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 imadugotrajnu primenu u prehrambenoj industriji kao starter kultura, najviše u proizvodnjitvrdih i polutvrdih sireva. Kako L. lactis ima ključnu ulogu u formiranju ukusa iteksture finalnog proizvoda, a doprinosi i produženju trajnosti namirnica, velikiekonomski i industrijski značaj doveo je do toga da su laktokoke predmet stalnogizučavanja. Iako se generalno smatraju bezbednim mikroorganizmima, mnogiistraživači ukazuju na činjenicu da laktokoke, kao i druge bakterije mlečne kiselinemogu biti rezervoar gena za rezistenciju. U cilju sprečavanja širenja ovakvih gena nadruge bakterije intestinalnog trakta ili patogene bakterije prisutne u hrani, od velikogznačaja je izučavanje mehanizama rezistencije, posebno kod laktokoka namenjenih zaprehrambenu industriju.Analizom genoma L. lactis detektovano je prisustvo 40 potencijalnih gena zaMDR (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 noviMDR transporter u soju L. lactis.Gen cmbT identifikovan je po prvi put prilikom izučavanja regulacijemetabolizma sumpora u L. lactis. Primenom različitih kompjuterskih programa,pokazano je da cmbT kodira transmembranski integralni efluks protein (veličine 454aminokiseline) 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 iekspresija cmbT gena pomoću precizno regulisanog NICE (eng. NIsin Controlled gene Expression) sistema...

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