Novi bakteriocinogeni sojevi Lactobacillus plantarum i njihova diferencijacija analizom slijeda 16S rDNA, 16S-23S i 23S-5S međugenskih razmaknica, te analizom nasumično umnožene polimorfne DNA

Abstract

Six strains of bacteriocinogenic Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 and RI11) isolated from Malaysian foods were investigated for their structural bacteriocin genes. A new combination of plantaricin EF and plantaricin W bacteriocin structural genes was successfully amplified from all studied strains, suggesting that they were novel bacteriocin-producing L. plantarum strains. A four-base pair variable region was detected in the short 16S-23S intergenic spacer regions of the studied strains by a comparative analysis with 17 L. plantarum strains deposited in the GenBank, implying they were new genotypes. The studied L. plantarum strains were subsequently differentiated into four groups on the basis of the detected four-base pair variable region of the short 16S-23S intergenic spacer region. Further analysis of the DNA sequence of 23S-5S intergenic spacer region revealed only one type of 23S-5S intergenic spacer region present in the studied strains, indicating it was highly conserved among the studied L. plantarum strains. Three randomly amplified polymorphic DNA experiments using three different combinations of arbitrary primers successfully differentiated the studied L. plantarum strains from each other, confirming they were different strains. In conclusion, the studied L. plantarum strains were shown to be novel bacteriocin producers and high level of strain discrimination could be achieved with a combination of randomly amplified polymorphic DNA analysis and the analysis of the variable region of short 16S-23S intergenic spacer region present in L. plantarum strains.Ispitana je prisutnost strukturalnih bakteriocinskih gena u šest sojeva Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 i RI11), izoliranih iz malezijske hrane. Nova kombinacija strukturalnih gena za plantaricin EF i plantaricin W uspješno je umnožena iz svih ispitanih sojeva, što upućuje na to da se radi o novim sojevima L. plantarum koji proizvode bakteriocine. Usporednom analizom sa 17 sojeva L. plantarum pohranjenih u GenBank otkrivena je varijabilna regija od četiri para baza u kratkim 16S-23S međugenskim razmaknicama tih sojeva, potvrđujući da se radi o novim genotipovima. Ispitani sojevi L. plantarum nakon toga su razvrstani u četiri skupine, na temelju uočene varijabilne regije od četiri para baza kratke 16S-23S međugenske razmaknice. Daljnjom analizom DNA slijeda 23S-5S međugenske razmaknice otkriven je samo jedan tip te razmaknice, pokazujući da je konzerviran u ispitanim sojevima L. plantarum. Trima pokusima nasumičnog umnožavanja polimorfne DNA, koristeći tri različite kombinacije proizvoljno odabranih početnica, uspješno su diferencirani ispitani sojevi L. plantarum, što potvrđuje da se radi o različitim sojevima. Dakle, može se zaključiti da novi sojevi L. plantarum proizvode bakteriocin i da se mogu uspješno razlikovati analizom nasumično umnožene polimorfne DNA i varijabilne regije kratke 16S-23S međugenske razmaknice