Analysis of the expression of a gene that provides resistance to antibiotic novobiocin in Escherichia coli K12

[s.n.]

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CysB protein je transkripcioni regulator koji deluje kao tetramer i pripada LysR familiji. On je aktivator gena cisteinskog regulona, cbl gena, tauABCD operona, ssuEADCB operona, adi gena i dr. Negativno reguliše ekspresiju sopstvenog cysB gena. U prethodnom radu selekcijom na azaserinu i novobiocinu su izolovani cysB mutanti koji su bili auksotrofi za cistein i pokazivali povećanu rezistenciju na antibiotik novobiocin. Primenom sistema λpJacZMu fuzija identifikovan je gen hslJ koji je negativno regulisan CysB proteinom I uključen u mehanizam rezistencije bakterije E. coli na novobiocin. U ovom radu su konstruisane operonske fuzije hs1J-lacZ koje nose različite dužine regulatomog regiona hslJ gena. Merenjem aktivnosti ovih fuzija determinisan je minimalni promotorski i regulatorni region. Određen je start transkripcije hslJ gena i definisan putativni σ70 promotor. CysB protein i mutirani CysB I33N izolovani su i prečišćeni na afinitetnoj Ni-NTA rezin koloni. Pokazano je da oba proteina imaju potencijal za oligomerizaciju stvarajući monomere, tetramere, heksamere i oligomere višeg reda. CysB protein i mutant CysB I33N vezuju se za minimalni regulatomi region hslJ gena a NAS povećava efikasnost vezivanja. Rezultati aktivnosti operonskih fuzija hslJ-lacZ u cysB+ i cysB- sojevima kao i in vitro analize vezivanja CysB proteina za hslJ lokus govore u prilog tome da je CysB protein represor hslJ transkripcije. Analiza mutacija u različitim cysB mutanatima pokazala je da oni sadrže defektan CysB protein koji može komplementacijom sa dmgim mutiranim CysB proteinom ostvariti funkcionalnu aktivnost što je praćeno rastom na minimalnoj podiozi bez cisteina I smanjenjem nivoa rezistencije na novobiocin. Ova interalelska komplementacija CysB mutanata pokazala je da se DNK vezujući mutant, CysB I33N, inače neaktivan u regulaciji cys regulona, aktivno vezuje za hslJ lokus i reprimira hslJ transkripciju kao i da je Cterminalni region CysB proteina odgovoran za oligomerizaciju i efikasno vezivanje CysB proteina u regulatornom regionu hslJ gena. Nizak nivo ekspresije hslJ gena strogo je kontrolisan; prisustvo hslJ na plazmidu sa većim brojem kopija letalno je za ćeliju. Ukoliko je hslJ gen prisutan na plazmidu sa malim brojem kopija dolazi do efekta genske doze I povećanja rezistencije na antibiotik novobiocin. Ovo je u direktnoj korelaciji sa povećanim nivoom transkripcije hslJ gena kada je represor CysB inaktivan ili slabo aktivan, što dovodi do povećanja rezistencije na novobiocin. Prema podacima kompjuterske analize, hslJ protein je najverovatnije lokalizovan u spoljašnjoj membrani ćelije. U prilog ovome idu i rezultati dobijeni merenjem aktivnosti operonske fuzije pspA-lacZ čija se aktivnost povećava oko pet puta ukoliko je u ćeliji na plazmidu umesto WT hslJ prisutan mutirani gen hslJ::ΩKan čiji izmenjeni produkt bi svojom nepravilnom lokalizacijom u membrani narušio njen integritet o aktivirao psp operon.

CysB is a tetrameric LysR-type transcription factor that acts as an activator of the cys regulon, cbl and adi genes, tau and ssu operons, etc., while cysB gene itself is under negative autoregulation. Previously, cysB auksotrophic mutants to cystein and mutants with increased resistance to novobiocin had been isolated by screening the azaserine and novobiocin resistant colonies. hslJ gene whose expression is negatively regulated by CysB was identified by generating random chromosomal lacZ gene fusions using the λplacZMu system. In this study, the operon fusions hslJ-lacZ were constructed by clonong different DNA fragments of the region upstream of the hs/J gene. Ву using these fusions placed on plasmids, the minimal promoter/regulatory region was defined. The hslJ transcription start site was determined and the inspection of DNA sequence revealed one putative σ70 promoter. WT CysB and CysB I33N fusion proteins, shown to be active in vivo, were purified on affinity chromatography columns and employed in /п vitro studies. These fusion proteins were able to interact with potential to form tetramers, hexsamers, and higher order structure oligomers. Both, CysB and its DNA-binding mutant CysB I33N bind hslJminimal regulatory region and ligand, NAS, increases the efficiency of this binding. Taking together the activities of hslJ-lacZ chromosomal/low сору plasmids placed operon fusions introduced to either cysB+ or cysB- strains, and /п vitro studies of CysB binding in hsIJ locus, it is very likely that CysB is a classical repressor of hslJtranscription. Analysis of different cysB mutants used in this study showed that interallelic complementation exists, and some cysB mutation can be reversed to functionality by the cysB allele mutated in different region. These results revealed that DNAbinding mutant CysB I33N otherwise inactive in regulating the cys regulon, binds hslJ minimal regulatory region and represses the transcription. Also, CysB S277Ter protein that carries the truncation of the C-terminal region, can not oligomerize, efficiently interact with DNA, and repress the hslJ transcription. The expression of hslJ is tightly controlled and very low. High number of intracellular hslJ gene copies is lethal but when present in low number of copies, the increased novobiocin resistance as a direct consequence exists. This is in direct correlation with the level of hslJ transcription and repression imposed by the CysB. When CysB is inactive the hslJ transcription is more effective and the resistance to novobiocin is elevated. Considering the deduced amino acid sequence and the computer analyses, the localization of the HslJ protein is predicted to be in the outer membrane of the cell. This assumption is supported by the fact that hslJ::ΩKan mutation activates the operon fusion pspA-lacZ shown to be sensitive to changes provoked by inappropriate outer membrane proteins localization.

hsU, cysB, negativna regulacija, represor, cysB mutanti, HslJ, rezistencija na novobiocin,

hslJ, cysB, negative regulation, repressor, cysB mutants, HslJ, novobiocin resistance

579.25(043.3)

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