Granulomorphometry: a suitable tool for identifying hydrophobic and disulfide bonds in ß-lactoglobulin aggregates. Application to the study of ß-lactoglobulin aggregation mechanism between 70 and 95°C.

This work deals with the investigation of ß-lactoglobulin (ß-LG) aggregation by granulomorphometry. In the first part of this study, we showed that the binding interactions involved in aggregate structure could be identified by their appearance in granulomorphometric pictures. The reliability of this analytical approach was demonstrated by comparing the appearance of ß-LG aggregates in the presence and absence of a thiol-blocking agent (N-ethylmaleimide). The translucency of the aggregates was associated with hydrophobic interactions and their opacity was associated with disulfide bonds. We state, based on the morphology of the aggregates, along with the color of protein aggregates and insoluble materials, that hydrophobic interactions had a better water-holding capacity than disulfide bonds. Additionally, our results suggest that disulfide and hydrophobic bonds compete for ß-LG aggregate shaping. In the second part of this work, interesting features of granulomorphometry useful for identifying aggregate binding interactions were highlighted to clarify the effect of temperature on the aggregation mechanisms occurring in a ß-LG concentrate with a moderate calcium content (6.6mmol·L(-1)). Heat treatment experiments were performed between 70 and 95°C, and granulomorphometric measurements (aggregate size, aggregate number, and gray level of the picture) were conducted at different sampling times up to 4h. Results, which were interpreted in light of calculated ß-LG denaturation levels, revealed that the aggregation mechanism could be split into 2 steps. Initially, ß-LG denatured quickly, leading to fast ß-LG aggregation by disulfide bonds. The denaturation rate then declined, which drastically slowed the disulfide aggregation mechanism. From that point on, a second aggregation path became preponderant. It consisted of the agglomeration of small aggregates by hydrophobic interactions and resulted in the formation of large aggregates containing both interaction types. This second aggregation mechanism was clearly favored at high temperatures because it was not detected in our experiments at temperatures below 85°C.

GenomeRing: alignment visualization based on SuperGenome coordinates.

MOTIVATION: The number of completely sequenced genomes is continuously rising, allowing for comparative analyses of genomic variation. Such analyses are often based on whole-genome alignments to elucidate structural differences arising from insertions, deletions or from rearrangement events. Computational tools that can visualize genome alignments in a meaningful manner are needed to help researchers gain new insights into the underlying data. Such visualizations typically are either realized in a linear fashion as in genome browsers or by using a circular approach, where relationships between genomic regions are indicated by arcs. Both methods allow for the integration of additional information such as experimental data or annotations. However, providing a visualization that still allows for a quick and comprehensive interpretation of all important genomic variations together with various supplemental data, which may be highly heterogeneous, remains a challenge. RESULTS: Here, we present two complementary approaches to tackle this problem. First, we propose the SuperGenome concept for the computation of a common coordinate system for all genomes in a multiple alignment. This coordinate system allows for the consistent placement of genome annotations in the presence of insertions, deletions and rearrangements. Second, we present the GenomeRing visualization that, based on the SuperGenome, creates an interactive overview visualization of the multiple genome alignment in a circular layout. We demonstrate our methods by applying them to an alignment of Campylobacter jejuni strains for the discovery of genomic islands as well as to an alignment of Helicobacter pylori, which we visualize in combination with gene expression data. AVAILABILITY: GenomeRing and example data is available at http://it.inf.uni-tuebingen.de/software/genomering/.

Influence of calcium on ß-lactoglobulin denaturation kinetics: Implications in unfolding and aggregation mechanisms.

Much research dealing with the processing of milk by-products in heat exchangers has noted the key role of calcium in ß-lactoglobulin (ß-LG) fouling behavior. Nevertheless, the manner by which Ca affects ß-LG denaturation has rarely been quantified using reliable kinetic and thermodynamic data. To this end, the influence of Ca on ß-LG denaturation mechanisms in simulated lactoserum concentrates was studied on the laboratory-scale under 100°C by HPLC analysis. The heat-treated solutions were composed of 53.3g/L ß-LG and were enriched in Ca at various concentrations (0, 66, 132, and 264 mg/kg). The kinetic parameters (reaction order, activation energy, and frequency factor) associated with ß-LG denaturation, along with the unfolding and aggregation thermodynamic parameters were deduced from these experiments and discussed with respect to Ca content. We found that the multistage process characterizing ß-LG thermal denaturation is not greatly affected by Ca addition. In fact, the general model subdividing ß-LG denaturation mechanisms in 2 steps, namely, unfolding and aggregation, remained valid for all tested Ca concentrations. The change in the predominant mechanism from unfolding to aggregation was observed at 80°C across the entire Ca concentration range. Moreover, the classical 1.5 reaction order value was unaffected by the presence of Ca. Interpretation of the acquired kinetic data showed that Ca addition led to a significant increase in kinetic rate, and more so in the aggregation temperature range. This indicates that Ca principally catalyzes ß-LG aggregation, by lowering the Coulombian repulsion between the negatively charged ß-LG reactive species, bridging ß-LG proteins, or via an ion-specific conformational change. To a lesser extent, Ca favors ß-LG unfolding, probably by disturbing the noncovalent binding network of native ß-LG. Simultaneously, Ca has a slight protective role on the native and unfolded ß-LG species, as shown by the increase in activation energy with Ca concentration. The calculation of thermodynamic parameters related to ß-LG denaturation confirmed this observation. A threshold effect in Ca influence was noted in this study: no further significant kinetic rate change was observed above 132 mg/kg of Ca; at this concentration, the studied solution was an almost equimolar mixture of ß-LG and Ca. Finally, we simulated the temporal evolution of ß-LG species concentrations at diverse Ca contents at 3 holding temperatures. The simulations were based on the acquired kinetic parameters. This permitted us to highlight the greater effect of Ca on ß-LG denaturation at high Ca content or for short-time heat treatments at temperatures near 100°C, as in heat exchangers.

A technical platform for generating reproducible expression data from Streptomyces coelicolor batch cultivations.

Streptomyces coelicolor, the model species of the genus Streptomyces, presents a complex life cycle of successive morphological and biochemical changes involving the formation of substrate and aerial mycelium, sporulation and the production of antibiotics. The switch from primary to secondary metabolism can be triggered by nutrient starvation and is of particular interest as some of the secondary metabolites produced by related Streptomycetes are commercially relevant. To understand these events on a molecular basis, a reliable technical platform encompassing reproducible fermentation as well as generation of coherent transcriptomic data is required. Here, we investigate the technical basis of a previous study as reported by Nieselt et al. (BMC Genomics 11:10, 2010) in more detail, based on the same samples and focusing on the validation of the custom-designed microarray as well as on the reproducibility of the data generated from biological replicates. We show that the protocols developed result in highly coherent transcriptomic measurements. Furthermore, we use the data to predict chromosomal gene clusters, extending previously known clusters as well as predicting interesting new clusters with consistent functional annotations.

Roles of metal ions and hydrogen peroxide in modulating the interaction of the Bacillus subtilis PerR peroxide regulon repressor with operator DNA.

The inducible response to H(2)O(2) stress in Bacillus subtilis is under the control of PerR, one of three Fur homologues in this organism. PerR was purified in both an inactive, metal-dependent form and an active, metal-containing form as determined using DNA-binding assays. Active PerR contains both zinc and iron and is designated PerR:Zn,Fe. Added manganous ion competes for binding to the iron site and can restore DNA-binding activity to the metal-dependent form of PerR, presumably generating PerR:Zn,Mn. The DNA-binding activity of PerR:Zn,Fe is eliminated by exposure to H(2)O(2) whereas PerR:Zn,Mn is comparatively resistant. DNA-binding activity can be restored by a thiol-reducing agent, suggesting that redox-active cysteines are involved in peroxide sensing. Experiments using reporter fusions demonstrate that elevated levels of manganese repress PerR regulon genes and prevent their full induction by H(2)O(2). In contrast, in cells grown with iron supplementation, a PerR-repressed gene is completely derepressed by H(2)O(2). These results are consistent with the idea that the intracellular form of the PerR metalloprotein, and therefore its hydrogen peroxide sensitivity, can be altered by growth conditions.

New perspectives on folate catabolism.

Folate catabolism has been assumed to result from the nonenzymatic oxidative degradation of labile folate cofactors. Increased rates of folate catabolism and simultaneous folate deficiency occur in several physiological states, including pregnancy, cancer, and when anticonvulsant drugs are used. These studies have introduced the possibility that folate catabolism may be a regulated cellular process that influences intracellular folate concentrations. Recent studies have demonstrated that the iron storage protein ferritin can catabolize folate in vitro and in vivo, and increased heavy-chain ferritin synthesis decreases intracellular folate concentrations independent of exogenous folate levels in cell culture models. Ferritin levels are elevated in most physiological states associated with increased folate catabolism. Therefore, folate catabolism is emerging as an important component in the regulation of intracellular folate concentrations and whole-body folate status.

Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors.

Fur (ferric uptake regulator) proteins control iron uptake in many Gram-negative bacteria. Although Fur homologues have been identified in Gram-positive bacteria, their roles in gene regulation are unknown. Genome sequencing has revealed three fur homologues in Bacillus subtilis: yqkL, yqfV and ygaG. We demonstrate that yqkL encodes an iron uptake repressor: both siderophore biosynthesis and transcription of ferri-siderophore uptake genes is constitutive in the yqkL mutant. Thus, yqkL encodes a repressor that is functionally as well as structurally related to Fur. B. subtilis peroxide stress genes are induced by either H2O2 or by metal ion limitation. Previous genetic studies defined a regulatory locus, perR, postulated to encode the peroxide regulon repressor. We demonstrate that a ygaG mutant has the perR phenotype: It is highly resistant to peroxides and overexpresses catalase, alkyl hydroperoxide reductase and the DNA binding protein MrgA. Nine spontaneous perR mutations, isolated by virtue of their ability to derepress mrgA transcription in the presence of managanous ion, all contain sequence changes in the ygaG locus and can be complemented by the cloned ygaG gene. Thus, ygaG encodes the peroxide regulon repressor and is allelic with perR.