Complete genome sequence of Oligella urethralis MSHR-50412PR, isolated from an ear discharge swab of a child with chronic suppurative otitis media.

Oligella urethralis are opportunistic pathogens typically associated with genitourinary infections. Here, we report the complete genome for an Oligella urethralis isolate recovered from ear discharge of a child with chronic suppurative otitis media (strain MSHR-50412PR). The genome comprises 2.58 Mb, with 2,448 coding sequences and 46.26% average GC content.

Bacteriophage infection of Escherichia coli leads to the formation of membrane vesicles via both explosive cell lysis and membrane blebbing.

Membrane vesicles (MVs) are membrane-bound spherical nanostructures that prevail in all three domains of life. In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane and are often referred to as outer membrane vesicles (OMVs). We have recently described another mechanism of MV formation in Pseudomonas aeruginosa that involves explosive cell-lysis events, which shatters cellular membranes into fragments that rapidly anneal into MVs. Interestingly, MVs are often observed within preparations of lytic bacteriophage, however the source of these MVs and their association with bacteriophage infection has not been explored. In this study we aimed to determine if MV formation is associated with lytic bacteriophage infection. Live super-resolution microscopy demonstrated that explosive cell lysis of Escherichia coli cells infected with either bacteriophage T4 or T7, resulted in the formation of MVs derived from shattered membrane fragments. Infection by either bacteriophage was also associated with the formation of membrane blebs on intact bacteria. TEM revealed multiple classes of MVs within phage lysates, consistent with multiple mechanisms of MV formation. These findings suggest that bacteriophage infection may be a major contributor to the abundance of bacterial MVs in nature.

Multifunctional Ground Control Points with a Wireless Network for Communication with a UAV.

Ground control points (GCPs) are commonly used for georeferencing in remote sensing. Precise position measurement of the GCPs typically requires careful ground surveying, which is time-consuming and labor-intensive and thus excessively costly if it needs to be repeated multiple times in a season. A system of multifunctional GCPs and a wireless network for communication with an unmanned aerial vehicle (UAV) was developed to improve the speed of GCP setup and provide GCP data collection in real-time during the flight. While testing the system, a single-board computer on a fixed-wing UAV used in the study successfully recorded position data from all the GCPs during the flight. The multifunctional GCPs were also tested for use as references for calibration of reflectance and height for field objects like crops. The test of radiometric calibration resulted in an average reflectance error of 2.0% and a strong relationship (R2 = 0.99) between UAV-based estimates and ground reflectance. Furthermore, the average height difference between UAV-based height estimates and ground measurements was within 10 cm.

Dbest1, a Drosophila homolog of human Bestrophin, is not required for viability or photoreceptor integrity.

Best macular dystrophy (BMD) is an autosomal dominant human disease characterized by macular degeneration with juvenile onset (OMIM 153700). The disease is most often associated with mutations in Bestrophin, which encodes a novel protein with four putative transmembrane domains. However, complete loss-of-function mutations in Bestrophin have not been reported in humans or mice. We have identified three homologs of human Bestrophin in the Drosophila genome (dbest1-3). The protein products of these three genes share significant homology to a 364 amino acid N-terminal domain of human Bestrophin. We used P-element mutagenesis to delete dbest1, which encodes a protein with the highest amino acid similarity to Bestrophin. Three independent dbest1 mutants were recovered from the mutagenesis screen. Homozygous null mutations in dbest1 do not significantly alter the viability or fertility of mutant flies. Moreover, dbest1 mutants have normal photoreceptor morphology and function.

A new metal-binding site for yeast phosphoglycerate kinase as determined by the use of a metal-ATP analog.

Suicide substrate beta, gamma-bidentate Rh(III)ATP (RhATP) was used to map the metal ion-binding site in yeast phosphoglycerate kinase (PGK). Cleavage of the RhATP-inactivated enzyme with pepsin and subsequent separation of peptides by reverse-phase high-performance liquid chromatography gave two Rh-nucleotide bound peptides. One of the peptides corresponded to the C-terminal residues of PGK, and the other to a part of helix V. Of the four glutamates present in the C-terminal peptide, Glu 398 may be a likely metal coordination site. Therefore, importance of the C-terminal residues in PGK catalysis may be attributed, in part to the coordination of metal ion of the metal-ATP substrate. Metal coordination may then align the C-terminal peptide to extend toward the N-terminal domain and form the "closed" active site. Results presented in this paper suggest that one or more side chains of the enzyme may be coordinated to the metal ion in the PGK.3-phospho-D-glycerate-RhATP complex, and that exchange-inert metal-ATP analogs could be used to determine metal coordination sites on kinases and other metal-ATP-utilizing enzymes.

Proton NMR studies of a large protein. pH, substrate titrations, and NOESY experiments with perdeuterated yeast phosphoglycerate kinase containing [1H]histidine residues.

Fully deuterated yeast phosphoglycerate kinase ([2H]PGK) was prepared biosynthetically with only histidine side chains of normal (1H) isotopic composition. The 1H NMR spectrum of this enzyme ([1H]His[2H]PGK) showed that the histidine side chains are clearly visible as sharp signals. Thus detailed structural studies by 1H NMR became feasible with isotope-hybrid phosphoglycerate kinase which is otherwise too large (M(r) approximately 46,000) for conventional 1H NMR studies. Proton signals of bound substrates were visible in the 1H NMR spectrum even with a substrate-to-enzyme ratio of less than 1/2 (mol/mol). The 2D NOESY spectrum of enzyme-MgdATP-glycerol 3-phosphate complex showed that, although protein concentration was very high (1.5 mM), no intraprotein cross peaks were observed other than those of intraresidue histidine NOE cross peaks. In addition, intrasubstrate NOEs and intermolecular NOEs between histidine and substrate protons were visible at a 1.5/1 substrate/enzyme (mol/mol) ratio. Paramagnetic effects of a substrate analog, Cr(III)ATP, on some of the histidine side chains indicated that the formation of the ternary enzyme-substrate complex causes large conformational changes in the enzyme.

Substrate activity of Rh(III)ATP with phosphoglycerate kinase and the role of the metal ion in catalysis.

An exchange-inert Rh(H2O)4ATP complex showed a one-turnover substrate activity with yeast phosphoglycerate kinase. Transfer of the phosphoryl group between ATP and 3-phosphoglycerate occurs with both substrates in the coordination sphere of the metal ion. Because of the slow ligand exchange rates of Rh3+, the reaction product 1,3-diphosphoglycerate (1,3-dPGA) remained coordinated to the metal ion. During the course of the reaction, the enzyme was inactivated, suggesting that the metal ion is coordinated to a protein side chain. Thus the product Rh(H2O)nADP.1,3-dPGA remained bound to the enzyme even after removal of excess substrate. These results suggested that the metal ion may not only act as an electron sink to activate the electrophile, but it may also help to optimally align both substrates for phosphoryl transfer by coordination to both substrates. It is therefore likely that entry of 3-phospho-D-glycerate into the coordination sphere of metal of a metal-ATP complex may start the proposed hinge-bending motion of yeast phosphoglycerate kinase to form a "closed" active site between the two substrate binding domains of the enzyme.