Time- and intensity-dependent broadband cavity-enhanced absorption spectroscopy with pulsed intra-cavity laser-induced plasmas

A pulsed laser-induced plasma (LIP) was generated in ambient air inside a high-finesse (F ≈ 5200) near-concentric optical cavity. The optical plasma emission was successfully trapped and sustained by the cavity, manifested by ring-down times in excess of 4 μs indicating effective mirror reflectivities of ∼0.9994. The light leaking from the cavity was used to measure broadband absorption spectra of gaseous azulene under ambient air conditions between 580 and 645 nm, employing (i) intensity-dependent cavity-enhanced, and (ii) time-dependent cavity-ring down methodologies. Minimum detectable absorption coefficients of 4.7 × 10−8 cm−1 and 7.4 × 10−8 cm−1 were achieved for the respective approaches. The two approaches were compared and implications of pulsed excitation for gated intensity-dependent measurements were discussed

Bacteriophage-Derived Peptidase CHAP

New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors

Crystallization Of The CHAP Domain Of The Endolysin From Staphylococcus Aureus Bacteriophage K

CHAPK is the N-terminal cysteine, histidine-dependent amidohydrolase/peptidase domain (CHAP domain) of the Staphylococcus aureus bacteriophage K endolysin LysK. It is formed from the first 165 residues of LysK and functions by cleaving specific peptidoglycan peptide bonds, causing bacterial lysis. CHAPK can lyse S. aureus when applied exogenously, making it a good candidate for the treatment of multidrug-resistant Staphylococcus aureus infections. Here, the crystallization of CHAPK and the collection of native and derivative data to high resolution, which allowed structure solution, are reported. The structure may help to elucidate the mechanism of action and in the design of chimeric proteins or mutants with improved antibacterial activity

The Eighth Central European Conference "Chemistry towards Biology": snapshot

The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August – 1 September 2016The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August-1 September 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting

Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K

[Background] Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge.[Methods] The CHAPK domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants.[Results] The structure of CHAPK revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAPK contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity.[Conclusions] The high-resolution structure of the CHAPK domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAPK with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X grant agreement no. 283570 and was sponsored by grant BFU2011-24843 (MJvR) from the Spanish Ministry of Economy and Competitiveness, a Masters fellowship (MSG) and an FPU Ph.D. fellowship (CGD) from the Spanish Ministry of Education, Culture and Sports. We also acknowledge financial support from TSR-StrandIII:CRS/07/CR03 and FIRM:08RDCIT600 of the Irish Department of Agriculture

Magnesium rescues the morphology of Bacillus subtilis mreB mutants through its inhibitory effect on peptidoglycan hydrolases

Cell wall homeostasis in bacteria is tightly regulated by balanced synthesis and degradation of peptidoglycan (PG), allowing cells to expand their sacculus during growth while maintaining physical integrity. In rod-shaped bacteria, actin-like MreB proteins are key players of the PG elongation machinery known as the Rod complex. In the Gram-positive model bacterium Bacillus subtilis depletion of the essential MreB leads to loss of rod shape and cell lysis. However, millimolar concentrations of magnesium in the growth medium rescue the viability and morphological defects of mreB mutants by an unknown mechanism. Here, we used a combination of cytological, biochemical and biophysical approaches to investigate the cell surface properties of mreB null mutant cells and the interactions of Mg2+ with the cell wall of B. subtilis. We show that ΔmreB cells have rougher and softer surfaces, and changes in PG composition indicative of increased DL- and DD-endopeptidase activities as well as increased deacetylation of the sugar moieties. Increase in DL-endopeptidase activity is mitigated by excess Mg2+ while DD-endopeptidase activity remains high. Visualization of PG degradation in pulse-chase experiments showed anisotropic PG hydrolase activity along the sidewalls of ΔmreB cells, in particular at the sites of increased cell width and bulging, while PG synthesis remained isotropic. Overall, our data support a model in which divalent cations maintain rod shape in ΔmreB cells by inhibiting PG hydrolases, possibly through the formation of crosslinks with carboxyl groups of the PG meshwork that affect the capacity of PG hydrolases to act on their substrate

Hydrogenated silicon nanoclusters with a permanent electric dipole moment for the controlled assembly of silicon-based nanostructures

While silicon nanoclusters have extensively been used for their outstanding properties for many decades, never before has their dipole moment been exploited for any application. Here, we have succeeded in producing hydrogenated silicon nanoclusters with a strong permanent electric dipole moment. This dipole moment allows us to use electric fields in order to orient and guide individual clusters. As a first example, we demonstrate the catalyst-free one-by-one self-assembly of one of the thinnest silicon nanowires yet observed. As a second example, we show that the simple presence of those nanoclusters on LaB6 cathodes leads to a 30-fold enhancement of the thermionic electron current density over pristine LaB6. Last but not least, the nanoclusters provide a protective layer against chemical and mechanical attack and largely prevent the evaporation of substrate materials, potentially increasing the operational lifetime of cathodes substantially

Hydrolysis of peptidoglycan is modulated by amidation of meso-diaminopimelic acid and Mg2+ in Bacillus subtilis

The ability of excess Mg2+ to compensate the absence of cell wall related genes in Bacillus subtilis has been known for a long time, but the mechanism has remained obscure. Here, we show that the rigidity of wild-type cells remains unaffected with excess Mg2+, but the proportion of amidated meso-diaminopimelic (mDAP) acid in their peptidoglycan (PG) is significantly reduced. We identify the amidotransferase AsnB as responsible for mDAP amidation and show that the gene encoding it is essential without added Mg2+. Growth without excess Mg2+ causes asnB mutant cells to deform and ultimately lyse. In cell regions with deformations, PG insertion is orderly and indistinguishable from the wild-type. However, PG degradation is unevenly distributed along the sidewalls. Furthermore, asnB mutant cells exhibit increased sensitivity to antibiotics targeting the cell wall. These results suggest that absence of amidated mDAP causes a lethal deregulation of PG hydrolysis that can be inhibited by increased levels of Mg2+. Consistently, we find that Mg2+ inhibits autolysis of wild-type cells. We suggest that Mg2+ helps to maintain the balance between PG synthesis and hydrolysis in cell wall mutants where this balance is perturbed in favor of increased degradation