In Staphylococcus aureus, virulence and colonization-associated surface proteins are covalently anchored to the cell wall by the transpeptidase Sortase A (SrtA). In order to better understand the contribution of specific active site residues to substrate recognition and catalysis, we performed mutational analysis of several key residues in the SrtA active site. Analysis of protein stability, kinetic parameters, solvent isotope effects, and pH-rate profiles for key SrtA variants are consistent with a reverse protonated Cys184-His120 catalytic dyad, and implicate a role for Arg197 in formation of an oxyanion hole to stabilize the transition state. In contrast, mutation of Asp185 and Asp186 produced negligible effects on catalysis, and no evidence was found to support the existence of a functional catalytic triad. Mutation of Thr180, Leu181, and Ile182 to alanine produced modest decreases in SrtA activity and led to substrate inhibition. Thermodynamic stability measurements by SUPREX (stability of unpurified proteins from rates of H/D exchange) revealed decreases in conformational stability that correlate with the observed substrate inhibition for each variant, signifying a potential role for the conserved 180TLITC184 motif in defining the active-site architecture of SrtA. In contrast, mutation of Thr183 to alanine led to a significant 1200-fold decrease in kcat, which appears to be unrelated to conformational stability. Potential explanations for these results are discussed, and a revised model for SrtA catalysis is presented.

The equilibrium unfolding properties of four model protein systems were characterized using SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is an H/D exchange- and mass spectrometry-based technique for measuring the free energy (DeltaGf) and m-value (deltaDeltaGf/delta[denaturant]) associated with the folding/unfolding reaction of a protein. The model proteins in this study (calmodulin, carbonic anhydrase II, RmlB, Bcl-xL) were chosen to test the applicability of SUPREX to the thermodynamic analysis of larger (> approximately 15 kDa) or multidomain proteins. In the absence of ligand, DeltaGf and m-values for these proteins could not be evaluated using the conventional data acquisition and analysis methods previously established for SUPREX. However, ligand-bound forms of the proteins were amenable to conventional SUPREX analyses, and it was possible to evaluate reasonably accurate and precise binding free energies of selected ligands. In some cases, protein-ligand dissociation constants (Kd values) could also be ascertained. The SUPREX-derived binding free energies and Kd values evaluated here were in good agreement with those reported on the same complexes using other techniques.

Six new p-terphenyl derivatives, named 4″-deoxy-3-hydroxyterphenyllin (1), 4″-deoxy-5'-desmethyl-terphenyllin (2), 5'-desmethylterphenyllin (3), 4″-deoxycandidusin A (4), 4,5-dimethoxycandidusin A (5), and terphenolide (6), four new diterpenoids with norcleistanthane (aspergiloid A (12) and aspergiloid B (13)), cleistanthane (aspergiloid C (14)), and isopimarane (aspergiloid D (15)) type skeletons, and five known p-terphenyl compounds (7-11) were isolated from the fermentation broth of the plant endophytic fungus Aspergillus sp. Their structures were elucidated on the basis of detailed spectroscopic analysis and by comparison of their NMR data with those reported in the literature. Compounds 4, 6, 7, and 9 displayed moderate neuraminidase inhibitory activity with IC(50) values ranging from 4.34 to 9.17 μM.

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The eddy-abundant circulation in the northern South China Sea (NSCS) tends to be dynamically complex due to monsoon forcing, Kuroshio intrusion, and emitted internal waves from the Luzon Strait. This study uses 13-year shipboard acoustic Doppler current profiler measurements (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016), orbital altimeter data, and high-resolution model output to perform wave-vortex decompositions and investigate the scale of transition from dominantly geostrophic flows to internal wave motions in the northern South China Sea. The upper ocean kinetic energy spectra transition on scales exceeds 200 km. This large scale of transition is attributed to the energetic low-mode internal waves (e.g., internal tides and inertia-gravity waves). However, inconsistencies in the decomposition reveal that the assumptions of homogeneity and isotropy required for the 1-D decomposition (Bühler et al., 2014) are sufficiently violated at smaller scales to affect the subdominant member of the decomposition on scales below 100 km. A method for direct quantification of the consequences by degree of violation using bootstrapping of the 2-D model data is developed and illustrated. Observed and modeled sea surface height spectra flatten at scales smaller than 125 km, which is found in the model to be due to the coherent, semidiurnal internal waves. The large scale of transition between geostrophic and wave motions in the South China Sea is an irreducible uncertainty for altimeter velocities (e.g., the Surface Water and Ocean Topography mission). Plain Language SummaryThe scale transition from balanced geostrophic flows to unbalanced motions is of great importance for understanding the circulation system of the South China Sea where both eddies and internal waves are particularly active. Based on observations and high-resolution simulation, this paper investigates the wave number spectra of kinetic energy and sea surface height variance in the northern South China Sea. The large transition length scale (>200 km) from balanced geostrophic flows to unbalanced motions is due to strong low-mode internal waves in the upper ocean. The violations of the assumptions for the 1-D decomposition (stationarity, homogeneity, and horizontal isotropy) are also assessed using the 2-D model data. With the model output, the low-mode semidiurnal internal tides are found to have significant impacts on the sea surface height variance at scales of~125 km. These results are directly relevant to the efficiency of diagnosing geostrophic flows from altimeters (e.g., the upcoming Surface Water and Ocean Topography mission).

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Each plant species in nature harbors endophytes, a community of microbes living within host plants without causing any disease symptom. However, the exploitation of endophyte-based phytoprotectants is hampered by the paucity of mechanistic understandings of endophyte-plant interaction. We here reported two endophytic Streptomyces isolates IFB-A02 and IFB-A03 recovered from a stress-tolerant dicotyledonous plant Artemisia annua L. After the determination of their non-pathogenicity at the genomic level and from the toxin (thaxtomin A, TXT) level, the endophytism of both isolates was supported by their successful colonization in planta. Of the two endophytes, IFB-A03 was further studied for the mechanism of endophyte-conferred phytoprotection owing to its plant growth promotion in model eudicot Arabidopsis thaliana. Using the endophyte-Arabidopsis co-cultivation system into which pathogenic Streptomyces scabies was introduced, we demonstrated that IFB-A03 pre-inoculation could activate the salicylic acid (SA)-mediated plant defense responses upon pathogen challenge. Moreover, IFB-A03 was shown to partially rescue the defense deficiency in eds5 (enhanced disease susceptibility 5) Arabidopsis mutants, putatively acting at the upstream of SA accumulation in the defense signaling pathway associated with the systemic acquired resistance (SAR). These data suggest that endophytic Streptomyces sp. IFB-A03 could be a promising candidate for biocontrol agents against S. scabies--a causative pathogen of common scab diseases prevailing in agronomic systems.