Funkcionalna karakterizacija CmbT transportera odgovornog za višestruke rezistencije na strukturno različite supstrate u soju Lactococcus lactis subsp. cremoris MG1363

Abstract

Lactococcus lactis pripada grupi bakterija mlečne kiseline (BMK) i ima dugotrajnu primenu u prehrambenoj industriji kao starter kultura, najviše u proizvodnji tvrdih i polutvrdih sireva. Kako L. lactis ima ključnu ulogu u formiranju ukusa i teksture finalnog proizvoda, a doprinosi i produženju trajnosti namirnica, veliki ekonomski i industrijski značaj doveo je do toga da su laktokoke predmet stalnog izučavanja. Iako se generalno smatraju bezbednim mikroorganizmima, mnogi istraživači ukazuju na činjenicu da laktokoke, kao i druge bakterije mlečne kiseline mogu biti rezervoar gena za rezistenciju. U cilju sprečavanja širenja ovakvih gena na druge bakterije intestinalnog trakta ili patogene bakterije prisutne u hrani, od velikog značaja je izučavanje mehanizama rezistencije, posebno kod laktokoka namenjenih za prehrambenu industriju. Analizom genoma L. lactis detektovano je prisustvo 40 potencijalnih gena za MDR (eng. multidrug resistance) transportere, od čega su svega dva ABC transportera, LmrA i LmrCD i jedan MFS transporter, LmrP funkcionalno okarakterisani. Istraživanja prikazana u ovom radu su imala za cilj da se okarakteriše CmbT (eng. Cysteine and Methionine Biosynthesis Transporter) protein, potencijalno novi MDR transporter u soju L. lactis. Gen cmbT identifikovan je po prvi put prilikom izučavanja regulacije metabolizma sumpora u L. lactis. Primenom različitih kompjuterskih programa, pokazano je da cmbT kodira transmembranski integralni efluks protein (veličine 454 aminokiseline) koji pokazuje homologiju sa članovima 2.A.1.3.X supstrat/H+ antiporter- 2 subfamilije. Prvi korak u funkcionalnoj karakterizaciji CmbT proteina bio je kloniranje i ekspresija cmbT gena pomoću precizno regulisanog NICE (eng. NIsin Controlled gene Expression) sistema...

Lactococcus lactis is a lactic acid bacterium (LAB) widely used as a constituent of many industrial and artisanal starter cultures in dairy industry, especially for fermentation of hard and semi hard cheeses. This organism plays a key role in the formation of flavour and texture of cheese and its preservation and because of the great industrial importance it has been the subject of numerous studies. Although they have acquired the „Generaly Regarded As Safe“ (GRAS) status, many investigators have speculated that lactococci as well as other LAB may act as reservoirs of antibiotic resistance genes. The main threat associated with these bacteria is that they can transfer resistance genes to intestinal microorganisms and food-associated pathogenic bacteria. In order to prevent and reduce the spreading of the resistance genes, more studies of multidrug resistance (MDR) transporters in lactococci intended for use in food systems are needed. The genome analysis of L. lactis indicated the presence of at least 40 putative drug transporter genes, but only two ABC transporters, LmrA, and LmrCD, and one MFS transporter, LmrP, have been studied in detail and all three have demonstrated MDR activity experimentally. The aim of this thesis was to investigate and characterize new MDR transporter CmbT (Cysteine and Methionine Biosynthesis Transporter) in L. lactis. The cmbT gene was originally identified from a random mutagenesis study of cysteine and methionine biosynthesis regulation in L. lactis. This gene is predicted to encode an integral membrane efflux protein (454 amino acids) with homology to members of the 2.A.1.3.X drug/H+ antiporter-2 subfamily of the major facilitator superfamily (MFS). In order to follow the transport function of the CmbT protein, the cmbT gene was cloned and over-expressed in L. lactis NZ9000 using the nisin controlled gene expression (NICE) system...