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

Liss, Petronella Francina (2003-12)

Thesis (MSc)--Stellenbosch University, 2003.

Thesis

ENGLISH ABSTRACT: Lactobacillus plantarum 285, isolated from sorghum beer, produces bacteriocin 285, which displays activity against several food spoilage organisms. For future application of bacteriocin 285 in the food industry, it was important to characterize the peptide and identify the genes encoding its production. The effect of bacteriocin 285 on sensitive cells was determined through the use of an indicator (sensitive) organism, Lactobacillus sakei DSM 20017. The indicator strain was genetically modified to express GFP (green fluorescent protein), with the aim of quantifying the antibacterial activity of bacteriocin 285 as a function of 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 broad spectrum of bacteria, including several food spoilage organisms. The sensitivity of L. sakei DSM 20017 towards antibacterial peptides produced by Lactobacillus curvatus DF38, L. plantarum 285, Lactobacillus casei LHS and Lactobacillus salivarius 241 is not limited to the growth stage of the organism. Cells remained sensitive to all four of these bacteriocins, from lag phase to late exponential growth. To inhibit growth of up to 90% of the cells of L. sakei DSM 20017, 1 AU/ml bacteriocin 285 (7 ng/ml) of partially purified bacteriocin 285 was required. However, to kill all viable cells of L. sakei DSM 20017, 16 AU/ml (110 ng/ml) of partially purified bacteriocin 285 was required. The gfpuv gene, encoding GFPuv, was cloned downstream of the Idh promoter and successfully expressed in L. sakei DSM 20017. However, GFPuv fluorescence could not be used as a direct method to quantify the antimicrobial activity of bacteriocin 285, since cells of strain DSM 20017 remained fluorescent for prolonged periods after treatment with lethal concentrations of the bacteriocin. The non-viability of the cells was confirmed with epifluorescence microscopy and a L1VE/DEAD® Baclight™ Bacterial Viability Probe. Cells that were stained with the viability probe indicated that the majority of untreated L. sakei DSM 20017 cells were viable. However, treatment of strain DSM 20017 with 16 AU/ml bacteriocin 285 rendered all visible cells non-viable.

AFRIKAANSE OPSOMMING: Lactobacillus plantarum 285 wat uit sorgumbier geïsoleer is, produseer bakteriosien 285. Die bakteriosien toon aktiwiteit teen verskeie organismes wat voedselbederi veroorsaak. Vir toekomstige aanwending van bakteriosien 285 in die voedselindustrie was dit belangrik om die peptied te karakteriseer en die gene wat vir die produksie daarvan kodeer, te identifiseer. Die effek van bakteriosien 285 op sensitiewe selle is bepaal deur die gebruik van 'n indikator (sensitiewe)-organisme, Lactobacillus sakei DSM 20017. Die indikator-organisme is geneties verander om die GFP (groen fluoreserende proteïen) uit te druk. Die doel was om die antibakteriese aktiwiteit van bakteriosien 285 te kwantifiseer as 'n funksie van GFP fluorisensie. Bakteriosien 285 is identies aan plantarisien 423 wat deur L. plantarum 423 produseer word. Plantarisien 423 is 'n klas Iia bakteriosien en vertoon antimikrobiese aktiwiteit teenoor 'n wye verskeidenheid bakterieë, insluitende verskeie organismes wat voedsel bederf. Die sensitiwiteit van L. sakei DSM 20017 teenoor antibakteriese peptiede wat deur Lactobacillus cutveius DF38, L. plantarum 285, Lactobacillus casei LHS en Lactobacillus salivarius 241 geproduseer word, word nie beïnvloed deur die groeifase van die organisme nie. Selle het sensitief gebly teenoor al vier die bakteriosiene van sloer- tot laat eksponensiële groei. Om groei van tot 90% van L. sakei DSM 20017 selle te inhibeer, word 1 AU/ml (7 ng/ml) gedeeltelik gesuiwerde bakteriosien 285 benodig. Om alle lewensvatbare L. sakei DSM 20017 selle te dood, word 16 AU/ml (110 ng/ml) gedeeltelik gesuiwerde bakteriosien 285 benodig. Die gfpuv-geen, wat GFPuv kodeer is stroomaf van die Idh-promoter gekloneer en suksesvol in L. sakei DSM 20017 uitgedruk. GFPuv fluoresensie kon nie as direkte metode gebruik word om die antimikrobiese aktiwiteit van bakteriosien 285 te bepaal nie, aangesien die selle van L. sakei DSM 20017 fluoreserend gebly het lank na behandeling met dodelike konsentrasies van die bakteriosien. Die lewensvatbaarheid van die selle is bevestig deur epifluoresensiemikroskopie en 'n LlVE/DEAD® Bac/ight™ bakteriese lewensvatbaarheidspeiler. Selle van L. sakei DSM 20017 wat deur die peiler gekleur is, het gewys dat die meeste selle wat nie deur bakteriosien 285 behandel was nie, lewensvatbaar was. Behandeling van L. sakei DSM 20017 met 16 AU/ml bakteriosien 285 het al die sigbare selle gedood.

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