Oral administration of transgenic biosafe microorganism containing antimicrobial peptide enhances the survival of tilapia fry infected bacterial pathogen.

To develop an alternative to conventional antibiotics used in the aquaculture and livestock industries, we employed Bacillus subtilis, considered a biosafe microorganism, to express the degradable antimicrobial peptide lactoferricin. An expression plasmid pP43-6LFBII-GFP, in which reporter GFP cDNA was fused downstream of lactoferricin cDNA driven by an endogenous constitutive P43 promoter was electroporated into B. subtilis, followed by regeneration and cultivation. The putative colonies harboring plasmids were primarily screened by PCR-amplification of lactoferricin cDNA. Four transformants which were stable inheritance of plasmid containing lactoferricin cDNA included strains T1, T4, T7 and T13. Based on Western blot and Southern blot analyses, we found that transgenic strains T1 and T13 not only highly expressed exogenous recombinant lactoferricin, but also exhibited more stable inheritance of plasmids with 931 and 647 copies per cell, respectively. In the antibacterial in vitro experiment, the bactericidal activity of each microliter of cell lysate from transgenic strains T1 and T13 (5 × 108 CFU) for Escherichia coli was equivalent to 56 and 53 ng of Ampicillin dosage, respectively, while for Staphylococcus epidermidis, the equivalency T1 and T13 was 154 and 130 ng of Ampicillin dosage, respectively. Equivalencies of bacterial activity for Vibrio parahaemolyticus and Edwardsiella tarda followed suit. In the antibacterial in vivo experiment, we oral-in-tube fed tilapia fry (Oreochromis mossambicus X O. niloticus) with cell lysate from transgenic strain T1 and T13 individually. After 1-h of incubation, we immersed these treated fish fry in a water tank containing E. tarda (5 × 1011 CFU) for a 5-hr bacterial challenge. After one month cultivation, an average survival rate of 63 and 67% was observed after having fed the fish fry with transgenic strains T1 and T13, respectively. However, the average survival rate of fish fry fed with B. subtilis WT strain and transgenic strain T19 without expressing recombinant lactoferricin reached only 5 and 9%, respectively. These data indicate that the survival of fish fry infected by the intestinal pathogen tested could be significantly enhanced by feeding transgenic B. subtilis containing antibacterial peptide. Therefore, we suggest that this strategy could be applied to both aquaculture and livestock industries to (i) reduce the dependency on conventional antibiotics during seasonal outbreaks and (ii) eliminate the problem of antibiotic resistance.

Purple chromoprotein gene serves as a new selection marker for transgenesis of the microalga Nannochloropsis oculata.

Among the methods used to screen transgenic microalgae, antibiotics selection has raised environmental and food safety concerns, while the observation of fluorescence proteins could be influenced by the endogenous fluorescence of host chloroplasts. As an alternative, this study isolated the purple chromoprotein (CP) from Stichodacyla haddoni (shCP). A plasmid in which shCP cDNA is driven by a heat-inducible promoter was linearized and electroporated into 2.5×10(8) protoplasts of Nannochloropsis oculata. Following regeneration and cultivation on an f/2 medium plate for two weeks, we observed 26 colonies that displayed a slightly dark green coloration. After individually subculturing and performing five hours of heat shock at 42°C, a dark brown color was mosaically displayed in five of these colonies, indicating that both untransformed and transformed cells were mixed together in each colony. To obtain a uniform expression of shCP throughout the whole colony, we continuously isolated each transformed cell that exhibited brown coloration and subcultured it on a fresh plate, resulting in the generation of five transgenic lines of N. oculata which stably harbored the shCP gene for at least 22 months, as confirmed by PCR detection and observation by the naked eye. As shown by Western blot, exogenous shCP protein was expressed in these transgenic microalgae. Since shCP protein is biodegradable and originates from a marine organism, both environmental and food safety concerns have been eliminated, making this novel shCP reporter gene a simple, but effective and ecologically safe, marker for screening and isolating transgenic microalgae.