 3.7 × 105. Experiments were carried out with different samples of S. gordonii DL1 competent cells.

FIG. 5

FIG. 5

Transformation of S. gordonii DL1 with pVACMC1 after competence induction by different growth supplements. The transformation protocol described in Materials and Methods was followed except that heat-inactivated horse serum was replaced with the other growth supplements.

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