Analiza regulatornih regiona i ekspresija sgm gena bakterije Micromonospora zionensis

Abstract

Proizvodjac aminoglikozidnog antibiotika G-52, Micromonospora zionensis, poseduje sgm gen koji kodira 16$ rRNK metilazu cijom se ak'uvnoscu ostvaruje rezistencija na 4,6-disupstituisane dezoksistreptaminske aminoglikozide. Identifikovana su dva promotora uzvodno od kodirajuce sekvence sgm gena. Jedan promoter je reiativno slab i on otpocinje transkripciju od G nukleotida koji se nalazi 72 bp uzvodno od translacionog start kodona (ATG). Drug: promotor je mnogo jaci i Iociran je na vecoj udaljenosti od ATG kodona (~250 nukleotida). Postojanje ovih tandemskih promotora bi moglo da omoguci diferencijalnu gensku ekspresiju pa je postulirana njihova razlicita funkcija kod proizvodjaca. Tako 1e predlozeno da je slabiji P1 promoter odgovoran za konstitutivnu transkripciju sgm gena, dok je P2 zaduzen za ekspresiju nizvodnih (biosintetskih) gena u vreme kada otpocinje sinteza antibiotika. Prema tome, P2 promotor bi mogao biti potencijalno koristan za favorizovanu ekspresiju kloniranih gena u stacionarnoj fazi rasta micromonospora. Konstruisana je serija transkripcionih i translacionih sgm-Iacz genskih fuzija na plazmidu pPLti7G. Transkripcija obe vrste fuzija se odvija pod kontrolom snaznog PLtI promotora, posto promotori sgm gena nisu funkcionalni u bakteriji E. coli. Nivo ekspresiie lacZ gena u pojedinim transformantima sa transkripcionim ili translacionim fuzijama odredjivan je merenjem B-galaktozidazne aktivnosti. Transformanti sa translacionom fuznjom koji imaju i ekstra kopiju sgm gena bilo u cis iii u trans poziciji, pokazuju izrazit pad B-galaktozidazne aktivnosti. U slicnim eksperimentima sa transkripcionim fuzijama nije uocen znacajan efekat ekstra kopije sgm gena. Ovi rezultati su pokazali da je ekspresija sgm gena autoregulisana transiacionom represijom, najverovatnije zbog vezivanja metilaze za sopstvenu iRNK. Ekspresijom sgm gena u razlicitim sojevima aktinomiceta utvrdjeno je da je visok nivo rezistenqije na higromicin B zaista determinisan sgm genom, ali da je ispoljavanje ove rezistencije zavisno od soja u kome se sgm gen eksprimira. Ovaj podatak sugerira na zakljucak da ispitivani sojevi mikromonospora poseduju identican mehanizam rezistencije na higromicin. Iz toga se moze zakljuciti da mikromonospore najverovatnije poseduju odredjenu specificnost u gradji male subjedinice ribozoma koja je. konzervirana medju sojevima ovog roda.

The sisomicin-gentamicin resistance methylase (sgm) gene from Micromonospora zionensis (producer of 6-52antibiotic), encodes an enzyme thatmodifies 16S rRNA, and thereby conferring resistance to 4,6-disubstituted deoxystreptamine aminoglycosides. Two promoters were identified upstream of the sgm coding sequence. One promoter (Pi) is relatively weak and initiates transcription at the G which is 72 nucleotides upstream of the translational initiation codon (ATG). A second (P2) promoter is much stronger and is located more upstream from the ATG codon (~250 nucleotides). These tandem promoters could enable differential gene expression of the sgm gene, and it has been postulated that promoters have specialized functions in the producing organism. The apparent weak P1 promoter is probably responsible for constitutive transcription of the sgm gene, whereas the P2 promoter may play a specialized role in expressron of the downstream (biosynthetic) genes, during the time that the antibiotic (3-52 is produced. Therefore, P2 promoter could be potentially used in expressing cloned genes especially during the stationary growth phase of micromonospora. A set of transcriptional and translational fusions oi the sgm gene and the feel reporter gene have been constructed by using the pPLtl7G plasmid. Both transcriptional and translational fusions were transcribed under the strong PLtl promoter,since it has been shown that the sgm promoters are non-functional in E. coli. Transformants harbouring either the transcriptional and translational fusions were assayed for B-gaiactosidase activity. Translational fusion transformants which also carried an extra copy of sgm gene either in cis or in trans position, exhibited a substantial decrease in B-galactosidase activity. No significant effect was observed in comparable experiments with the transcriptional fusions. These results demonstrate that the expression of the sgm gene is regulated by translational autorepression, presumably due to methylase binding to the specific site/s on its own mFiNA. The translational repression model is discussed in light of overall control of the sgm gene expression. The expression of the sgm gene in various actinomycetes strains revealed that expression of high-level resistance to hygromycin B, determined by sgm gene, is background dependent. This evidence suggests that it may well be that ail tested micromonospora strains share a common mode of hygromycin B resistance. Factors that might be contributed to background-dependent expression of hygromycin B resistance are discussed.