Kyu-Yeon Lee

Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity

VapD-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3D structure of these proteins. Recently, a relationship between the Cas2 family of ribonucleases associated with the CRISPR system of microbial immunity and VapD was suggested. Here, we show for the first time the structure of a member of the VapD family and present a relationship of VapD with Cas2 family and toxin–antitoxin (TA) systems. The crystal structure of HP0315 from Helicobacter pylori was solved at a resolution of 2.8 Å. The structure of HP0315, which has a modified ferredoxin-like fold, is very similar to that of the Cas2 family. Like Cas2 proteins, HP0315 shows endoribonuclease activity. HP0315-cleaved mRNA, mainly before A and G nucleotides preferentially, which means that HP0315 has purine-specific endoribonuclease activity. Mutagenesis studies of HP0315 revealed that D7, L13, S43 and D76 residues are important for RNase activity, in contrast, to the Cas2 family. HP0315 is arranged as an operon with HP0316, which was found to be an antitoxin-related protein. However, HP0315 is not a component of the TA system. Thus, HP0315 may be an evolutionary intermediate which does not belong to either the Cas2 family or TA system.

Evaluation of Air Injection and Extraction Tests at a Petroleum Contaminated Site, Korea

Air injection and extraction tests were conducted at a site where soils and groundwater were contaminated by petroleum hydrocarbons, mainly composed of TEX (toluene, ethylbenzene,and xylene). Storage tanks of petroleum hydrocarbons located less than 20 m away from the center of the test site were suspected to be the contamination source. Six injection/extraction wells and 21 monitoring wells were installed to evaluate performance and radius of influence with respect tosoil vapor extraction and air injection. Effective radius ofinfluence of the air injection tests based on pressure changes ranged from 3.3 to 10.5 m. Soil gas pressures, concentrationsof O2, CH4 and CO2, and temperatures were measured during the tests. Air permeability and radii of influence with respect to gas pressure and oxygen supply by air injection were estimated. Dynamic changes in the concentrations of O2, CH4 and CO2 gases and temperature around the extent of their ranges were detected asair was injected. We evaluated extensively the variations ofthe geochemical parameters that were indicative of active microbial degradation in this study.

Ecological Risk Characterization in a Military Heavy Metals– and Explosives-Contaminated Site

ABSTRACT Potential ecological risks of two heavy metals (Cu, Pb) and three explosives (TNT, RDX, HMX) were determined for a military gunnery range. Since a portion of the site will be submerged after the construction of a flood control reservoir, risk assessment of the site was conducted over two time points: at the current state and after the construction of the flood control reservoir. Terrestrial plants, terrestrial invertebrates, birds, mammals, and aquatic invertebrates were selected as affected ecological receptors at the study site. Potential noncarcinogenic risk was found only in one terrestrial site for Cu and RDX at both time points: For terrestrial plants, ecological hazard quotient (EHQ) by Cu was 1.22. For terrestrial invertebrates, EHQs for Cu and RDX were 1.38 and 6.47, respectively. EHQs in the aquatic environment increased to some extents after the reservoir construction, but the values were less than 1.0. A comparison between EHQs before and after the construction for the same site shows that the reservoir construction would not significantly increase the overall ecological risk, suggesting that the reservoir construction influenced the potential ecological risks at the study site, but the changes were acceptable. Uncertainties involved in the assessment process were identified and discussed.

Evaluation of groundwater chemistry affected by an abandoned metal mine within a dam construction site, South Korea

Groundwater chemistry in and around an abandoned metal mine within a large dam construction site was evaluated. For this purpose, a total of 50 groundwater samples were collected and chemically analysed for major ions and toxic metals. The pHs of the groundwaters were near neutral to slightly basic, with an apparent increase along the flow direction away from the mine waste dump. Higher values of electrical conductivity with a mean of 532 μS cm−1 were observed in the mine area. Dissolved oxygen concentrations over 5 mg l−1 clearly indicated an oxygenated groundwater environment. The high concentration of sulphate was derived from weathering and oxidation of sulphide minerals. Hydrochemistry of the groundwater samples is characterized by the relatively significant enrichment of Ca2+ and SO42−. Results of factor analysis also indicated that the dominant mechanisms or continuing processes affecting the distribution of the chemical parameters in the study area are various geochemical reactions including dissolution of sulphide and carbonate minerals, and removal of calcium and sulphate by dilution through mixing with Ca2+− and SO42−-poor groundwaters. In the mine area, substantial groundwater contamination by toxic metals including Zn, Al, Fe and Mn was observed. Most of the toxic metals decreased gradually or dramatically with distance from the mine, although some metals were also found in high concentrations in the downstream areas. Compared with the contamination levels of the surrounding farmland soils and stream sediments, the levels of contamination of groundwater by toxic metals were relatively low. The low concentrations are ascribed to the high pH and highly oxygenated conditions, which reduce toxic metal mobility. Significant groundwater contamination with toxic metals was strictly limited within the immediate vicinity of the mine waste dump but the ubiquitous distribution of the toxic metals with slightly elevated levels all over the study area may be attributed to the same geology and mineralogy as in the mine area.

Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking and RNase activities

HP0268 is a conserved, uncharacterized protein from Helicobacter pylori. Here, we determined the solution structure of HP0268 using three-dimensional nuclear magnetic resonance (NMR) spectroscopy, revealing that this protein is structurally most similar to a small MutS-related (SMR) domain that exhibits nicking endonuclease activity. We also demonstrated for the first time that HP0268 is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. The nuclease activities for DNA and RNA were maximally increased by Mn2+ and Mg2+ ions, respectively, and decreased by Cu2+ ions. Using NMR chemical shift perturbations, the metal and nucleotide binding sites of HP0268 were determined to be spatially divided but close to each other. The lysine residues (Lys7, Lys11 and Lys43) are clustered and form the nucleotide binding site. Moreover, site-directed mutagenesis was used to define the catalytic active site of HP0268, revealing that this site contains two acidic residues, Asp50 and Glu54, in the metal binding site. The nucleotide binding and active sites are not conserved in the structural homologues of HP0268. This study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases.

Hydrogeological investigation and discharge control of a nutrient-rich acidic solution from a coastal phosphogypsum stack at Yeocheon, Korea

Nutrient-rich, highly acidic leachate draining from a coastalphosphogypsum storage site located at a phosphate fertilizer manufacturing company posed potentially harmful environmental effects to the coastal ecosystem. This study evaluated the chemical characteristics of the phosphogypsum and the hydrogeological characteristics of the surroundings. Hydraulic and chemical properties of the leachate draining from the site were also evaluated. The leachate is nutrient-rich and very acidic and discharges into the sea. The leachate and seawater are hydraulically connected through highly-permeable riprap placed at the toe of the stack. The chemical quality of the leachate and its drainage rate showed large variations depending on the location of the well, indicating heterogeneous hydraulic conditions. A vertical barrier system consisting of horizontal drainage wells and vertical leachate-collecting wells is suggested, in order to contain and collect the leachate. A back-up system of soil-bentonite cut-off walls is suggested to control leachate discharge to the sea.

Structural characterization of HP1264 reveals a novel fold for the flavin mononucleotide binding protein.

Complex I (NADH-quinone oxidoreductase) is an enzyme that catalyzes the initial electron transfer from nicotinamide adenine dinucleotide (NADH) to flavin mononucleotide (FMN) bound at the tip of the hydrophilic domain of complex I. The electron flow into complex I is coupled to the generation of a proton gradient across the membrane that is essential for the synthesis of ATP. However, Helicobacter pylori has an unusual complex I that lacks typical NQO1 and NQO2 subunits, both of which are generally included in the NADH dehydrogenase domain of complex I. Here, we determined the solution structure of HP1264, one of the unusual subunits of complex I from H. pylori, which is located in place of NQO2, by three-dimensional nuclear magnetic resonance (NMR) spectroscopy and revealed that HP1264 can bind to FMN through UV-visible, fluorescence, and NMR titration experiments. This result suggests that FMN-bound HP1264 could be involved in the initial electron transfer step of complex I. In addition, HP1264 is structurally most similar to Escherichia coli TusA, which belongs to the SirA-like superfamily having an IF3-like fold in the SCOP database, implying that HP1264 adopts a novel fold for FMN binding. On the basis of the NMR titration data, we propose the candidate residues Ile32, Met34, Leu58, Trp68, and Val71 of HP1264 for the interaction with FMN. Notably, these residues are not conserved in the FMN binding site of any other flavoproteins with known structure. This study of the relationship between the structure and FMN binding property of HP1264 will contribute to improving our understanding of flavoprotein structure and the electron transfer mechanism of complex I.

Slowly released molasses barrier system for controlling nitrate plumes in groundwater: A pilot-scale tank study

A well-type barrier system containing solidified molasses as a reactive medium was developed to promote the indigenous denitrifying activity and to treat nitrate plumes in groundwater. Three slowly released molasses (SRM) barrier systems harboring 60, 120, and 120 SRM rods, which were named System A, B, and C, respectively, were operated to examine nitrate removal efficiency in a pilot-scale sandy tank. These SRM systems induced a consistent removal of nitrate without pore clogging and hydraulic disturbance during the test period. The initial nitrate concentration was 142mgL(-1), and the concentrations decreased by 80%, 84%, and 79% in System A, B, and C, respectively. In particular, System C was inoculated with heterotrophic denitrifiers, but the nitrate removal efficiency was not enhanced compared to System B, probably due to the prior existence of indigenous denitrifiers in the sandy tank. The presence of nitrite reductase-encoding gene (i.e. nirK) at the site was confirmed by denatured gradient gel electrophoresis analysis.

Backbone 1 H, 15 N, and 13 C resonance assignments and secondary structure prediction of NifU-like protein, HP1492 from Helicobacter Pylori

HP1492 is a NifU-like protein of Helicobacter pylori (H. pylori) and plays a role as a scaffold which transfer Fe-S cluster to Fe-S proteins like Ferredoxin. To understand how to bind to iron ion or iron-sulfur cluster, HP1492 was expressed and purified in Escherichia coli (E. coli). From the NMR measurement, we could carry out the sequence specific backbone resonance assignment of HP1492. Approximately 91% of all resonances could be assigned unambiguously. By analyzing results of CSI and TALOS from NMR data, we could predict the secondary structure of HP1492, which consists of three -helices and three -sheets. This study is an essential step towards the structural characterization of HP1492.

Regulatory mechanisms of thiol-based redox sensors: lessons learned from structural studies on prokaryotic redox sensors

Oxidative stresses, such as reactive oxygen species, reactive electrophilic species, reactive nitrogen species, and reactive chlorine species, can damage cellular components, leading to cellular malfunction and death. In response to oxidative stress, bacteria have evolved redox-responsive sensors that enable them to simultaneously monitor and eradicate potential oxidative stress. Specifically, redox-sensing transcription regulators react to oxidative stress by means of modifying the thiol groups of cysteine residues, functioning as part of an efficient survival mechanism for many bacteria. In general, oxidative molecules can induce changes in the three-dimensional structures of redox sensors, which, in turn, affects the transcription of specific genes in detoxification pathways and defense mechanisms. Moreover, pathogenic bacteria utilize these redox sensors for adaptation and to evade subsequent oxidative attacks from host immune defense. For this reason, the redox sensors of pathogenic bacteria are potential antibiotic targets. Understanding the regulatory mechanisms of thiol-based redox sensors in bacteria will provide insight and knowledge into the discovery of new antibiotics.