RESUMEN
The ribosome-binding GTPase HflX is required for manganese homeostasis in E. coli. While under normal conditions ΔhflX cells behave like wild type E. coli with respect to growth pattern and morphology, deletion of hflX makes E. coli cells extremely sensitive to manganese, characterized by arrested cell growth and filamentation. Here we demonstrate that upon complementation by hflX, manganese stress is relieved. In phenotypic studies done in a manganese-rich environment, ΔhflX cells were highly sensitive to antibiotics that bind the penicillin binding protein 3 (PBP3), suggesting that the manganese stress led to impaired peptidoglycan biosynthesis. An irregular distribution of dark bands of constriction along filaments, delocalization of the dark bands from midcell towards poles and subpoles, lack of septum formation and arrested cell division were observed in ΔhflX cells under manganese stress. However, chromosome replication and segregation of nucleoids were unaffected under these conditions, as observed from confocal microscopy imaging and FACS studies. We conclude that absence of HflX leads to manganese accumulation in E. coli cells, affecting cell septum formation, probably by modulating the activity of the cell division protein PBP3 (FtsI), a major component of the divisome apparatus. We propose that HflX acts as a gatekeeper, regulating the influx of manganese into the cell.
Asunto(s)
Cloruros/farmacología , Proteínas de Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Proteínas de Unión al GTP/genética , Regulación Bacteriana de la Expresión Génica , Compuestos de Manganeso/farmacología , Proteínas de Unión a las Penicilinas/genética , Estrés Fisiológico/genética , Antibacterianos/farmacología , División Celular/efectos de los fármacos , Cloruros/metabolismo , Segregación Cromosómica/efectos de los fármacos , Cromosomas Bacterianos/metabolismo , Cromosomas Bacterianos/ultraestructura , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Unión al GTP/deficiencia , Eliminación de Gen , Prueba de Complementación Genética , Homeostasis/genética , Compuestos de Manganeso/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/biosíntesis , Estrés Fisiológico/efectos de los fármacosRESUMEN
Manganese is a micronutrient required for activities of several important enzymes under conditions of oxidative stress and iron starvation. In Escherichia coli, the manganese homeostasis network primarily constitutes a manganese importer (MntH) and an exporter (MntP), which are regulated by the MntR dual regulator. In this study, we find that deletion of E. coli hflX, which encodes a ribosome-associated GTPase with unknown function, renders extreme manganese sensitivity characterized by arrested cell growth, filamentation, lower rate of replication, and DNA damage. We demonstrate that perturbation by manganese induces unprecedented influx of manganese in ΔhflX cells compared to that in the wild-type E. coli strain. Interestingly, our study indicates that the imbalance in manganese homeostasis in the ΔhflX strain is independent of the MntR regulon. Moreover, the influx of manganese leads to a simultaneous influx of zinc and inhibition of iron import in ΔhflX cells. In order to review a possible link of HflX with the λ phage life cycle, we performed a lysis-lysogeny assay to show that the Mn-perturbed ΔhflX strain reduces the frequency of lysogenization of the phage. This observation raises the possibility that the induced zinc influx in the manganese-perturbed ΔhflX strain stimulates the activity of the zinc-metalloprotease HflB, the key determinant of the lysis-lysogeny switch. Finally, we propose that manganese-mediated autophosphorylation of HflX plays a central role in manganese, zinc, and iron homeostasis in E. coli cells.