Structural features of halophilicity derived from the crystal structure of dihydrofolate reductase from the Dead Sea halophilic archaeon, Haloferax volcanii

AbstractBackground: The proteins of halophilic archaea require high salt concentrations both for stability and for activity, whereas they denature at low ionic strength. The structural basis for this phenomenon is not yet well understood. The crystal structure of dihydrofolate reductase (DHFR) from Haloferax volcanii (hv-DHFR) reported here provides the third example of a structure of a protein from a halophilic organism. The enzyme is considered moderately halophilic, as it retains activity and secondary structure at monovalent salt concentrations as low as 0.5 M.Results: The crystal structure of hv-DHFR has been determined at 2.6 å resolution and reveals the same overall fold as that of other DHFRs. The structure is in the apo state, with an open conformation of the active-site gully different from the open conformation seen in other DHFR structures. The unique feature of hv-DHFR is a shift of the α helix encompassing residues 46–51 and an accompanied altered conformation of the ensuing loop relative to other DHFRs. Analysis of the charge distribution, amino acid composition, packing and hydrogen-bonding pattern in hv-DHFR and its non-halophilic homologs has been performed.Conclusions: The moderately halophilic behavior of hv-DHFR is consistent with the lack of striking structural features expected to occur in extremely halophilic proteins. The most notable feature of halophilicity is the presence of clusters of non-interacting negatively charged residues. Such clusters are associated with unfavorable electrostatic energy at low salt concentrations, and may account for the instability of hv-DHFR at salt concentrations lower than 0.5 M. With respect to catalysis, the open conformation seen here is indicative of a conformational transition not reported previously. The impact of this conformation on function and/or halophilicity is unknown

Development of Anti-Fouling, Anti-Microbial Membranes by Chemical Patterning

Over 1 billion people lack access to clean drinking water. Membranes are a tool that can help provide clean water to these people. However, treatment of impaired waters for beneficial use exposes the membranes to feed waters containing biological and abiotic species, which leads to fouling and loss of membrane productivity over time. Since reduction in flux due to fouling is one of the largest costs associated with membrane processes in water treatment, new coatings that limit fouling would have significant economic and societal impacts. Developing these advanced coatings is the focus of our work

Development of Anti-Fouling, Anti-Microbial Membranes by Chemical Patterning

Membranes are a tool that can help provide clean water to people. However, treatment of impaired waters exposes the membranes to feed waters containing biological and abiotic species, which leads to fouling and loss of membrane productivity over time. Since flux reduction due to fouling is one of the largest costs associated with membrane processes in water treatment, new coatings that limit fouling would have significant economic and societal impacts. Prior studies in this area largely have focused on chemical modifications to the membrane surface, which can be effective but not sufficient for controlling biofouling. A more recent area of research is nano-patterning the membrane surface, inspired by nature (i.e., shark skin). Our hypothesis is combining these two methods (chemical coating and patterning) will yield membrane surfaces that are more effective at biofouling control than either method alone. We will introduce the methodology used to coat membrane surfaces with polymer nanolayers designed to combat biofouling and the methodology used to pattern membrane surfaces. We will explain the chemical switching mechanism and use FTIR to support the reversible switching of the polymer nanolayer between its antifouling and antimicrobial states. We will demonstrate the feasibility of the patterning methodology through AFM

Osmotic effects of biofouling in reverse osmosis (RO) processes: Physical and physiological measurements and mechanisms

Abstract Microbial biofilm formation on reverse osmosis (RO) membranes is known to reduce permeate flux and, in most cases to reduce salt rejection. These effects are consequences of increased overall hydraulic resistance for water permeation through the membrane and a hindered back-diffusion of salts through the biofilm. In return, salt concentration near the membrane is elevated, a phenomenon known as "biofilm enhanced osmotic pressure" (BEOP), resulting in enhanced salt passage. While the effect of elevated hydraulic resistance is clear, the effect of salt passage increase is counterintuitive. In most cases tested, the typical increase of salt passage due to biofouling using commercial high-flux RO membranes cannot be attributed just to RO transport (permeate flux and salt rejection relation), and the typical values of salt passage elevation are too high under biofouling conditions and can only be explained by enhanced concentration polarization effects. Delineating biofouling mechanisms on RO membranes and analyzing the interrelated effects of the biofouling layer on the system performance as well as further changes in the biofouling layer physiology are important for monitoring the extent of biofouling in desalination processes. The BEOP phenomena is enlightened by both synthetic biofouling controlled experiments as well as more realistic studies using tertiary wastewater and brackish water desalination laboratory systems

Real-time analysis of atrazine biodegradation and sessile bacterial growth : A quartz crystal microbalance with dissipation monitoring study

Biodegradation is a fundamental process for removal of the environmentally prevalent herbicide, atrazine, from contaminated waters. Biodegradation is more efficient when bacteria are attached on surface of an adsorbing carrier that supports the microbial population. However, for various reasons, biodegradation is almost always monitored in the liquid phase. In this study, we employ a novel Quartz Crystal Microbalance with Dissipation technique (QCM-D) for continuous, real-time monitoring of the attachment of atrazine-degrading bacteria to the surface, atrazine adsorption and degradation, and the consequent proliferation of the irreversibly attached sessile bacteria. The effect of atrazine biodegradation was observed in a batch mode of operation, in which a significant frequency decrease of the piezoelectric sensor was observed in the QCM-D, due to the proliferation of atrazine-degrading bacteria on the expense of atrazine. The latter was confirmed microscopically. Results also suggest that the viscoelastic properties of the atrazine-degrading consortium immediately changed in response to the presence of atrazine, whereas those of the non-degrading consortium were not affected. Importantly though, atrazine adsorption was similar regardless of the sessile consortia layers. When the QCM-D flow cell was exposed to a continuous flow of saturated atrazine solution, the degrading consortium layer was significantly more fluidic compared to batch mode conditions. The magnitude and kinetics of atrazine adsorption, which were monitored using QCM-D, were higher on bacterial cells comparing to the pristine, polystyrene-coated sensor. Findings from the current study can improve bioremediation design and open an avenue for studies on biodegradation and adsorption of micro-pollutants using QCM-D technology.</p

YdgG (TqsA) Controls Biofilm Formation in Escherichia coli K-12 through Autoinducer 2 Transport

YdgG is an uncharacterized protein that is induced in Escherichia coli biofilms. Here it is shown that deletion of ydgG decreased extracellular and increased intracellular concentrations of autoinducer 2 (AI-2); hence, YdgG enhances transport of AI-2. Consistent with this hypothesis, deletion of ydgG resulted in a 7,000-fold increase in biofilm thickness and 574-fold increase in biomass in flow cells. Also consistent with the hypothesis, deletion of ydgG increased cell motility by increasing transcription of flagellar genes (genes induced by AI-2). By expressing ydgG in trans, the wild-type phenotypes for extracellular AI-2 activity, motility, and biofilm formation were restored. YdgG is also predicted to be a membrane-spanning protein that is conserved in many bacteria, and it influences resistance to several antimicrobials, including crystal violet and streptomycin (this phenotype could also be complemented). Deletion of ydgG also caused 31% of the bacterial chromosome to be differentially expressed in biofilms, as expected, since AI-2 controls hundreds of genes. YdgG was found to negatively modulate expression of flagellum- and motility-related genes, as well as other known products essential for biofilm formation, including operons for type 1 fimbriae, autotransporter protein Ag43, curli production, colanic acid production, and production of polysaccharide adhesin. Eighty genes not previously related to biofilm formation were also identified, including those that encode transport proteins (yihN and yihP), polysialic acid production (gutM and gutQ), CP4-57 prophage functions (yfjR and alpA), methionine biosynthesis (metR), biotin and thiamine biosynthesis (bioF and thiDFH), anaerobic metabolism (focB, hyfACDR, ttdA, and fumB), and proteins with unknown function (ybfG, yceO, yjhQ, and yjbE); 10 of these genes were verified through mutation to decrease biofilm formation by 40% or more (yfjR, bioF, yccW, yjbE, yceO, ttdA, fumB, yjiP, gutQ, and yihR). Hence, it appears YdgG controls the transport of the quorum-sensing signal AI-2, and so we suggest the gene name tqsA