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Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is the first known phage infecting P. putida S12, a strain increasingly used as a synthetic biology chassis. MiCath was isolated from garden soil under a tomato plant using P. putida S12 as a host and was also found to infect four other P. putida strains. MiCath has a ~ 61 kbp double-stranded DNA genome which encodes 97 predicted open reading frames (ORFs); functions could only be predicted for 48 ORFs using comparative genomics. Functions include structural phage proteins, other common phage proteins (e.g., terminase), a queuosine gene cassette, a cas4 exonuclease, and an endosialidase. Restriction digestion analysis suggests the queuosine gene cassette encodes a pathway capable of modification of guanine residues. When compared to other phage genomes, MiCath shares at most 74% nucleotide identity over 2% of the genome with any sequenced phage. Overall, MiCath is a novel phage with no close relatives, encoding many unique gene products. [ABSTRACT FROM AUTHOR]

Emerging and re-emerging viral pathogens present a unique challenge for anti-viral therapeutic development. Anti-viral approaches with high flexibility and rapid production times are essential for combating these high-pandemic risk viruses. CRISPR-Cas technologies have been extensively repurposed to treat a variety of diseases, with recent work expanding into potential applications against viral infections. However, delivery still presents a major challenge for these technologies. Lipid-coated mesoporous silica nanoparticles (LCMSNs) offer an attractive delivery vehicle for a variety of cargos due to their high biocompatibility, tractable synthesis, and amenability to chemical functionalization. Here, we report the use of LCMSNs to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) that target the Niemann–Pick disease type C1 gene, an essential host factor required for entry of the high-pandemic risk pathogen Ebola virus, demonstrating an efficient reduction in viral infection. We further highlight successful in vivo delivery of the RNP-LCMSN platform to the mouse liver via systemic administration. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories. [ABSTRACT FROM AUTHOR]

Recent work using chemical cross-linking to define interresidue distance constraints in proteins has shown that these constraints are useful for testing tertiary structural models. We applied this approach to the G-protein-coupled receptor bovine rhodopsin in its native membrane using lysine- and cysteine-targeted bifunctional cross-linking reagents. Cross-linked proteolytic peptides of rhodopsin were identified by combined liquid chromatography and FT-ICR mass spectrometry with automated data-reduction and assignment software. Tandem mass spectrometry was used to verify cross-link assignments and locate the exact sites of cross-link attachment. Cross-links were observed to form between 10 pairs of residues in dark-state rhodopsin. For each pair, cross-linkers with a range of linker lengths were tested to determine an experimental distance-of-closest-approach (DCA) between reactive side-chain atoms. In all, 28 cross-links were identified using seven different cross-linking reagents. Molecular mechanics procedures were applied to published crystal structure data to calculate energetically achievable theoretical DCAs between reactive atoms without altering the position of the protein backbone. Experimentally measured DCAs are generally in good agreement with the theoretical DCAs. However, a cross-link between C316 and K325 in the C-terminal region cannot be rationalized by DCA simulations and suggests that backbone reorientation relative to the crystal coordinates occurs on the timescale of cross-linking reactions. Biochemical and spectroscopic data from other studies have found that the C-terminal region is highly mobile in solution and not fully represented by X-ray crystallography data. Our results show that chemical cross-linking can provide reliable three-dimensional structural information and insight into local conformational dynamics in a membrane protein. [ABSTRACT FROM AUTHOR]

We present a two-step approach to modeling the transmembrane spanning helical bundles of integral membrane proteins using only sparse distance constraints, such as those derived from chemical cross-linking, dipolar EPR and FRET experiments. In Step 1, using an algorithm, we developed, the conformational space of membrane protein folds matching a set of distance constraints is explored to provide initial structures for local conformational searches. In Step 2, these structures refined against a custom penalty function that incorporates both measures derived from statistical analysis of solved membrane protein structures and distance constraints obtained from experiments. We begin by describing the statistical analysis of the solved membrane protein structures from which the theoretical portion of the penalty function was derived. We then describe the penalty function, and, using a set of six test cases, demonstrate that it is capable of distinguishing helical bundles that are close to the native bundle from those that are far from the native bundle. Finally, using a set of only 27 distance constraints extracted from the literature, we show that our method successfully recovers the structure of dark-adapted rhodopsin to within 3.2 Å of the crystal structure. [ABSTRACT FROM AUTHOR]