RESUMEN
Various rod-shaped bacteria mysteriously glide on surfaces in the absence of appendages such as flagella or pili. In the deltaproteobacterium Myxococcus xanthus, a putative gliding motility machinery (the Agl-Glt complex) localizes to so-called focal adhesion sites (FASs) that form stationary contact points with the underlying surface. Here we show that the Agl-Glt machinery contains an inner-membrane motor complex that moves intracellularly along a right-handed helical path; when the machinery becomes stationary at FASs, the motor complex powers a left-handed rotation of the cell around its long axis. At FASs, force transmission requires cyclic interactions between the molecular motor and the adhesion proteins of the outer membrane via a periplasmic interaction platform, which presumably involves contractile activity of motor components and possible interactions with peptidoglycan. Our results provide a molecular model of bacterial gliding motility.
Asunto(s)
Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Adhesiones Focales/metabolismo , Myxococcus xanthus/fisiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Movimiento Celular , Proteínas Motoras Moleculares/metabolismo , Myxococcus xanthus/citología , Periplasma/metabolismo , RotaciónRESUMEN
Eukaryotic morphogenesis is seeded with the establishment and subsequent amplification of polarity cues at key times during the cell cycle, often using (cyclic) nucleotide signals. We discovered that flagellum de- and repolarization in the model prokaryote Caulobacter crescentus is precisely orchestrated through at least three spatiotemporal mechanisms integrated at TipF. We show that TipF is a cell cycle-regulated receptor for the second messenger--bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)--that perceives and transduces this signal through the degenerate c-di-GMP phosphodiesterase (EAL) domain to nucleate polar flagellum biogenesis. Once c-di-GMP levels rise at the G1 â S transition, TipF is activated, stabilized, and polarized, enabling the recruitment of downstream effectors, including flagellar switch proteins and the PflI positioning factor, at a preselected pole harboring the TipN landmark. These c-di-GMP-dependent events are coordinated with the onset of tipF transcription in early S phase and together enable the correct establishment and robust amplification of TipF-dependent polarization early in the cell cycle. Importantly, these mechanisms also govern the timely removal of TipF at cell division coincident with the drop in c-di-GMP levels, thereby resetting the flagellar polarization state in the next cell cycle after a preprogrammed period during which motility must be suspended.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/citología , Caulobacter crescentus/metabolismo , Ciclo Celular/fisiología , Flagelos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Caulobacter crescentus/genética , Polaridad Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Activación Enzimática , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Alineación de Secuencia , Transducción de SeñalRESUMEN
Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena.
Asunto(s)
Genes Bacterianos/fisiología , Locomoción/genética , Modelos Biológicos , Myxococcus xanthus/genética , Myxococcus xanthus/fisiología , Esporas Bacterianas/genética , Evolución Biológica , Filogenia , Esporas Bacterianas/metabolismoRESUMEN
INTRODUCTION: This study evaluated student reported achievement of essential elements (EE) across three required advanced pharmacy practice experiences (APPEs) to identify differences in the frequency of each EE during different delivery modalities. METHODS: APPE students from three different programs were assigned a self-assessment EE inventory after required acute care, ambulatory care, and community pharmacy APPEs between May 2018 and December 2020. Using a four-point frequency scale, students reported exposure to and completion of each EE. Pooled data were analyzed to compare differences in frequencies of EE during standard and disrupted delivery. All standard delivery APPEs were in-person, but during the study period APPEs shifted to a disrupted delivery using hybrid and remote formats. Frequency changes were reported as combined data and compared between programs. RESULTS: A total of 2191 of 2259 (97%) evaluations were completed. Acute care APPEs had a statistically significant change in frequency of evidence-based medicine elements. Ambulatory care APPEs had a statistically significant decrease in the frequency of reported pharmacist patient care elements. Community pharmacy had a statistically significant decrease in frequency in each category of EE except practice management. Statistically significant differences between programs were observed for select EEs. CONCLUSIONS: The frequency of EE completion during disrupted APPEs revealed minimal change. Acute care was the least impacted whereas community APPEs experienced the greatest change. This may be attributable to shifts in direct patient interactions during the disruption. Ambulatory care was impacted to a lesser degree, potentially due to utilization of telehealth communications.
Asunto(s)
Educación en Farmacia , Servicios Farmacéuticos , Farmacias , Farmacia , Humanos , Atención AmbulatoriaRESUMEN
The uncharacterized protein family UPF0042 of the Swiss-Prot database is predicted to be a member of the conserved group of bacterium-specific P-loop-containing proteins. Here we show that two of its members, YvcJ from Bacillus subtilis and YhbJ, its homologue from Escherichia coli, indeed bind and hydrolyze nucleotides. The cellular function of yvcJ was then addressed. In contrast to results recently obtained for E. coli, which indicated that yhbJ mutants strongly overproduced glucosamine-6-phosphate synthase (GlmS), comparison of the wild type with the yvcJ mutant of B. subtilis showed that GlmS expression was quite similar in the two strains. However, in mutants defective in yvcJ, the transformation efficiency and the fraction of cells that expressed competence were reduced. Furthermore, our data show that YvcJ positively controls the expression of late competence genes. The overexpression of comK or comS compensates for the decrease in competence of the yvcJ mutant. Our results show that even if YvcJ and YhbJ belong to the same family of P-loop-containing proteins, the deletion of corresponding genes has different consequences in B. subtilis and in E. coli.
Asunto(s)
Adenosina Trifosfato/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Secuencia Conservada , Regulación Bacteriana de la Expresión Génica , Guanosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Conformación Proteica , Proteínas de Unión al ARN , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We have recently shown that the Bacillus subtilis GTPase YvcJ is involved in the phosphorylation of an unidentified cellular component and that the deletion of yvcJ induced a decrease in competence efficiency. In this paper, we report that growth conditions influence both the YvcJ-dependent phosphorylation event and the localization of this protein. More precisely, we have observed that YvcJ can be localized in the cell either as a helical-like pattern or as foci close to the poles and the septa depending on growth phase and on growth medium. In addition, we show that the mutation of the catalytic lysine residue (K22) located in the Walker A motif of YvcJ, and necessary for its GTPase activity, induces a decrease in competence efficiency similar to that observed for the yvcJ null mutant. This mutation also inhibits the YvcJ-dependent phosphorylation event. Furthermore, a phylogenetic analysis of the YvcJ homologues shows that this protein is ancient in Bacteria (being possibly present in their last common ancestor) and has been conserved in a number of major bacterial phyla, suggesting that this protein has an important function in this domain of life. To sum up, even if the precise cellular role of this ancient protein remains unknown, our data show that the GTPase activity of B. subtilis YvcJ and its function in the phosphorylation of a cellular component are influenced by the growth conditions, and are important for the effect of YvcJ on competence efficiency.
Asunto(s)
Bacillus subtilis/enzimología , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/genética , Secuencia Conservada , GTP Fosfohidrolasas/química , Familia de Multigenes , Mutación , Fosforilación , Filogenia , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Eliminación de SecuenciaRESUMEN
In Bacillus subtilis cells, we identified a new partner of HPr, an enzyme of the glycolysis pathway, the glyceraldehyde-3-phosphate dehydrogenase GapA. We showed that, in vitro, phosphorylated and unphosphorylated forms of HPr and its homologue, Crh, could interact with GapA, but only their seryl-phosphorylated forms were able to inhibit its activity.