Cloning of the gfp (green fluorescent protein) gene downstream of the ldh promoter in a bacteriocin-sensitive strain of Lactobacillus sakei to serve as a reporter strain in bacteriocin studies

[摘要] ENGLISH ABSTRACT: Lactobacillus plantarum 285, isolated from sorghum beer, produces bacteriocin 285, whichdisplays activity against several food spoilage organisms. For future application ofbacteriocin 285 in the food industry, it was important to characterize the peptide and identifythe genes encoding its production. The effect of bacteriocin 285 on sensitive cells wasdetermined through the use of an indicator (sensitive) organism, Lactobacillus sakei DSM20017. The indicator strain was genetically modified to express GFP (green fluorescentprotein), with the aim of quantifying the antibacterial activity of bacteriocin 285 as a functionof GFP fluorescence.Bacteriocin 285 proved to be identical to plantaricin 423 produced by L. plantarum 423.Plantaricin 423 is a class lIa bacteriocin and displays antimicrobial activity towards a broadspectrum of bacteria, including several food spoilage organisms. The sensitivity of L. sakeiDSM 20017 towards antibacterial peptides produced by Lactobacillus curvatus DF38, L.plantarum 285, Lactobacillus casei LHS and Lactobacillus salivarius 241 is not limited to thegrowth stage of the organism. Cells remained sensitive to all four of these bacteriocins, fromlag phase to late exponential growth. To inhibit growth of up to 90% of the cells of L. sakeiDSM 20017, 1 AU/ml bacteriocin 285 (7 ng/ml) of partially purified bacteriocin 285 wasrequired. However, to kill all viable cells of L. sakei DSM 20017, 16 AU/ml (110 ng/ml) ofpartially purified bacteriocin 285 was required.The gfpuv gene, encoding GFPuv, was cloned downstream of the Idh promoter andsuccessfully expressed in L. sakei DSM 20017. However, GFPuv fluorescence could not beused as a direct method to quantify the antimicrobial activity of bacteriocin 285, since cells ofstrain DSM 20017 remained fluorescent for prolonged periods after treatment with lethalconcentrations of the bacteriocin. The non-viability of the cells was confirmed withepifluorescence microscopy and a L1VE/DEAD® Baclight™ Bacterial Viability Probe. Cellsthat were stained with the viability probe indicated that the majority of untreated L. sakeiDSM 20017 cells were viable. However, treatment of strain DSM 20017 with 16 AU/mlbacteriocin 285 rendered all visible cells non-viable.

[发布机构] Stellenbosch University