Genomics and Biochemistry of Metabolic Pathways for the C<Subscript>1</Subscript> Compounds Utilization in Colorless Sulfur Bacterium <Emphasis Type="Italic">Beggiatoa leptomitoformis</Emphasis> D-402

The metabolic pathways of one-carbon compounds utilized by colorless sulfur bacterium Beggiatoa leptomitoformis D-402 were revealed based on comprehensive analysis of its genomic organization, together with physiological, biochemical and molecular biological approaches. Strain D-402 was capable of aerobic methylotrophic growth with methanol as a sole source of carbon and energy and was not capable of methanotrophic growth because of the absence of genes of methane monooxygenases. It was established that methanol can be oxidized to CO₂ in three consecutive stages. On the first stage methanol was oxidized to formaldehyde by the two PQQ (pyrroloquinolinequinone)-dependent methanol dehydrogenases (MDH): XoxF and Mdh2. Formaldehyde was further oxidized to formate via the tetrahydromethanopterin (H₄MPT) pathway. And on the third stage formate was converted to CO₂ by NAD⁺-dependent formate dehydrogenase Fdh2. Finally, it was established that endogenous CO₂, formed as a result of methanol oxidation, was subsequently assimilated for anabolism through the Calvin–Benson–Bassham cycle. The similar way of one-carbon compounds utilization also exists in representatives of another freshwater Beggiatoa species—B. alba.     

The coup de grâce for the nested clade phylogeographic analysis?

Nested clade phylogeographic analysis (NCPA) has become a popular method for reconstructing the history of populations across species ranges. Ever since its invention in 1995, criticisms have been formulated, but the method, which has been regularly updated, continues to attract investigators. Molecular Ecology has published a large fraction of the literature on the topic — both pro and con. A recent study by Panchal and Beaumont (2007) finally allows a precise evaluation of the method by developing software that automates the somewhat complicated NCPA procedure. Using simulations of random-mating populations, Panchal and Beaumont find a high frequency of false-positives with their automated NCPA procedure (over 75%). These findings, which echo and amplify earlier warnings, appear serious enough to suggest to researchers to await further evaluation of the method. Although no other all-encompassing method such as the NCAP currently exists to evaluate phylogeographic data sets, researchers have many alternative methods to test ever more refined hypotheses.     

Ecophysiology of lithotrophic sulfur-oxidizing <Emphasis Type="Italic">Sphaerotilus</Emphasis> species from sulfide springs in the Northern Caucasus

Six strains of sulfur-oxidizing bacteria of the known organotrophic species Sphaerotilus natans were isolated from two North Caucasian sulfide springs. Similar to known colorless sulfur bacteria, all the strains accumulated elemental sulfur when grown in media with sulfide. Unlike previously isolated S. natans strains, new isolates had higher temperature growth optimum (33–37°C) and variable metabolism. All the strains were capable of organotrophic, lithoheterotrophic, and mixotrophic growth with sulfur compounds as electron donors for energy metabolism. Variable metabolism of new Sphaerotilus isolates is a highly important adaptation mechanism which facilitates extension of their geographic range and supports their mass development in new habitats, e.g. sulfide springs. Within the cluster of new isolates, the physiological heterogeneity was shown to result from the inducible nature of the enzymes of oxidative sulfur metabolism and from their resistance to aerobic cultivation.     

Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca²⁺/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.     

Carbon and Sulfur Metabolism in Representatives of Two Clusters of Bacteria of the Genus Leucothrix: A Comparative Study

Major pathways of carbon and sulfur metabolisms were studied in representatives of two clusters of bacteria: Leucothrix thiophila (cluster I, strains 2WS, 4WS, and 6WS) and Leucothrix sp. (cluster II, strains 1WS, 3WS, and 5WS). All strains were capable of chemoorganoheterotrophic growth, as well as of chemolithoheterotrophic growth in the presence of reduced sulfur compounds. The bacteria were found to possess a complete set of the enzymes of the tricarboxylic acid cycle and glyoxylate cycle. The dehydrogenase activity in cells of cluster I strains was an order of magnitude lower than in cluster II strains and in other known heterotrophic bacteria. Cells of bacteria of both clusters exhibited high activity levels of enzymes involved in the energy metabolism of sulfur. The oxidation of sulfur compounds and the operation of the electron-transport chain were shown to be related. Cluster II bacteria more efficiently use organic compounds in their energy metabolism, whereas cluster I bacteria are characterized by more efficient utilization of reduced sulfur compounds. During sulfite oxidation, cluster I bacteria can synthesize ATP both via substrate-level phosphorylation and oxidative phosphorylation, whereas cluster II bacteria synthesize ATP only via the latter process.     

The nonlinear effect of the composite influence of red and blue light on <Emphasis Type="Italic">Escherichia coli</Emphasis> viability

A nonlinear dependence of E. coli cell inhibition was found during the simultaneous irradiation by the blue and red spectral regions at a power density of 100 mW/cm². This dependence can be explained by the cascade two-photon light absorption by DNA molecules with intermediate resonance at the cellular chromophores, which leads to excitation and subsequent DNA damage similar to the damage that is caused by exposure to UV quanta.     

Contributions of different mosquito species to the transmission of lymphatic filariasis in central Nigeria: implications for monitoring infection by PCR in mosquito pools

Background

Monitoring and evaluation are essential to the successful implementation of mass drug administration programmes for LF elimination. Monitoring transmission when it is low requires both large numbers of mosquito vectors and sensitive methods for detecting Wuchereria bancrofti infections in them. PCR-based methods are preferred over classical dissections but the best protocol so far achieved detection of one L₃ Wuchereria bancrofti larva in a pool of 35–50 Anopheles mosquitoes. It also lacks consistency and remains still a costly tool. Hence we decided to improve upon this to achieve detection in a pool of 100 or more by enhancing the quality of the template DNA. Prior to this we also evaluated three vector sampling methods in the context of numbers for monitoring.

Methods

Human landing, pyrethrium spray and light traps catches were conducted concurrently at sites in an LF endemic district in Ghana and the numbers obtained compared. Two DNA extraction methods; Bender buffer and phenol/chloroform purification, and DNAeasy Tissue kit (Quaigen Inc) were used on pools of 25, 50, 75 100 and 150 mosquitoes each seeded with one L₃ or its quivalent amount of DNA. Then another set of extracted DNA by the two methods was subjected to Dynal bead purification method (using capture oligonucleotide primers). These were used as template DNA in PCR to amplify W. bancrofti sequences. The best PCR result was then evaluated in the field at five sites by comparing its results (infections per 1000 mosquitoes) with that of dissection of roughly equal samples sizes.

Results

The largest numbers of mosquitoes were obtained with the human landing catches at all the sites sampled. Although PCR detection of one L₃ in pools of 25, 50 and 75 mosquitoes was consistent irrespective of the extraction method, that of one L₃ in 100 was only achieved with the kit-extracted DNA/Dynal bead purification method. Infections were found at only two sites by both dissection and pool-screening being 14.3 and 19 versus 13.4 and 20.1 per 1000 Anopheles mosquitoes respectively, which were not statistically significant

Discussion and conclusion

HLC still remains the best option for sampling for the large numbers of mosquitoes required for monitoring transmission during MDA programmes, when vector population densities are high and classical indices of transmission are required. One – in – 100 detection is an improvement on previous PCR pool-screening methods, which in our opinion was a result of the introduction of the extra step of parasite DNA capture using Dynal/beads. As pool sizes increase the insects DNA will swamp parasite DNA making the latter less available for an efficient PCR, therefore we propose either additional steps of parasite DNA capture or real-time PCR to improve further the pool screening method. The study also attests also to the applicability of Katholi et al's algorithm developed for determining onchocerciasis prevalence in LF studies.     

Oxidative stress and antioxidant cell protection systems in the microaerophilic bacterium Spirillum winogradskii

The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined. The metabolic adjustments caused by transition from microaerobic (2% O2) aerobic conditions (21% O2 of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen. This resulted in a twofold decline of the growth yield coefficient. Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes protecting from toxic forms of O2--peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)--massive lysis of cells starts in the mid-exponential phase and leads to culture death in the stationary phase because of H2O2 accumulation in the periplasm (up to 10 micrograms/mg protein). The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H2O2, was shown. It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H2O2 accumulates in the periplasm due to the lack of cytochrome-c-peroxidase. The addition to the medium of thiosulfate promotes elimination of H2O2, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield.