The effect of HtpG chaperone on Cas3 protein levels and protection against phage lambda infection depending on the temperature in Escherichia coli

Sustav CRISPR-Cas je važan mehanizam obrane mnogih prokariota od infekcije stranom DNA poput faga i plazmida. Sastoji se od lokusa CRISPR koji sadrži ponavljajuće palindromske sekvence DNA odvojene razmaknicama koje potječu od strane DNA faga ili plazmida te gena cas čijom ekspresijom nastaju proteini koji sudjeluju u obrani od infekcije. U bakteriji Escherichia coli efikasnost zaštite bakterija od faga sustavom CRISPR-Cas ovisi o aktivnosti proteina Cas3 koja pak ovisi o temperaturi inkubiranja i šaperonu HtpG. U ovom radu je istraživano utječe li šaperon HtpG na količinu ili konformaciju proteina Cas3 u ovisnosti o temperaturi. Utjecaj suviška proteina HtpG na količinu proteina Cas3 određen je metodom Western-blot, a utjecaj na konformaciju proteina Cas3 određen je mjerenjem efikasnosti zaštite određenih točkastih mutanata cas3 od infekcije fagom λ. Dobiveni rezultati ukazuju da šaperon HtpG povećava količinu proteina Cas3 te da potiče smatanje točkastih mutanata Cas3, ali samo na 30 °C.For many prokaryotes, the CRISPR-Cas system is an important defense mechanism against foreign DNA infections, such as phages or plasmids. It is composed of the locus CRISPR, which consists of short palindromic DNA sequences separated by spacers derived from foreign DNA phages or plasmids, and cas genes whose expression produces Cas proteins that participate in the defense against infection. In bacteria Escherichia coli the efficiency of protection of the bacteria against phages by the CRISPR-Cas system depends on the activity of the Cas3 protein, which depends on the incubation temperature and HtpG chaperone. In this work, we investigated whether the HtpG chaperone influences the amount or conformation of the Cas3 protein as a function of temperature. The influence of elevated HtpG levels on the amounts of the Cas3 protein is determined using the Western blot method, and the influence on the conformation of the Cas3 protein is determined by measuring the efficiency of protection of specific cas3 point mutants against infection with the λ phage. The results suggest that the HtpG chaperone increases the amounts of Cas3 protein and induces the folding of Cas3 point mutants, but only at 30 °C