Metabolomics-based profiling of three terminal alkene-producing Jeotgalicoccus spp. during different growth phase.

Production of terminal alkenes by microbes has gained importance due to its role as a chemical feedstock in commercial industries. Jeotgalicoccus species has been widely unexplored despite being well-known as a natural producer of terminal alkene, catalyzing the one-step fatty acid decarboxylation reaction by OleTJE cytochrome P450. In this study, widely targeted ion-pair LC-MS/MS was used to monitor central carbon metabolism of Jeotgalicoccus halotolerans JCM 5429, Jeotgalicoccus huakuii JCM 8176, and Jeotgalicoccus psychrophilus JCM 5429 at logarithmic and stationary phases. Growth and production profile of terminal alkene, alcohols and organic acids were also measured. Among the three strains used in this study, J. halotolerans and J. psychrophilus showed higher terminal alkene production compared to J. huakuii. All strains achieved maximum terminal alkene production at logarithmic phase and therefore, detailed analysis of the metabolite profiles of the three strains were performed in logarithmic phase. PCA analysis showed that the strains were discriminated based on their ability to produce terminal alkene along PC1 and some of the important metabolites corresponding to this separation is the acetyl-CoA and 2-oxoglutarate. This study is the first report on metabolite profiling of three Jeotgalicoccus spp. in different growth phases. The results from this study can provide a better understanding of the changes that occur in the metabolome level during growth and production of terminal alkene in Jeotgalicoccus species.

Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE.

The Jeotgalicoccus sp. peroxygenase cytochrome P450 OleTJE (CYP152L1) is a hydrogen peroxide-driven oxidase that catalyzes oxidative decarboxylation of fatty acids, producing terminal alkenes with applications as fine chemicals and biofuels. Understanding mechanisms that favor decarboxylation over fatty acid hydroxylation in OleTJE could enable protein engineering to improve catalysis or to introduce decarboxylation activity into P450s with different substrate preferences. In this manuscript, we have focused on OleTJE active site residues Phe79, His85, and Arg245 to interrogate their roles in substrate binding and catalytic activity. His85 is a potential proton donor to reactive iron-oxo species during substrate decarboxylation. The H85Q mutant substitutes a glutamine found in several peroxygenases that favor fatty acid hydroxylation. H85Q OleTJE still favors alkene production, suggesting alternative protonation mechanisms. However, the mutant undergoes only minor substrate binding-induced heme iron spin state shift toward high spin by comparison with WT OleTJE, indicating the key role of His85 in this process. Phe79 interacts with His85, and Phe79 mutants showed diminished affinity for shorter chain (C10-C16) fatty acids and weak substrate-induced high spin conversion. F79A OleTJE is least affected in substrate oxidation, whereas the F79W/Y mutants exhibit lower stability and cysteine thiolate protonation on reduction. Finally, Arg245 is crucial for binding the substrate carboxylate, and R245E/L mutations severely compromise activity and heme content, although alkene products are formed from some substrates, including stearic acid (C18:0). The results identify crucial roles for the active site amino acid trio in determining OleTJE catalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.

Catalytic strategy for carbon-carbon bond scission by the cytochrome P450 OleT.

OleT is a cytochrome P450 that catalyzes the hydrogen peroxide-dependent metabolism of Cn chain-length fatty acids to synthesize Cn-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe(4+)-oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C-H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe(4+)-OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon-carbon scission reaction catalyzed by OleT.

A high-sensitivity fiber-optic evanescent wave sensor with a three-layer structure composed of Canada balsam doped with GeO2.

In this paper, we present a high-sensitivity polymer fiber-optic evanescent wave (FOEW) sensor with a three-layer structure that includes bottom, inter-, and surface layers in the sensing region. The bottom layer and inter-layer are POFs composed of standard cladding and the core of the plastic optical fiber, and the surface layer is made of dilute Canada balsam in xylene doped with GeO2. We examine the morphology of the doped GeO2, the refractive index and composition of the surface layer and the surface luminous properties of the sensing region. We investigate the effects of the content and morphology of the GeO2 particles on the sensitivity of the FOEW sensors by using glucose solutions. In addition, we examine the response of sensors incubated with staphylococcal protein A plus mouse IgG isotype to goat anti-mouse IgG solutions. Results indicate very good sensitivity of the three-layer FOEW sensor, which showed a 3.91-fold improvement in the detection of the target antibody relative to a conventional sensor with a core-cladding structure, and the novel sensor showed a lower limit of detection of 0.2ng/l and a response time around 320s. The application of this high-sensitivity FOEW sensor can be extended to biodefense, disease diagnosis, biomedical and biochemical analysis.

Probing sequence dependence of folding pathway of α-helix bundle proteins through free energy landscape analysis.

A comparative study on the folding of multiple three-α-helix bundle proteins including α3D, α3W, and the B domain of protein A (BdpA) is presented. The use of integrated-tempering-sampling molecular dynamics simulations achieves reversible folding and unfolding events in individual short trajectories, which thus provides an efficient approach to sufficiently sample the configuration space of protein and delineate the folding pathway of α-helix bundle. The detailed free energy landscape analyses indicate that the folding mechanism of α-helix bundle is not uniform but sequence dependent. A simple model is then proposed to predict folding mechanism of α-helix bundle on the basis of amino acid composition: α-helical proteins containing higher percentage of hydrophobic residues than charged ones fold via nucleation-condensation mechanism (e.g., α3D and BdpA) whereas proteins having opposite tendency in amino acid composition more likely fold via the framework mechanism (e.g., α3W). The model is tested on various α-helix bundle proteins, and the predicted mechanism is similar to the most approved one for each protein. In addition, the common features in the folding pathway of α-helix bundle protein are also deduced. In summary, the present study provides comprehensive, atomic-level picture of the folding of α-helix bundle proteins.

Nosocomiicoccus ampullae gen. nov., sp. nov., isolated from the surface of bottles of saline solution used in wound cleansing.

Strains TRF-1(T) and TC-9 were isolated from transfer spikes of two separate bottles containing 0.9 % NaCl (physiological saline) solution used repeatedly to wash wounds in hospital wards, months apart. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains TRF-1(T) and TC-9 formed a distinct branch within the family Bacillaceae most closely related to the members of the genus Jeotgalicoccus. The two strains, with identical 16S rRNA gene sequences, showed sequence similarities of 89.8-93.9 % with species of Jeotgalicoccus, 91.1-91.8 % with species of Salinicoccus and 89.1-89.7 % with species of Staphylococcus. Strains TRF-1(T) and TC-9 are Gram-positive, non-spore-forming and non-motile cocci, with an optimum growth temperature of about 37 degrees C. Strain TRF-1(T) grew optimally in medium containing 3 % (w/v) NaCl (maximum about 14 % NaCl), while strain TC-9 grew optimally in medium with 1 % (w/v) NaCl. Both strains produce a brown pigment when grown in the presence of NaCl. The cell-wall peptidoglycan is of the A3alpha type with a cross-linkage containing the peptide l-Lys-Gly(4)-l-Ser-Gly. The major respiratory quinones are menaquinone 7 (MK-7) and menaquinone 8 (MK-8), the major fatty acids are straight-chain C(14 : 0) and C(16 : 0) (more than 85 % of the total) and the major polar lipid is an unknown aminophospholipid. The DNA G+C content is 33.5 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics, we are of the opinion that strains TRF-1(T) and TC-9 represent a novel species of a new genus, for which we propose the name Nosocomiicoccus ampullae gen. nov., sp. nov. The type strain of Nosocomiicoccus ampullae is strain TRF-1(T) (=LMG 24060(T) =CIP 109506(T)).

A covalent S-F heterodimer of leucotoxin reveals molecular plasticity of beta-barrel pore-forming toxins.

Staphylococcal leucotoxins, leucocidins, and gamma-hemolysins are bicomponent beta-barrel pore-forming toxins (beta-PFTs). Their production is associated with several clinical diseases. They have cytotoxic activity due to the synergistic action of a class S component and a class F component, which are secreted as water-soluble monomers and form hetero-oligomeric transmembrane pores, causing the lysis of susceptible cells. Structural information is currently available for the monomeric S and F proteins and the homoheptamer formed by the related alpha-hemolysin. These structures illustrate the start and end points in the mechanistic framework of beta-PFT assembly. Only limited structural data exist for the intermediate stages, including hetero-oligomeric complexes of leucotoxins. We investigated the protein-protein interactions responsible for maintaining the final bipartite molecular architecture and describe here the high-resolution crystal structure and low-resolution solution structure of a site-specific cross-linked heterodimer of gamma-hemolysin (HlgA T28C-HlgB N156C), which were solved by X-ray crystallography and small angle X-ray scattering, respectively. These structures reveal a molecular plasticity of beta-PFTs, which may facilitate the transition from membrane-bound monomers to heterodimers.

Salinicoccus luteus sp. nov., isolated from a desert soil.

A moderately halophilic bacterium, strain YIM 70202(T), was isolated from a desert soil sample collected from Egypt and was subjected to a taxonomic investigation. In a phylogenetic dendrogram based on 16S rRNA gene sequence analysis, strain YIM 70202(T) was affiliated to the Salinicoccus clade, showing 94.5-96.8 % 16S rRNA gene sequence similarity to the recognized species of the genus Salinicoccus, in which Salinicoccus roseus CCM 3516(T) was the nearest neighbour. The DNA-DNA relatedness value of the novel isolate with S. roseus CCM 3516(T) was 12.7 %. The novel isolate grew at temperatures between 4 and 45 degrees C and at pH values ranging from 7.0 to 11.0, with an optimum of 30 degrees C and pH 8.0-9.0, respectively. Strain YIM 70202(T) grew optimally in the presence of 10 % NaCl (w/v) and growth was observed at NaCl concentrations in the range 1-25 % (w/v). Chemotaxonomic data revealed that strain YIM 70202(T) contained MK-6 as the predominant respiratory quinone, possessed L-Lys-Gly(5) as the cell-wall peptidoglycan, had phosphatidylglycerol, diphosphatidylglycerol and an unknown glycolipid as the polar lipids and contained i-C(15 : 0) and ai-C(15 : 0) as the predominant fatty acids. The DNA G+C content was 49.7 mol%. The biochemical and chemotaxonomic properties demonstrate that strain YIM 70202(T) represents a novel species of the genus Salinicoccus. The name Salinicoccus luteus sp. nov. is proposed with strain YIM 70202(T) (=CGMCC 1.6511(T)=KCTC 3941(T)) as the type strain.