The influence of emrBAR efflux system on symbiosis between Medicago truncatula and Sinorhizobium meliloti

Dušik je bitan element biomolekula i potreban je za metabolizam i razvoj svih biljaka. Simbiotske interakcije između bakterija i biljaka razvile su se kako bi se omogućila apsorpcija dušika u biljkama. Glavni cilj ovog istraživanja bio je učinak sustava izbacivanja EmrBAR na simbiozu mahunarke Medicago truncatula i bakterije Sinorhizobium meliloti. Rezultati su pokazali da su geni emrA i emrR eksprimirani u ranim fazama simbiotskih interakcija, tijekom nastajanja infekcijskih niti. Gen emrA je eksprimiran u infekcijskoj zoni i zoni fiksacije dušika, a gen emrR samo u infekcijskoj zoni korijenove kvržice. Analiza nodulacijskog fenotipa emrBAR mutanata i divljeg tipa u nakošenim epruvetama ispunjenim agarom sugerira da je stvaranje novih korijenovih kvržica bio odgovor na neučinkovitu fiksaciju dušika. Analiza nodulacijskog fenotipa emrBAR mutanata i divljeg tipa u Magenta posudama sugerira da delecija emrBAR utječe na rast simbiontskih biljaka smanjujući učinkovitost fiksacije dušika u korijenovim kvržicama. Ispitivanje rasta bakterija je pokazalo da 2-fenilfenol inhibira rast dvaju sojeva emrBAR mutanta u odnosu na divlji tip. To bi moglo ukazivati na važnost sustava izbacivanja EmrBAR u izbacivanju otrovnih tvari iz bakterijskih stanica.Nitrogen is an essential part of biomolecules and is needed for the metabolism and development of all plants. Symbiotic interactions between bacteria and plants have evolved in order to enable the absorption of nitrogen to plants. The main focus of this study was the effect of the EmrBAR efflux system on the symbiosis between the legume Medicago truncatula and the rhizobium Sinorhizobium meliloti. The results showed that emrA and emrR are expressed in early symbiotic interactions, during the formation of infection threads. EmrA is expressed in the infection and nitrogen-fixation zones, and emrR only in the infection zone of the nodule. Analysis of nodulation phenotype of the emrBAR deletion mutants and the wild type in sloped agar tubes might suggest that the creation of new nodules was a response to inefficient nitrogen fixation. Analysis of nodulation phenotype of the emrBAR deletion mutants and the wild type in Magenta boxes suggests that the emrBAR deletion affects symbiotic plant growth by decreasing nodule efficiency. A bacterial growth assay showed that 2-phenylphenol inhibits the growth of the two emrBAR deletion mutant strains in comparison with the wild type. This seems to indicate the importance of the EmrBAR efflux system in the extrusion of toxic substances

CRISPR-Cas in Escherichia coli: regulation by H-NS, LeuO and temperature

CRISPR-Cas adaptive immune systems are present in many bacteria and archaea and provide protection against invading DNA such as phages and plasmids. These systems are very versatile and complex in their gene composition and genomic architecture. CRISPR-Cas systems are classified into 2 classes, 6 types and 33 subtypes although this number is not definitive and the research is ongoing. All CRISPR-Cas systems have been thoroughly investigated in order to better understand the mechanism of CRISPR immunity enabling its use as a tool in genome editing and other biotechnological applications. However, regulation of the CRISPR-Cas system is also very complex and still not fully understood; it must provide optimal protection without introducing harmful consequences to the host. In this review we give an overview on the regulation of the CRISPR-Cas system Class 1 Type I-E in Escherichia coli with the emphasis on the role of temperature in regulation of the CRISPR-Cas activity and the interplay of the key regulators H-NS and StpA repressors and LeuO antirepressor in regulation of cas gene expression and HtpG chaperone in maintaining functional levels of Cas3.</p

A tryptophan ‘gate’ in the CRISPR-Cas3 nuclease controls ssDNA entry into the nuclease site, that when removed results in nuclease hyperactivity

Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30C to 37C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp- 406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells

Mechanisms regulating the CRISPR-Cas system in Escherichia coli

Sustav CRISPR-Cas je adaptivni imunosni sustav za obranu prokariota od stranih genetičkih elemenata. Regulacija sustava CRISPR-Cas u bakteriji E. coli je kompleksna i nije u potpunosti razjašnjena. Poznato je da protein H-NS djeluje na razini transkripcije, šaperon HtpG na razini post-translacije, dok temperatura utječe na aktivnost proteina Cas3 na nepoznati način. U ovom radu su detaljnije istraženi mehanizmi regulacije aktivnosti sustava CRISPR-Cas na razini transkripcije i post-translacije. Praćenjem otpornosti stanica na infekciju fagom λvir i određivanjem količine transkripata gena cas utvrđeno je da protein StpA može inhibirati promotore Pcas8e i anti-Pcas u nedostatku proteina H-NS, te da pojačana ekspresija ili inaktivacija proteaze Lon u mutantu hns aktivira zaštitu od infekcije neovisno o sustavu CRISPR-Cas. Na aktivnost proteina Cas3 utječe ''anti-sense'' mRNA na nepoznat način, dok temperatura uzrokuje alosteričku promjenu konformacije ovog proteina čime se inhibira njegova nukleazna aktivnost. Šaperon HtpG nije utjecao na količine proteina Cas3, već vjerojatno na zauzimanje njegove pravilne konformacije.The CRISPR-Cas system is a prokaryotic adaptive immune system that provides defense against foreign DNA. The regulation of the CRISPR-Cas system in E. coli is complex and not fully understood. The repressor H-NS acts at the transcriptional level, HtpG posttranslationally, while the activity of Cas3 is affected by temperature in an unknown way. The aim of this research was to further elucidate the mechanisms regulating the activity of the CRISPR-Cas system at the transcriptional and post-translational level. Phage sensitivity assays and quantification of cas transcripts showed that StpA can inhibit Pcas8e and anti-Pcas promoters in the absence of H-NS. Overexpression or inactivation of Lon protease in hns mutants provides protection against phage infection independent of CRISPR-Cas. Cas3 protein activity is affected by an "anti-sense" mRNA in an unknown way, while allosteric change in Cas3 structure by temperature inhibits its nuclease activity. HtpG did not affect Cas3 levels, but the acquisition of the correct conformation

Mechanisms regulating the CRISPR-Cas system in Escherichia coli

Sustav CRISPR-Cas je adaptivni imunosni sustav za obranu prokariota od stranih genetičkih elemenata. Regulacija sustava CRISPR-Cas u bakteriji E. coli je kompleksna i nije u potpunosti razjašnjena. Poznato je da protein H-NS djeluje na razini transkripcije, šaperon HtpG na razini post-translacije, dok temperatura utječe na aktivnost proteina Cas3 na nepoznati način. U ovom radu su detaljnije istraženi mehanizmi regulacije aktivnosti sustava CRISPR-Cas na razini transkripcije i post-translacije. Praćenjem otpornosti stanica na infekciju fagom λvir i određivanjem količine transkripata gena cas utvrđeno je da protein StpA može inhibirati promotore Pcas8e i anti-Pcas u nedostatku proteina H-NS, te da pojačana ekspresija ili inaktivacija proteaze Lon u mutantu hns aktivira zaštitu od infekcije neovisno o sustavu CRISPR-Cas. Na aktivnost proteina Cas3 utječe ''anti-sense'' mRNA na nepoznat način, dok temperatura uzrokuje alosteričku promjenu konformacije ovog proteina čime se inhibira njegova nukleazna aktivnost. Šaperon HtpG nije utjecao na količine proteina Cas3, već vjerojatno na zauzimanje njegove pravilne konformacije.The CRISPR-Cas system is a prokaryotic adaptive immune system that provides defense against foreign DNA. The regulation of the CRISPR-Cas system in E. coli is complex and not fully understood. The repressor H-NS acts at the transcriptional level, HtpG posttranslationally, while the activity of Cas3 is affected by temperature in an unknown way. The aim of this research was to further elucidate the mechanisms regulating the activity of the CRISPR-Cas system at the transcriptional and post-translational level. Phage sensitivity assays and quantification of cas transcripts showed that StpA can inhibit Pcas8e and anti-Pcas promoters in the absence of H-NS. Overexpression or inactivation of Lon protease in hns mutants provides protection against phage infection independent of CRISPR-Cas. Cas3 protein activity is affected by an "anti-sense" mRNA in an unknown way, while allosteric change in Cas3 structure by temperature inhibits its nuclease activity. HtpG did not affect Cas3 levels, but the acquisition of the correct conformation

Mechanisms regulating the CRISPR-Cas system in Escherichia coli

Sustav CRISPR-Cas je adaptivni imunosni sustav za obranu prokariota od stranih genetičkih elemenata. Regulacija sustava CRISPR-Cas u bakteriji E. coli je kompleksna i nije u potpunosti razjašnjena. Poznato je da protein H-NS djeluje na razini transkripcije, šaperon HtpG na razini post-translacije, dok temperatura utječe na aktivnost proteina Cas3 na nepoznati način. U ovom radu su detaljnije istraženi mehanizmi regulacije aktivnosti sustava CRISPR-Cas na razini transkripcije i post-translacije. Praćenjem otpornosti stanica na infekciju fagom λvir i određivanjem količine transkripata gena cas utvrđeno je da protein StpA može inhibirati promotore Pcas8e i anti-Pcas u nedostatku proteina H-NS, te da pojačana ekspresija ili inaktivacija proteaze Lon u mutantu hns aktivira zaštitu od infekcije neovisno o sustavu CRISPR-Cas. Na aktivnost proteina Cas3 utječe ''anti-sense'' mRNA na nepoznat način, dok temperatura uzrokuje alosteričku promjenu konformacije ovog proteina čime se inhibira njegova nukleazna aktivnost. Šaperon HtpG nije utjecao na količine proteina Cas3, već vjerojatno na zauzimanje njegove pravilne konformacije.The CRISPR-Cas system is a prokaryotic adaptive immune system that provides defense against foreign DNA. The regulation of the CRISPR-Cas system in E. coli is complex and not fully understood. The repressor H-NS acts at the transcriptional level, HtpG posttranslationally, while the activity of Cas3 is affected by temperature in an unknown way. The aim of this research was to further elucidate the mechanisms regulating the activity of the CRISPR-Cas system at the transcriptional and post-translational level. Phage sensitivity assays and quantification of cas transcripts showed that StpA can inhibit Pcas8e and anti-Pcas promoters in the absence of H-NS. Overexpression or inactivation of Lon protease in hns mutants provides protection against phage infection independent of CRISPR-Cas. Cas3 protein activity is affected by an "anti-sense" mRNA in an unknown way, while allosteric change in Cas3 structure by temperature inhibits its nuclease activity. HtpG did not affect Cas3 levels, but the acquisition of the correct conformation