Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.     

Olfactory and Behavioral Mechanisms Underlying Enhanced Mating Competitiveness Following Exposure to Ginger Root Oil and Orange Oil in Males of the Mediterranean Fruit Fly, Ceratitis capitata (Diptera: Tephritidae)

Males of the Mediterranean fruit fly, Ceratitis capitata, are strongly attracted to various plant odors, and previous work has demonstrated that male exposure to certain odors, including the scent of orange oil (OO) and ginger root oil (GRO), increases their mating success relative to non-exposed males. However, the mechanism(s) underlying this mating increase is not known. Here, we describe several experiments that further investigate the association between GRO- and OO-exposure and male signaling activity, pheromone attractiveness, and mating success in male medflies. Exposure to GRO or OO increased time spent pheromone calling but did not accelerate the rate of male sexual maturation. Using a wind tunnel, we compared female attraction to the pheromone of control, non-exposed males versus males previously exposed to OO or GRO. There was no evidence that GRO exposure enhanced the attractiveness of the male pheromone. The data for OO were inconclusive: females tended to spend more time on spheres emanating pheromone from OO-exposed males than on spheres emanating pheromone from non-exposed males, but the number of female landings did not differ between the two types of pheromone sources. Female choice tests confirmed that GRO- and OO-exposure boost male mating success relative to non-exposed males. Application of GRO directly to the abdomen reduced male mating success, whereas similar application of OO boosted male mating success. The potential role and mode of action of plant chemicals in the mating behavior of male medflies are evaluated in light of these findings.     

Neutral and cationic mononuclear copper(II) complexes with enrofloxacin: structure and biological activity

The mononuclear copper complexes with the quinolone antibacterial drug enrofloxacin (=Herx) in the presence or not of a nitrogen donor heterocyclic ligand 1,10-phenanthroline (=phen) and 2,2'-bipyridine (=bipy) have been prepared and characterized. Interaction of copper(II) with deprotonated enrofloxacin leads to the formation of the neutral complex Cu(erx)2(H2O), 1, while the presence of phen or bipy leads to the formation of a neutral or a cationic mononuclear complex, respectively. The crystal structures of (chloro)(1,10-phenanthroline)(enrofloxacinato)copper(II), 2, and (aqua)(2,2'-bipyridine)(enrofloxacinato)copper(II) chloride, 3, have been determined with X-ray crystallography. The complexes have been studied with X-band electron paramagnetic resonance in aqueous solutions at liquid helium temperature. The study of the interaction of the complexes with calf-thymus DNA has been performed with diverse spectroscopic techniques and has showed that all complexes are bound to DNA by the intercalative mode. The antimicrobial efficiency of the complexes has been tested on three different microorganisms and the available evidence supports that the best inhibition is provided by Cu(erx)2(H2O) (minimum inhibitory concentration=0.125 microg mL(-1)) against Escherichia coli and Pseudomonas aeruginosa.     

Synthesis, structure and biological activity of copper(II) complexes with oxolinic acid

The neutral mononuclear copper complexes with the quinolone antibacterial drug oxolinic acid in the presence or not of a nitrogen donor heterocyclic ligand 1,10-phenanthroline, 2,2'-bipyridine or 2,2'-dipyridylamine have been synthesized and characterized with infrared, UV-visible and electron paramagnetic resonance spectroscopies. The experimental data suggest that oxolinic acid acts as a deprotonated bidentate ligand and is coordinated to the metal ion through the pyridone and one carboxylate oxygen atoms. The crystal structure of (chloro)(1,10-phenanthroline)(oxolinato) copper(II), 2, has been determined with X-ray crystallography. For all complexes a distorted square pyramidal environment around Cu(II) is suggested. The EPR (electron paramagnetic resonance) behavior of 2 in aqueous solutions indicates mixture of dimeric and monomeric species. The investigation of the interaction of the complexes with calf-thymus DNA has been performed with diverse spectroscopic techniques and showed that the complexes are bound to calf-thymus DNA. The antimicrobial activity of the complexes has been tested on three different microorganisms. The complexes show a decreased biological activity in comparison to the free oxolinic acid.     

Cloning and structural characterization of the 6-phosphogluconate dehydrogenase locus of the medfly Ceratitis capitata and the olive fruit fly Bactrocera oleae

The pentose phosphate cycle is considered as a major source of NADPH and pentose needed for nucleic acid biosynthesis. 6-Phosphogluconate dehydrogenase (6PGD), an enzyme participating in this cycle, catalyzes the oxidative decarboxylation of 6PGD to ribulose 5-phosphate with the subsequent release of CO(2) and the reduction of NADP. We have determined the genomic sequences of 6PGD of two species of Tephritidae, the medfly Ceratitis capitata and olive fruit fly Bactrocera oleae, and constructed a three-dimensional model of 6PGD of C. capitata based on the homologous known sheep structure. In a comparative study of 6PGD sequences from seven species, all the conserved and variable sites of the enzyme were analyzed and the regions of functional importance were localized, an attempt promoted also by the direct involvement of the enzyme in various human diseases. The enzymes between the two species of Tephritidae have a very high homology and further examination of the variable positions with respect to the highly conserved binding site residues enabled their grouping in three distinct categories, with possible association to dimer formation, functional specificity, and antigenicity. Moreover, placement of sequence differences on the 3-D model suggests probable sites accommodating variations appearing at the allozymic variants of both species.     

Role of integrin-linked kinase in vascular smooth muscle cells: regulation by statins and angiotensin II

Our goal was to characterize the role of integrin-linked kinase (ILK) in vascular smooth muscle cells (VSMC), which play a crucial role in atherogenesis. Transfection of VSMC with wild-type and dominant-negative ILK cDNA constructs revealed that ILK mediates migration and proliferation of VSMC but has no effect on VSMC survival. The pro-atherogenic mediator angiotensin II increases ILK protein expression and kinase activity while statin treatment down-regulates ILK in VSMC. Functionally, ILK is necessary for angiotensin II-mediated VSMC migration and proliferation. In VSMC transduced with dominant-negative ILK, statins mediate an additive inhibition of VSMC migration and proliferation, while transfection with wild-type ILK is sufficient to overcome the inhibitory effects of statin treatment on VSMC migration and proliferation. In vivo, ILK is expressed in VSMC of aortic sections from wild-type mice where it is down-regulated following statin treatment and up-regulated following induction of atherosclerosis in apoE-/- mice. These data identify ILK as a novel target in VSMC for anti-atherosclerotic therapy.     

Analysis of allelic variants in the catalase gene in patients with the skin depigmenting disorder vitiligo

Vitiligo is an acquired hypomelanotic skin disorder characterised by circumscribed depigmented macules resulting from the loss of functional melanocytes from the cutaneous epidermis. Conditions that might result in epidermal oxidative stress and consequently damage to pigment cells have been reported in the skin of vitiligo patients, including low catalase activity and increases in hydrogen peroxide levels. However, the cause of the decrease in catalase activity has not been equivocally determined. Several allelic variants in the catalase gene, a number of which have deleterious effects upon the expression or function of the enzyme, have been described and the aim of the present work was to assess the relevance of catalase gene variants in patients with vitiligo. Associations between ten separate allelic variants in the catalase gene and a predisposition to vitiligo were investigated in case-control studies with 166 English patients and 169 ethnically-matched controls using DNA sequencing and restriction fragment length polymorphism-polymerase chain reaction methods. Of the ten allelic variants analysed, only a C/T single nucleotide polymorphism in exon 9 of the catalase gene was associated with vitiligo. The C/T genotype was significantly over-represented in the vitiligo patient group compared with the control cohort. Of 166 vitiligo genotypes, 66 (39.8%) had the C/T variant compared to 45/169 (26.6%) control genotypes (P = 0.030). No evidence for an association between other allelic variants in the catalase gene and vitiligo susceptibility was found. The low catalase activity in vitiligo patient epidermis is more likely to result from environmental conditions such as inhibitory levels of hydrogen peroxide rather than allelic variations in the catalase gene which affect either expression or function of the enzyme.     

Evidence for a dimeric assembly of two titin/telethonin complexes induced by the telethonin C-terminus

The Z-disk region defines the lateral boundary of the sarcomere and requires a high level of mechanical strength to provide a stable framework for large filamentous muscle proteins. The level of complexity at this area is reflected by a large number of protein-protein interactions. Recently, we unraveled how the N-terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the N-terminal titin-binding segment of the Z-disk ligand telethonin/T-cap [Zou, P., Pinotsis, N., Lange, S., Song, Y.H., Popov, A., Mavridis, I., Mayans, O.M., Gautel, M., Wilmanns, M., 2006. Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. Nature 439, 229-233]. In this contribution, we present structural data of a related complex of the titin N-terminus with full-length telethonin. The C-terminus of telethonin remains invisible, suggesting that it does not fold into a defined structure even in the presence of titin. In contrast to the structure with truncated telethonin, a dimer of two titin/telethonin complexes is formed within the crystal environment, potentially indicating the formation of higher oligomers. We further investigated the structure and dynamics of this assembly by small-angle X-ray scattering, circular dichroism, and in vivo complementation data. The data consistently indicate the involvement of the C-terminal part of telethonin into the assembly of two titin/telethonin complexes.