Efficient purification and reconstitution of ATP binding cassette transporter B6 (ABCB6) for functional and structural studies.

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (Kd = 0.18 µM) and an ATPase activity with a Km of 0.99 mM. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.

Attachment surface energy effects on nitrification and estrogen removal rates by biofilms for improved wastewater treatment.

Submerged biofilm systems, such as integrated fixed-film activated sludge (IFAS) and moving bed bioreactors (MBBRs), are increasingly being used for domestic wastewater treatment, often to improve nitrification. Little is known about whether and how biofilm attachment surface chemical properties affect treatment performance, although surface chemistry is known to affect attachment in other systems, and work with pure strains has suggested that attachment of nitrifying bacteria may be enhanced on high surface energy surfaces. The objective of this research was to systematically evaluate the effects of surface chemistry on biofilm quantity and rates of nitrification and estrogen removal. Biofilms were grown on four plastic attachment surfaces with a range of hydrophobicity and surface energy values (nylon, melamine, high-density-polyethylene [HDPE], and acetal polymeric plastic) by immersing them in a full scale nitrifying activated sludge wastewater treatment system, followed by batch test experiments. The attachment surface water contact angles ranged from 53° to 98° and surface energies ranged from 48.9 to 20.9 mJ/m(2). Attachment surface hydrophilicity and surface energy were positively correlated with total biomass attachment, with more than twice as much biomass on the highest surface energy, most hydrophilic surface (nylon) than on the lowest surface energy, least hydrophilic surface (acetal plastic). Absolute and specific nitrification rates were also correlated with hydrophilicity and surface energy (varying by factors of 5 and 2, respectively), as were absolute and specific removal first order rate constants of the hormones estrone (E1), ß-estradiol (E2) and 17α-ethynylestradiol (EE2). These results suggested that attachment surface chemistry may be a useful design parameter for improving biofilm performance for removal of ammonia and endocrine disrupting hormones from wastewater. Further research is required to verify these results at longer time scales and with typical media geometries.

Combined effects of EPS and HRT enhanced biofouling on a submerged and hybrid PAC-MF membrane bioreactor.

The goal of this study was to quantify and demonstrate the dynamic effects of hydraulic retention time (HRT), organic carbon and various components of extracellular polymeric substances (EPS) produced by microorganisms on the performance of submersed hollow-fiber microfiltration (MF) membrane in a hybrid powdered activated carbon (PAC)-MF membrane bioreactor (MBR). The reactors were operated continuously for 45 days to treat surface (river) water before and after pretreatment using a biofiltration unit. The real-time levels of organic carbon and the major components of EPS including five different carbohydrates (D(+) glucose and D(+) mannose, D(+) galactose, N-acetyl-D-galactosamine and D-galactose, oligosaccharides and L(-) fucose), proteins, and polysaccharides were quantified in the influent water, foulants, and in the bulk phases of different reactors. The presence of PAC extended the filtration cycle and enhanced the organic carbon adsorption and removal more than two fold. Biological filtration improved the filtrate quality and decreased membrane fouling. However, HRT influenced the length of the filtration cycle and had less effect on organic carbon and EPS component removal and/or biodegradation. The abundance of carbohydrates in the foulants on MF surfaces was more than 40 times higher than in the bulk phase, which demonstrates that the accumulation of carbohydrates on membrane surfaces contributed to the increase in transmembrane pressure significantly and PAC was not a potential adsorbent of carbohydrates. The abundance of N-acetyl-d-galactosamine and d-galactose was the highest in the foulants on membranes receiving biofilter-treated river water. Most of the biological fouling compounds were produced inside the reactors due to biodegradation. PAC inside the reactor enhanced the biodegradation of polysaccharides up to 97% and that of proteins by more than 95%. This real-time extensive and novel study demonstrates that the PAC-MF hybrid MBR is a sustainable technology for treating river water.

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

Characterization and effect of biofouling on polyamide reverse osmosis and nanofiltration membrane surfaces.

Biofouling is a major reason for flux decline in the performance of membrane-based water and wastewater treatment plants. Initial biochemical characterization of biofilm formation potential and biofouling on two commercially available membrane surfaces from FilmTec Corporation were investigated without filtration in laboratory rotating disc reactor systems. These surfaces were polyamide aromatic thin-film reverse osmosis (RO) (BW30) and semi-aromatic nanofiltration (NF270) membranes. Membrane swatches were fixed on removable coupons and exposed to water with indigenous microorganisms supplemented with 1.5 mg l(-1) organic carbon under continuous flow. After biofilms formed, the membrane swatches were removed for analyses. Staining and epifluorescence microscopy revealed more cells on the RO than on the NF surface. Based on image analyses of 5-µm thick cryo-sections, the accumulation of hydrated biofoulants on the RO and NF surfaces exceeded 0.74 and 0.64 µm day(-1), respectively. As determined by contact angle the biofoulants increased the hydrophobicity up to 30° for RO and 4° for NF surfaces. The initial difference between virgin RO and NO hydrophobicities was ∼5°, which increased up to 25° after biofoulant formation. The initial roughness of RO and NF virgin surfaces (75.3 nm and 8.2 nm, respectively) increased to 48 nm and 39 nm after fouling. A wide range of changes of the chemical element mass percentages on membrane surfaces was observed with X-ray photoelectron spectroscopy. The initial chemical signature on the NF surface was better restored after cleaning than the RO membrane. All the data suggest that the semi-aromatic NF surface was more biofilm resistant than the aromatic RO surface. The morphology of the biofilm and the location of active and dead cell zones could be related to the membrane surface properties and general biofouling accumulation was associated with changes in the surface chemistry of the membranes, suggesting the validity of the combination of these novel approaches for initial assessment of membrane performance.

Experimental and theoretical examination of surface energy and adhesion of nitrifying and heterotrophic bacteria using self-assembled monolayers.

Biofilm-based systems, including integrated fixed-film activated sludge and moving bed bioreactors, are becoming increasingly popular for wastewater treatment, often with the goal of improving nitrification through the enrichment of ammonia and nitrite oxidizing bacteria. We have previously demonstrated the utility of self-assembled monolayers (SAMs) as tools for studying the initial attachment of bacteria to substrata systematically varying in physicochemical properties. In this work, we expanded these studies to bacteria of importance in wastewater treatment systems and we demonstrated attachment rates were better correlated with surface energy than with wettability (water contact angle). Toward the long-term goal of improving wastewater treatment performance through the strategic design of attachment substrata, the attachment rates of two autotrophic ammonia-oxidizing bacteria (Nitrosomonas europaea and Nitrosospira multiformis) and a heterotroph (Escherichia coli) were evaluated using SAMs with a range of wettabilities, surface energies, and functional properties (methyl, hydroxyl, carboxyl, trimethylamine, and amine terminated). Cell attachment rates were somewhat correlated with the water contact angles of the SAMs with polar terminal groups (hydroxyl, carboxyl, trimethylamine, and amine). Including all SAM surfaces, a better correlation was found for all bacteria between attachment rates and surface free energy, as determined using the Lewis Acid-Base approach. The ammonia-oxidizers had higher adhesion rates on the SAMs with higher surface energies than did the heterotroph. This work demonstrated the successful application of SAMs to determine the attachment surface preferences of bacteria important to wastewater treatment, and it provides guidance for a new area of research aimed at improving treatment performance through rational attachment surface design.