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 ؋ 10 ؊6 to 5.8 ؋ 10 ؊7 transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 ؋ 10 ؊9 ). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 ؋ 10 ؊10 ), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 ؋ 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.

Safety Assessment of Transgenic Organisms, Volume 4

“…Marker rescue based on restoration of deleted nptII was also used to study transfer of bacterial or transgenic plant DNA in Streptococcus gordonii. While in vitro transformation could be achieved, no marker rescue was observed in gnotobiotic rats, presumably due to the absence of competence-stimulating factors like serum proteins (Kharazmi et al, 2003). A peptidepheromone system which controls genetic competence in Streptococcus mutans functions optimally when cells are living in actively growing biofilms (Li et al, 2001).…”

Stimulation of bacterial DNA transformation by cattle saliva: implications for using genetically modified plants in animal feed

“…In situ transfer has not been detected under experimental conditions but cannot be excluded since the hypothetical transformation rate of transgenic DNA to B. subtilis was calculated to be in the range of 8.5 9 10 -19 to 1.2 9 10 -27 per cell for homologous and non-homologous recombination, respectively (Kharazmi et al 2003b). However, in this situation it must be considered that a region of homology between donor and recipient DNA is a prerequisite for stable integration (Kharazmi et al 2003a) or, as demonstrated for S. pneumoniae, foreign DNA fragments have to be rescued by homology-directed illegitimate recombination during transformation (Prudhomme et al 2002). Non-homologous integration of foreign DNA occurs at detectable frequencies only with the aid of highly specialized integrases, such as the int gene product of transposons (Malanowska et al 2006).…”

“…In the case of shortened and therefore defective genes, marker rescue (repair of homologous DNA) can occur (Kharazmi et al 2003a). However, DNA is rapidly degraded in the rumen into small fragments Wiedemann et al 2006) which are unlikely to retain the length necessary for biological activity.…”

“…However, DNA is rapidly degraded in the rumen into small fragments Wiedemann et al 2006) which are unlikely to retain the length necessary for biological activity. Kharazmi et al (2003a) showed by marker rescue transformation that plasmid and chromosomal DNA of bacteria as well as the DNA of transgenic potatoes was transferred to bacteria under laboratory conditions. In situ transfer has not been detected under experimental conditions but cannot be excluded since the hypothetical transformation rate of transgenic DNA to B. subtilis was calculated to be in the range of 8.5 9 10 -19 to 1.2 9 10 -27 per cell for homologous and non-homologous recombination, respectively (Kharazmi et al 2003b).…”

Lack of detectable DNA uptake by bacterial gut isolates grownin vitroand byAcinetobacter baylyicolonizing rodentsin vivo

“…For instance, it has been reported that the naturally competent Streptococcus gordonii DL1 could be transformed by plasmid DNA in saliva (Mercer et al, 1999b;. Other studies examining in vivo transfer in gut samples from different mice and rat models have not been able to detect bacterial transformants (Kharazmi et al, 2003;Wilcks et al, 2004). One DNA consumption study in humans (including ileostomists; individuals in which the terminal ileum is resected and the digesta is diverted via a stoma to a colostomy bag) indicated that horizontal transfer of recombinant DNA from GM soya to the microbiota of the small bowel had taken place in the past.…”