Ramseyrosenkilde9443

Genome transfer from a virus into a cell is a critical early step in viral replication. Enveloped viruses achieve the delivery of their genomes into the cytoplasm by merging the viral membrane with the cellular membrane via a conceptually simple mechanism called membrane fusion. In contrast, genome translocation mechanisms in nonenveloped viruses, which lack viral membranes, remain poorly understood. Although cellular assays provide useful information about cell entry and genome release, it is difficult to obtain detailed mechanistic insights due both to the inherent technical difficulties associated with direct visualization of these processes and to the prevalence of nonproductive events in cellular assays performed at a very high multiplicity of infection. To overcome these issues, we developed an in vitro single-particle fluorescence assay to characterize genome release from a nonenveloped virus (poliovirus) in real time using a tethered receptor-decorated liposome system. Our results suggest that poliovicellular assays have provided useful information about cell entry, the mechanism used to deliver the viral genomes across the cellular membrane into the cytoplasm is not well characterized for nonenveloped viruses such as poliovirus. In this study, we developed a fluorescence imaging assay to visualize poliovirus genome release using a synthetic vesicle system. Our results not only provide new mechanistic insights into poliovirus genome translocation but also offer a cell-free assay to bridge gaps in understanding of this process in other nonenveloped viruses.Acinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide. Multidrug efflux within these highly drug-resistant strains and other opportunistic pathogens is a major cause of failure of drug-based treatments of infectious diseases. The best-characterized multidrug efflux system in A. baumannii is the prevalent Acinetobacter drug efflux B (AdeB) pump, which is a member of the resistance-nodulation-cell division (RND) superfamily. Here, we report six structures of the trimeric AdeB multidrug efflux pump in the presence of ethidium bromide using single-particle cryoelectron microscopy (cryo-EM). These structures allow us to directly observe various novel conformational states of the AdeB trimer, including the transmembrane region of trimeric AdeB can be associated with form a trimer assembly or dissociated into "dimer plus monomer" and "monomer plus monomer plus monomer" configurations. We also discover that a single AdeB protomer can simdently.Despite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida strain mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid, and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they colocalize with ribosome-rich domains of the intracellular milieu. This arrangement was maintained even when the xyl genes were artificially relocated to different chromosomal locations. The same held true when genes were expressed through a heterologous T7 polymerase-based system, which likewise led to mRNA foci outside the DNA. In contrast, rifampin treatment, known to ease crowding,zation and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida strain KT2440 through the nonhomogeneous milieu of the bacterial cytoplasm. The results expose that-similarly to computers-the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code and accompanies the environmental lifestyle of this bacterium.Chaperone-usher (CU) fimbriae are the most abundant Gram-negative bacterial fimbriae, with 38 distinct CU fimbria types described in Escherichia coli alone. Some E. coli CU fimbriae have been well characterized and bind to specific glycan targets to confer tissue tropism. For example, type 1 fimbriae bind to α-d-mannosylated glycoproteins such as uroplakins in the bladder via their tip-located FimH adhesin, leading to colonization and invasion of the bladder epithelium. Despite this, the receptor-binding affinity of many other E. coli CU fimbria types remains poorly characterized. Here, we used a recombinant E. coli strain expressing different CU fimbriae, in conjunction with glycan array analysis comprising >300 glycans, to dissect CU fimbria receptor specificity. find more We initially validated the approach by demonstrating the purified FimH lectin-binding domain and recombinant E. coli expressing type 1 fimbriae bound to a similar set of glycans. This technique was then used to map the glycan binding affinity of sinteractions with glycans were quantified by surface plasmon resonance. This novel, dual-level analysis, where screening for the repertoire of glycan binding and the hierarchy of affinity of the identified ligands is determined directly from a natively expressed fimbrial structure on the bacterial cell surface, is superior in both throughput and biological relevance.A number of computational or experimental tools have been developed to identify targets of small RNA (sRNA) regulation. Here, we modified one of these methods, based on in vivo proximity ligation of sRNAs bound to their targets, referred to as rGRIL-seq, that can be used to capture sRNA regulators of a gene of interest. Intracellular expression of bacteriophage T4 RNA ligase leads to a covalent linking of sRNAs base-paired with mRNAs, and the chimeras are captured using oligonucleotides complementary to the mRNA, followed by sequencing. This allows the identification of known as well as novel sRNAs. We applied rGRIL-seq toward finding sRNA regulators of expression of the stress response sigma factor RpoS in Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae In E. coli, we confirmed the regulatory role of known sRNAs and discovered a new negative regulator, asYbiE. When applied to P. aeruginosa and V. cholerae, we identified two novel sRNAs (s03661 and s0223) in P. aeruginosa and two known sRNAs (TfoR and Vcr043) in V.