Glycerate 2-kinase of Thermotoga maritima and genomic reconstruction of related metabolic pathways

Members of a novel glycerate-2-kinase (GK-II) family were tentatively identified in a broad range of species, including eukaryotes and archaea and many bacteria that lack a canonical enzyme of the GarK (GK-I) family. The recently reported three-dimensional structure of GK-II from Thermotoga maritima (TM1585; PDB code 2b8n) revealed a new fold distinct from other known kinase families. Here, we verified the enzymatic activity of TM1585, assessed its kinetic characteristics, and used directed mutagenesis to confirm the essential role of the two active-site residues Lys-47 and Arg-325. The main objective of this study was to apply comparative genomics for the reconstruction of metabolic pathways associated with GK-II in all bacteria and, in particular, in T. maritima. Comparative analyses of ~400 bacterial genomes revealed a remarkable variety of pathways that lead to GK-II-driven utilization of glycerate via a glycolysis/gluconeogenesis route. In the case of T. maritima, a three-step serine degradation pathway was inferred based on the tentative identification of two additional enzymes, serine-pyruvate aminotransferase and hydroxypyruvate reductase (TM1400 and TM1401, respectively), that convert serine to glycerate via hydroxypyruvate. Both enzymatic activities were experimentally verified, and the entire pathway was validated by its in vitro reconstitution.     

Estimation of protein aggregation propensity with a melting point apparatus

A method for studying protein aggregation with an automated melting point apparatus is described. The method employs thermal ramping and can generate a series of protein aggregation curves. The midpoint aggregation curve-associated temperature (T_a) is used to evaluate the difference between the curves where the lower T_a value corresponds to a higher aggregation propensity. The applicability of the method was demonstrated with human interleukin-1 receptor antagonist (IL-1ra) as a protein aggregation model. The method could be employed for rapid evaluation of various factors such as mutations, buffers, and excipients influencing protein aggregation propensity under the thermal stress.     

Morphometric difference between the Novosibirsk and Tomsk chromosome races of<Emphasis Type="Italic">Sorex araneus</Emphasis> in a zone of parapatry

A hybrid zone between the Novosibirsk and Tomsk chromosome races of the common shrewSorex araneus Linnaeus, 1758 was found near Novosibirsk city (West Siberia, Russia) in an area unimpeded by geographic barriers. In this zone, the shrews of both races and their hybrids were trapped and karyotyped and 22 features of their cranial and postcranial skeleton were measured. Canonical discriminant analysis revealed 3 distinct groups of individuals, which corresponded to the 3 karyotypic categories involved in the analysis. The first discriminant function reflected the differences in the size of skeletal elements. The Novosibirsk shrews and the hybrids were significantly smaller than the Tomsk shrews. The second discriminant function was interpreted as a parameter of skeletal proportionality. The hybrids were significantly less proportional than the parental races. This study revealed one of the clearest examples of morphological differentiation between chromosome races of the common shrew.     

Legumains - a family of asparagine-specific cysteine endopeptidases involved in propolypeptide processing and protein breakdown in plants

Legumains are a recently discovered family of plant and animal cysteine endopeptidases with a cleavage specificity for Asn in the P1 position of peptide bonds. Asp-flanked peptide bonds also are cleaved, but with a much lower efficiency. Legumains evolved from GPI transamidase-like progenitors. Sequence analysis revealed three major groups of plant legumains corresponding to differences in the developmental and organ-specific gene expression. With the exception of a single cell wall specific representative, all legumains occur in the vacuolar compartment. Legumains are either involved in protein degradation or play a role in the processing of precursor proteins by Asn/Asp-specific limited proteolysis. Which function legumains perform depends on the conformational state of the substrate protein. A legumain acts as a vacuolar processing enzyme when it only has access to the regular processing sites of a precursor polypeptide, but it acts as a degradative enzyme when an altered conformation opens the substrate for unlimited proteolysis. The specificity of these interactions seems to be the result of a co-evolution of enzyme and substrate. The double function of legumains is particularly evident in the events of deposition and mobilisation of storage globulins during seed maturation and germination/seedling growth and in senescing and dying cells.     

Structural and genetic characterization of Escherichia coli O99 antigen

The influence of antibiotic exposure in the early postnatal period on the development of intestinal microbiota was monitored in 26 infants including five antibiotic-treated (AT) subjects orally administered a broad-spectrum antibiotic for the first 4 days of life and three caesarean-delivered (CD) subjects whose mothers were intravenously injected by the similar type of antibiotics in the same period. The faecal bacterial composition was analysed daily for the first 5 days and monthly for the first 2 months. Terminal restriction fragment length polymor-phisms in the AT subjects showed less diversity with the attenuation of the colonization of some bacterial groups, especially in Bifidobacterium and unusual colonization of Enterococcus in the first week than the control antibiotic-free infants (AF, n =18). Quantitative real-time PCR showed overgrowth of enterococci (day 3, P =0.01; day 5, P =0.003; month 1, P =0.01) and arrested growth of Bifidobacterium (day 3, P =0.03) in the AT group. Furthermore, after 1 month, the Enterobacteriaceae population was markedly higher in the AT group than in the AF group (month 1, P =0.02; month 2, P =0.02). CD infants sustained similar, although relatively weaker, alteration in the developing microbiota. These results indicate that antibiotic exposure at the beginning of life greatly influences the development of neonatal intestinal microbiota.     

Density-dependent regulation in populations of potato-colonizing aphids

Scarcity of long-term (over 30 years) data series represents a major challenge for an accurate estimation of the role of density-dependent processes in population regulation. We analyzed population densities of the wingless parthenogenic morphs of buckthorn aphid (BA), Aphis nasturtii Kaltenbach, potato aphid (PA), Macrosiphum euphorbiae (Thomas), and green peach aphid (GPA), Myzus persicae (Sulzer) from 1949 to 2003 for signs of density-dependent regulation. We found strong evidence of density-dependent regulation, with detection of density dependence being fairly consistent among the different statistical techniques. Direct density dependence was detected for the populations of all three species. There was also evidence of delayed density dependence for PA. The periodicity of population fluctuations for BA and GPA was 6.1 years and 3.9 years, respectively. The periodicity for PA was not explicit, being highly variable throughout the time series. Effects of density-independent weather factors were relatively minor compared to density-dependent regulation. The BA populations experienced a significant reduction in both density and annual oscillations starting in 1995, while GPA populations experienced a similar reduction in 1991. No such change was apparent for PA. The most likely explanation for the observed phenomenon is a change in the composition of the lady beetle community following the establishment of two alien coccinellid species, and/or changes in insecticide use by commercial growers in the area of the study.     

Towards environmental systems biology of Shewanella

Bacteria of the genus Shewanella are known for their versatile electron-accepting capacities, which allow them to couple the decomposition of organic matter to the reduction of the various terminal electron acceptors that they encounter in their stratified environments. Owing to their diverse metabolic capabilities, shewanellae are important for carbon cycling and have considerable potential for the remediation of contaminated environments and use in microbial fuel cells. Systems-level analysis of the model species Shewanella oneidensis MR-1 and other members of this genus has provided new insights into the signal-transduction proteins, regulators, and metabolic and respiratory subsystems that govern the remarkable versatility of the shewanellae.