Crystallization and preliminary X-ray analysis of the sporulation factor SpoIIAA in its native and phosphorylated forms

Sporulation in Bacillus begins with an asymmetric cell division producing two progeny with identical chromosomes but different developmental fates. As such, it is a simple example of cellular differentiation. The establishment of cell type is controlled by a series of alternate RNA polymerase sigma subunits. The first compartment-specific sigma factor is sigma (F), whose activity is controlled by SpoIIAB, an anti-sigma factor, and SpoIIAA, an anti-sigma factor antagonist which is phosphorylated by the kinase activity of SpoIIAB. Here, the preliminary crystallographic analysis of SpoIIAA and phosphorylated SpoIIAA from B. sphaericus in forms suitable for high-resolution structure determination are reported

Synthesis and evaluation of mucoadhesive acryloyl-quaternized PDMAEMA nanogels for ocular drug delivery

Poly((2-dimethylamino)ethyl methacrylate) (PDMAEMA) nanogels were synthesized via surfactant-free free-radical polymerization technique in aqueous conditions utilizing N,N’-methylene-bis-acrylamide (MBA) as a crosslinking agent. The PDMAEMA nanogels were subsequently quaternized with acryloyl chloride in order to yield mucoadhesive materials which incorporate two mucoadhesive concepts; electrostatic interactions and covalent bond forming acrylate groups. The native PDMAEMA nanogels were found to exhibit good mucoadhesive properties on ex vivo bovine conjunctival tissues, which was found to increase proportionally with the degree of quaternization. With a view to determine the ocular drug delivery capabilities of the materials, both quaternized and native nanogels were loaded with pilocarpine hydrochloride via an absorption method, and their in vitro release profiles were analysed. The nanogels were found to exhibit a high loading capacity (>20% of total weight) and a sustained release over 6 h

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

The crystallization of three catalytically inactive mutants of penicillin Vacylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide threedimensional structures of precursor PVA forms, plus open a route to the study of enzyme-substrate complexes for this industrially important enzyme

Crystallization of the regulatory and effector domains of the key sporulation response regulator Spo0A

The key response-regulator gene of sporulation, spo0A, has been cloned from Bacillus stearothermophilus and the encoded protein purified. The DNA-binding and phospho-acceptor domains of Spo0A have been prepared by tryptic digestion of the intact protein and subsequently crystallized in forms suitable for X-ray crystallographic studies. The DNA-binding domain has been crystallized in two forms, one of which diffracts X-rays to beyond 2.5 Angstrom spacing. The crystals of the phospho-acceptor domain diffract X-rays beyond 2.0 Angstrom spacing using synchrotron radiation

The seasonal cycle of submesoscale flows

The seasonal cycle of submesoscale flows in the upper ocean is investigated in an idealised model domain analogous to mid-latitude open ocean regions. Submesoscale processes become much stronger as the resolution is increased, though with limited evidence for convergence of the solutions. Frontogenetical processes increase horizontal buoyancy gradients when the mixed layer is shallow in summer, while overturning instabilities weaken the horizontal buoyancy gradients as the mixed layer deepens in winter. The horizontal wavenumber spectral slopes of surface temperature and velocity are steep in summer and then shallow in winter. This is consistent with stronger mixed layer instabilities developing as the mixed layer deepens and energising the submesoscale. The degree of geostrophic balance falls as the resolution is made finer, with evidence for stronger non-linear and high-frequency processes becoming more important as the mixed layer deepens. Ekman buoyancy fluxes can be much stronger than surface cooling and are locally dominant in setting the stratification and the potential vorticity at fronts, particularly in the early winter. Up to 30% of the mixed layer volume in winter has negative potential vorticity and symmetric instability is predicted inside mesoscale eddies as well as in the frontal regions outside of the vortices

Evolution of oceanic near surface stratification in response to an autumn storm

Understanding the processes that control the evolution of the ocean surface boundary layer (OSBL) is a prerequisite for obtaining accurate simulations of air-sea fluxes of heat and trace gases. Observations of the rate of dissipation of turbulent kinetic energy (ɛ), temperature, salinity, current structure and wave-field over a period of 9.5 days in the NE Atlantic during the Ocean Surface Mixing, Ocean Submesoscale Interaction Study (OSMOSIS), are presented. The focus of this study is a storm which passed over the observational area during this period. The profiles of ɛ in the OSBL are consistent with profiles from large eddy simulation (LES) of Langmuir turbulence. In the transition layer (TL), at the base of the OSBL, ɛ was found to vary periodically at the local inertial frequency. A simple bulk model of the OSBL and a parametrisation of shear driven turbulence in the TL are developed. The parametrisation of ɛ is based on assumptions about the momentum balance of the OSBL and shear across the TL. The predicted rate of deepening, heat budget and the inertial currents in the OSBL were in good agreement with the observations, as is the agreement between the observed value of ɛ and that predicted using the parametrisation. A previous study reported spikes of elevated dissipation related to enhanced wind-shear alignment at the base of the OSBL after this storm. The spikes in dissipation are not predicted by this new parametrisation, implying that they are not an important source of dissipation during the storm

Crystal structures of the GH18 domain of the bifunctional peroxiredoxin-chitinase CotE from Clostridium difficile

CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349-712 has been crystallized and its structure has been determined to reveal a core eight-stranded β-barrel fold with a neighbouring subdomain containing a five-stranded β-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission

Tracing the origins of permitted emission lines in RU Lupi down to AU scales

Most of the observed emission lines and continuum excess from young accreting low mass stars (Classical T Tauri stars -- CTTSs) take place in the star-disk or inner disk region. These regions have a complex emission topology still largely unknown. In this paper the magnetospheric accretion and inner wind contributions to the observed permitted He and H near infrared (NIR) lines of the bright southern CTTS RU Lupi are investigated for the first time. Previous optical observations of RU Lupi showed a large H-alpha profile, due to the emission from a wind in the line wings, and a micro-jet detected in forbidden lines. We extend this analysis to NIR lines through seeing-limited high spectral resolution spectra taken with VLT/ISAAC, and adaptive optics (AO) aided narrow-band imaging and low spectral resolution spectroscopy with VLT/NACO. Using spectro-astrometric analysis we investigate the presence of extended emission down to very low spatial scales (a few AU). The HeI 10830 line presents a P Cygni profile whose absorption feature indicates the presence of an inner stellar wind. Moreover the spectro-astrometric analysis evidences the presence of an extended emission superimposed to the absorption feature and likely coming from the micro-jet detected in the optical. On the contrary, the origin of the Hydrogen Paschen and Brackett lines is difficult to address. We tried tentatively to explain the observed line profiles and flux ratios with both accretion and wind models showing the limits of both approaches. The lack of spectro-astrometric signal indicates that the HI emission is either compact or symmetric. Our analysis confirms the sensitivity of the HeI line to the presence of faint extended emission regions in the close proximity of the star.Comment: 11 pages, 4 figures, accepted for publication on A&

Seasonality of submesoscale flows in the ocean surface boundary layer

A signature of submesoscale flows in the upper ocean is skewness in the distribution of relative vorticity. Expected to result for high Rossby-number flows, such skewness has implications for mixing, dissipation and stratification within the upper ocean. An array of moorings deployed in the Northeast Atlantic for one year as part of the OSMOSIS experiment reveals that relative vorticity is positively skewed during winter even though the scale of the Rossby number is less than 0.5. Furthermore, this skewness is reduced to zero during spring and autumn. There is also evidence of modest seasonal variations in the gradient Rossby number. The proposed mechanism by which relative vorticity is skewed is that the ratio of lateral to vertical buoyancy gradients, as summarized by the inverse gradient Richardson number, restricts its range during winter but less so at other times of the year. These results support recent observations and model simulations suggesting the upper ocean is host to a seasonal cycle in submesoscale turbulence