Assessment of horizontal gene transfer-mediated destabilization of Synechococcus elongatus PCC 7942 biocontainment system.

Biological containment is a biosafety strategy that prevents the dispersal of genetically modified organisms in natural ecosystems. We previously established a biocontainment system that makes bacterial growth dependent on the availability of phosphite (Pt), an ecologically rare form of phosphorus (P), by introducing Pt metabolic pathway genes and disrupting endogenous phosphate and organic phosphate transporter genes. Although this system proved highly effective, horizontal gene transfer (HGT) mediated recovery of a P transporter gene is considered as a potential pathway to abolish the Pt-dependent growth, resulting in escape from the containment. Here, we assessed the risk of HGT driven escape using the Pt-dependent cyanobacterium Synechococcus elongatus PCC 7942. Transformation experiments revealed that the Pt-dependent strain could regain phosphate transporter genes from the S. elongatus PCC 7942 wild-type genome and from the genome of the closely related strain, S. elongatus UTEX 2973. Transformed S. elongatus PCC 7942 became viable in a phosphate-containing medium. Meanwhile, transformation of the Synechocystis sp. PCC 6803 genome or environmental DNA did not yield escape strains, suggesting that only genetic material derived from phylogenetically-close species confer high risk to generate escape. Eliminating a single gene necessary for natural competence from the Pt-dependent strain reduced the escape occurrence rate. These results demonstrate that natural competence could be a potential risk to destabilize Pt-dependence, and therefore inhibiting exogenous DNA uptake would be effective for enhancing the robustness of the gene disruption-dependent biocontainment.

Biodiversity risk assessment of genetically modified Chaetoceros gracilis for outdoor cultivation.

A genetically modified (GM) strain of the diatom Chaetoceros gracilis expressing the phosphite dehydrogenase gene (ptxD), which is a useful gene both for the biological containment and the avoidance of microbial contamination, was characterized to estimate the risk against the biodiversity by laboratory experiments. GM strain could grow in the medium containing phosphite as a sole source of phosphorus, while its general characteristics such as growth, salt tolerance, heat and dehydration resistance in the normal phosphate-containing medium were equivalent to those of wild type (WT) strain. The increase in potential toxicity of GM strain against plant, crustacean, fish and mammal was also disproved. The dispersal ability of WT strain cultured in an outdoor raceway pond was investigated for 28 days by detecting the psb31 gene in vessels, settled at variable distances (between 5 and 60 m) from the pond. The diatom was detected only in one vessel placed 5 m apart. To estimate the influence on the environment, WT and GM strains were inoculated into freshwater, seawater and soil. The influence on the microbiome in those samples was assessed by 16S rRNA gene amplicon sequencing, in addition to the analysis of the survivability of those strains in the freshwater and the seawater. The results indicated that the effect to the microbiome and the survivability were comparable between WT and GM strains. All results showed that the introduction of the ptxD gene into the diatom had a low risk on biodiversity.