The Crystal Structure of OprG from Pseudomonas aeruginosa, a Potential Channel for Transport of Hydrophobic Molecules across the Outer Membrane

Background: The outer membrane (OM) of Gram-negative bacteria provides a barrier to the passage of hydrophobic and hydrophilic compounds into the cell. The OM has embedded proteins that serve important functions in signal transduction and in the transport of molecules into the periplasm. The OmpW family of OM proteins, of which P. aeruginosa OprG is a member, is widespread in Gram-negative bacteria. The biological functions of OprG and other OmpW family members are still unclear. Methodology/Principal Findings: In order to obtain more information about possible functions of OmpW family members we have solved the X-ray crystal structure of P. aeruginosa OprG at 2.4 A ˚ resolution. OprG forms an eightstranded b-barrel with a hydrophobic channel that leads from the extracellular surface to a lateral opening in the barrel wall. The OprG barrel is closed off from the periplasm by interacting polar and charged residues on opposite sides of the barrel wall. Conclusions/Significance: The crystal structure, together with recent biochemical data, suggests that OprG and other OmpW family members form channels that mediate the diffusion of small hydrophobic molecules across the OM by a latera

Thermal Stabilization of an Endoglucanase by Cyclization

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins

Metagenomic Analysis of the Bioremediation of Diesel-Contaminated Canadian High Arctic Soils

As human activity in the Arctic increases, so does the risk of hydrocarbon pollution events. On site bioremediation of contaminated soil is the only feasible clean up solution in these remote areas, but degradation rates vary widely between bioremediation treatments. Most previous studies have focused on the feasibility of on site clean-up and very little attention has been given to the microbial and functional communities involved and their ecology. Here, we ask the question: which microorganisms and functional genes are abundant and active during hydrocarbon degradation at cold temperature? To answer this question, we sequenced the soil metagenome of an ongoing bioremediation project in Alert, Canada through a time course. We also used reverse-transcriptase real-time PCR (RT-qPCR) to quantify the expression of several hydrocarbon-degrading genes. Pseudomonas species appeared as the most abundant organisms in Alert soils right after contamination with diesel and excavation (t = 0) and one month after the start of the bioremediation treatment (t = 1m), when degradation rates were at their highest, but decreased after one year (t = 1y), when residual soil hydrocarbons were almost depleted. This trend was also reflected in hydrocarbon degrading genes, which were mainly affiliated with Gammaproteobacteria at t = 0 and t = 1m and with Alphaproteobacteria and Actinobacteria at t = 1y. RT-qPCR assays confirmed that Pseudomonas and Rhodococcus species actively expressed hydrocarbon degradation genes in Arctic biopile soils. Taken together, these results indicated that biopile treatment leads to major shifts in soil microbial communities, favoring aerobic bacteria that can degrade hydrocarbons

Insight from the draft genome of Dietzia cinnamea P4 reveals mechanisms of survival in complex tropical soil habitats and biotechnology potential

The draft genome of Dietzia cinnamea strain P4 was determined using pyrosequencing. In total, 428 supercontigs were obtained and analyzed. We here describe and interpret the main features of the draft genome. The genome contained a total of 3,555,295 bp, arranged in a single replicon with an average G+C percentage of 70.9%. It revealed the presence of complete pathways for basically all central metabolic routes. Also present were complete sets of genes for the glyoxalate and reductive carboxylate cycles. Autotrophic growth was suggested to occur by the presence of genes for aerobic CO oxidation, formate/formaldehyde oxidation, the reverse tricarboxylic acid cycle and the 3-hydropropionate cycle for CO2 fixation. Secondary metabolism was evidenced by the presence of genes for the biosynthesis of terpene compounds, frenolicin, nanaomycin and avilamycin A antibiotics. Furthermore, a probable role in azinomycin B synthesis, an important product with antitumor activity, was indicated. The complete alk operon for the degradation of n-alkanes was found to be present, as were clusters of genes for biphenyl ring dihydroxylation. This study brings new insights in the genetics and physiology of D. cinnamea P4, which is useful in biotechnology and bioremediation