Exploitation of the semi-homothallic life cycle of Saccharomyces cerevisiae for the development of breeding strategies

A strain of Saccharomyces cerevisiae having desirable winemaking properties and high spore viability was bred from a semi-homothallic parent strain with similar winemaking properties but that produced sixfold fewer viable spores. Because the parent was homozygous for HO and for the MATa allele at both silent HMR and HML loci, it produced two MATa and two nonmating progeny per ascus. To obtain a segregant able to mate with the stable MATa progeny, a strain of the nonmating progeny, previously subjected to HO distruption with a KanMX4 cassette, was used. The resultant MATalphaho::KanMX4 transformant was mated to a MATa HO segregant and the diploid produced was sporulated to allow the isolation of a semi-homothallic diploid segregant designated 2D that lacked the KanMX4-disrupted HO allele as confirmed by sequence analysis. Genetic analysis indicated greater homozygosity in 2D than in the parent as assessed by PCR at five loci. The sugar consumption profiles of both 2D and the parent in grape juice fermentations were the same. Acetaldehyde levels and postfermentation biofilm formation were higher in 2D than in the parent. Because 2D has acceptable winemaking characteristics but produces significantly more viable spores than the parent strain, it will be useful in future breeding efforts.     

Microbiology in the post-genomic era

Genomics has revolutionized every aspect of microbiology. Now, 13 years after the first bacterial genome was sequenced, it is important to pause and consider what has changed in microbiology research as a consequence of genomics. In this article, we review the evolving field of bacterial typing and the genomic technologies that enable comparative analysis of multiple genomes and the metagenomes of complex microbial environments, and address the implications of the genomic era for the future of microbiology.     

Molecular dynamic investigation of the interaction of supported affinity ligands with monoclonal antibodies

Diagnostics and therapeutic treatments based on monoclonal antibodies have been attaining an increasing importance in the past decades, but their large scale employment requires the optimization of purification processes. To obtain this goal, research is focusing on affinity chromatography techniques and the development of new synthetic ligands. In this work we present a computational investigation aimed at obtaining some guidelines for the rational design of affinity ligands, through the study of their interactions with both monoclonal antibodies (modeled as the FC domain of human IgG) and a model support material (agarose). The study was carried out performing molecular dynamics simulations of the support-spacer-ligand-IgG complex in explicit water. Binding energies between IgG and two supported ligands, a disubstituted derivative of trichlorotriazine and a tetrameric peptide, were determined with the linear interaction energy and MM-GBSA approaches. A detailed study of the possible binding sites of the considered ligands was performed exploiting docking protocols and MD simulations. It was found that both ligands bind IgG in the same site as protein A, which is the hinge region between the CH2 and CH3 domains of IgG. However this site is not easily accessible and requires a high mobility of the ligands. The energetic analysis revealed that van der Waals and electrostatic energies of interaction of the triazine ligand with the support are significant and comparable to those with the protein, so that they limit its capability to reach the protein binding site. A similar result was found also for the tetrameric peptide, which is however able to circumvent the problem; for steric reasons only two of its arms can interact at the same time with the agarose support, thus leaving the remaining two available to bind the protein. These results indicate that the interaction between ligand and support material is an important parameter, which should be considered in the computational and experimental design of ligands for affinity chromatography.     

"Candidatus Midichloria" endosymbionts bloom after the blood meal of the host, the hard tick Ixodes ricinus

"Candidatus Midichloria mitochondrii," an intracellular symbiont of the tick Ixodes ricinus, is the only described organism able to invade the mitochondria of any multicellular organism. We used quantitative PCR to examine cycles of bacterial growth and death throughout the host's development and found that they correspond with the phases of engorgement and molt, respectively.     

Influences of temperature and threshold effect of NaCl concentration on Alpias vulpinus OCT

Thermodynamic, circular dichroism (CD), and activity measurements have been used to characterize the different conformational states and the effects of NaCl concentrations (0.0-3.0 M) on thermal unfolding of ornithine carbamoyltransferase (OCT) from Alopias vulpinus. Furthermore conformational changes in whole enzyme structure have been monitored by titration of SH-groups. OCT unfolding process follows an irreversible two-state mechanism with a first-order kinetic of denaturation, without breaking-point. NaCl shows very little stabilization effects at low concentration and its action become very important over 1.5 M concentration. The presence of 3.0 M NaCl completely avoids OCT unfolding at 60, 64 and 66 degrees C. Kinetic and thermodynamic parameters are strongly influenced by the presence of high NaCl concentration. Our experiments showed that NaCl stabilization process involved changes in preferential binding, in electrostatic and van der Waals interactions and exposure of buried site and SH-groups. During thermal denaturation, UV-vis and CD spectroscopy show that high salts concentration preserves OCT activity, avoiding exposure of hydrophobic site and destruction of secondary and tertiary structure elements.     

Partial persistence of exercise-induced myocardial angiogenesis following 4-week detraining in the rat

Enhanced angiogenesis, or capillary growth, has a prominent role among the various beneficial effects of exercise training on the myocardium. The aim of the present study is to assess if training-induced increases in capillarity and vascularization persist after 4 weeks of detraining. Adult male rats were trained to run on a treadmill for 10 weeks at ∼60% VO_2max, which did not induce cardiac hypertrophy, but increased (P < 0.05) the soleus/body weight ratio, left ventricle capillarity and von Willebrand-positive cell density (n = 6). In another group of animals (n = 6) subjected to training followed by 4-week detraining, the soleus/body weight ratio returned to normal, with only partial reversal of left ventricle capillarity and von Willebrand-positive cell density. Markers of angiogenesis (VEGF, KDR/VEGF-R2 and HIF-1α mRNA, studied by real-time RT-PCR) were upregulated at the end of training, and returned to baseline value after detraining. Electron microscopy highlighted some morphological features in trained hearts (endothelial cell sprouting and bridges and pericyte detachment), suggestive of endothelial cell proliferation and capillary growth that were absent in untrained and detrained hearts. We conclude that the training-induced increase in cardiac capillarity and vascularization are retained for some time upon cessation of the training program even in the absence of angiogenic stimuli.     

Genetic variability within isolates of Colletotrichum lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil

Colletotrichum lindemuthianum, the causal agent of anthracnose in the common bean (Phaseolus vulgaris), presents a wide genetic and pathogenic variability that gives rise to complications in the development of resistant bean cultivars. The aim of this study was to identify the variability within race 65 of C. lindemuthianum, the race most commonly encountered in Brazil, through randomly amplified polymorphic DNA (RAPD) and anastomosis analyses. Thirteen isolates of race 65, collected in different years and from various host cultivars located in diverse areas of the state of Minas Gerais, Brazil, were investigated. Twenty-four RAPD primers were employed and 83 polymorphic bands amplified. Genetic similarities were estimated from the Sorensen-Dice coefficient and ranged from 0.54 to 0.82. The dendrogram obtained by cluster analysis classified the isolates into 11 separate groups. For the purposes of the analysis of anastomosis, isolates were considered to be compatible when the fusion of hyphae from different isolates could be observed. The proportion of compatible reactions for each isolate was estimated and similarity estimates, based on the Russel & Rao coefficient, ranged from 0.28 to 0.85. Isolates were classified into 11 anastomosis groups, 10 of which were formed by only one isolate. Although isolates LV61, LV73 and LV58 were classified in the same anastomosis group, they were genetically distinct according to RAPD analysis. Results from both RAPD and anastomosis analyses revealed great variability within C. lindemuthianum race 65.     

Spatial proximity between two host plant species influences oviposition and larval distribution in a leaf beetle

Everything else being equal, insect herbivores can be expected to oviposit on host plants that provide the qualitatively and quantitatively best food for larvae. However, the selection of a plant for oviposition may be influenced by such ecological factors as natural enemies, host distribution, host patch size or host patch density. We performed a field study to test whether spatial proximity between two host plant species influences the oviposition patterns and larval distribution of the alpine leaf beetle Oreina elongata. In the population studied, O. elongata oviposits and feeds on two host plants, that belong to the same family (Asteraceae): Adenostyles alliariae and Cirsiumspinosissimum. The first species contains pyrrolizidine alkaloids that are sequestered by the beetle as a chemical defence, whereas the second plant does not contain any alkaloids but has hairy and spiny leaves that might give some mechanical protection to beetle larvae.
During two consecutive summers, we quantified oviposition and larval distribution on randomly chosen C. spinosissimum that grew spatially isolated from A. alliariae, on C. spinosissimum that grew in leaf contact with A. alliariae and on A. alliariae that grew in leaf contact with C. spinosissimum (isolated A. alliariae was not considered, because it is rare in the study population). In both years, more eggs were laid on C. spinosissimum than on A. alliariae and more on those C. spinosissimum that were growing close to A. alliariae than on those growing isolated. Large numbers of larvae moved from C. spinosissimum to A. alliariae during the season. Patch size did not influence egg and larval numbers. Eggs survived better on C. spinosissimum than on A. alliariae in the field. The data suggest that C. spinosissimum may provide eggs with better protection against stormy weather. In a separate study of the same population, we found that larval performance was better on A. alliariae than on C. spinosissimum. Our present data suggest that O. elongata preferentially oviposits on plants of the species that maximizes egg survival and that grow in close proximity to plants of the species that provides better food and chemical defence.     

Organization and Regulation of meta Cleavage Pathway Genes for Toluene and o-Xylene Derivative Degradation in Pseudomonas stutzeri OX1

Pseudomonas stutzeri OX1 meta pathway genes for toluene and o-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the Pseudomonas sp. strain CF600 dmp operon for phenol catabolism and was found to be coregulated by the tou operon activator TouR. A hypothesis about the evolution of the toluene and o-xylene catabolic pathway in P. stutzeri OX1 is discussed.