Marker rescue studies of the transfer of recombinant DNA to Streptococcus gordonii in vitro, in foods and gnotobiotic rats

Abstract

A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 x 10(-6) to 5.8 x 10(-7) transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 x 10(-9)). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 x 10(-10)), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 x 10(-11). No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.

FIG. 1.

Average daily uptake of drinking water (closed symbols) and plasmid DNA (open symbols) for the animals in days 1 to 6.

FIG. 2.

Counts of S. gordonii LTH 5597 in fecal (closed symbols) and saliva (open symbols) samples of animals 1 to 6 receiving the plasmid DNA (A) and of the control animals (7 to 10) receiving no DNA (B). Data are expressed as mean log₁₀ CFU per gram of sample (± standard error of the mean).

FIG. 3.

Counts of S. gordonii LTH 5597 in different compartments of the gastrointestinal tracts of the gnotobiotic rats 1 to 6 (white bars) and 7 to 10 (grey bars). Data are mean log₁₀ CFU per gram of sample (± standard error of the mean).

FIG. 4.

Verification of the origin of the 792-bp amplification fragments of nptII generated with the DNA isolated from colon samples. The fragments were digested with NcoI. Lane 1, colon sample of animal 1 spiked with DNA of plasmid pMR2 (donor DNA); lane 2, colon sample of animal 1; lane 3, fragment amplified with DNA of E. coli Sure; Lane 4, nondigested fragment of nptII. L, 1-kb DNA ladder (MBI Fermentas).

FIG. 5.

Transformation of S. gordonii LTH 5597 with the reisolated pMR2 DNA which had been exposed to the saliva of germfree rats.