A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage ?29

Protein p6 of the Bacillus subtilis phage ø29 is essential for in vivo viral DNA replication. This protein activates the initiation of ø29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su ^–) cells to support growth of a ø29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

The C-terminal extension of bacterial flavodoxin-reductases: Involvement in the hydride transfer mechanism from the coenzyme

To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP⁺ than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding.     

Assessing the nucleotide diversity of three aphid species by RAPD

A method is presented for the estimation of nucleotide diversity and genetic structure of populations from RAPD (random amplified polymorphic DNA) data. It involves a modification of the technique developed by Lynch and Crease (1990) for the case of restriction sites as survey data. As new elements the method incorporates (i) dominance correction, (ii) values of asexual reproduction of the populations sampled, and (iii) an analytical variance of the number of nucleotide substitutions per site. Sampling was carried out at two geographic scales for three aphid species. At a macrogeographic scale, populations of Rhopalosiphum padi did not show statistical genetic differentiation. Aphis gossypii and Myzus persicae, which were sampled at a microgeographic scale, showed a higher genetic differentiation than R. padi, it being statistically significant in M. persicae. The major sources of sampling variance within- and between-populations were found to be nucleotide (i.e., the number of alleles used as a function of the number of primers used) and population (i.e., sample size) sampling. Extremely low estimates of nucleotide diversity were obtained for the species studied here. This result is consistent with previous reports on genetic diversity for the same or other aphid species which were based on allozyme polymorphism, mitochondrial DNA variation and qualitative analyses of RAPDs.     

Freshwater ecosystems could become the biggest losers of the Paris Agreement

Securing access to energy for a growing population under the international commitment of reduction of greenhouse emissions requires increasing the contribution of renewable sources to the global share. Hydropower energy, which accounts for >80% of green energy, is experiencing a boom fostered by international investment mainly in developing countries. This boom could be further accelerated by the recent climate agreement reached in Paris. Despite its flexibility, hydropower production entails social, economic and ecological risks that need to be carefully considered before investing in the development of potentially thousands of planned hydropower projects worldwide. This is especially relevant given the weak or nonexistent legislation that regulates hydropower project approval and construction in many countries. I highlight the need for adequate policy to provide the Paris Agreement with new financial and planning mechanisms to avoid further and irreversible damage to freshwater ecosystem services and biodiversity.     

The Impact of the West Africa Ebola Outbreak on Obstetric Health Care in Sierra Leone

Background

As Sierra Leone celebrates the end of the Ebola Virus Disease (EVD) outbreak, we can begin to fully grasp its impact on already weak health systems. The EVD outbreak in West Africa forced many hospitals to close down or reduce their activity, either to prevent nosocomial transmission or because of staff shortages. The aim of this study is to assess the potential impact of EVD on nationwide access to obstetric care in Sierra Leone.

Methods and Findings

Community health officers collected weekly data between January 2014—May 2015 on in-hospital deliveries and caesarean sections (C-sections) from all open facilities (public, private for-profit and private non-profit sectors) offering emergency obstetrics in Sierra Leone. This was compared to official data of EVD cases per district. Logistic and Poisson regression analyses were used to compute risk and rate estimates. Nationwide, the number of in-hospital deliveries and C-sections decreased by over 20% during the EVD outbreak. The decline occurred early on in the EVD outbreak and was mainly attributable to the closing of private not-for-profit hospitals rather than government facilities. Due to difficulties in collecting data in the midst of an epidemic, limitations of this study include some missing data points.

Conclusions

Both the number of in-hospital deliveries and C-sections substantially declined shortly after the onset of the EVD outbreak. Since access to emergency obstetric care, like C-sections, is associated with decreased maternal mortality, many women are likely to have died due to the reduced access to appropriate care during childbirth. Future research on indirect health effects of health system breakdown should ideally be nationwide and continue also into the recovery phase. It is also important to understand the mechanisms behind the deterioration so that important health services can be reestablished.     

Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA

Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by ¹H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27–36 is essential to bind purine- or pyrimidine-based nucleosides.Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence.     

Role of a cluster of hydrophobic residues near the FAD cofactor in Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal complex formation and electron transfer to ferredoxin

In the ferredoxin-NADP(+) reductase (FNR)/ferredoxin (Fd) system, an aromatic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction. We have investigated further the role of hydrophobic interactions in complex stabilization and ET between these proteins by replacing three hydrophobic residues, Leu-76, Leu-78, and Val-136, situated on the FNR surface in the vicinity of its FAD cofactor. Whereas neither the ability of FNR to accept electrons from NADPH nor its structure appears to be affected by the introduced mutations, different behaviors with Fd are observed. Thus, the ET interaction with Fd is almost completely lost upon introduction of negatively charged side chains. In contrast, only subtle changes are observed upon conservative replacement. Introduction of Ser residues produces relatively sizable alterations of the FAD redox potential, which can explain the modified behavior of these mutants. The introduction of bulky aromatic side chains appears to produce rearrangements of the side chains at the FNR/Fd interaction surface. Thus, subtle changes in the hydrophobic patch influence the rates of ET to and from Fd by altering the binding constants and the FAD redox potentials, indicating that these residues are especially important in the binding and orientation of Fd for efficient ET. These results are consistent with the structure reported for the Anabaena FNR.Fd complex.     

The crystal structure of tetrameric methionine adenosyltransferase from rat liver reveals the methionine-binding site

Most of the transmethylation reactions use the same methyl donor, S-adenosylmethionine (SAM), that is synthesised from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, two MAT enzymes have been detected, one ubiquitous and another liver specific. The liver enzyme exists in two oligomeric forms, a tetramer (MAT I) and a dimer (MAT III), MAT I being the one that shows a higher level of affinity for methionine but a lower SAM synthesis capacity. We have solved the crystal structure of rat liver MAT I at 2.7 A resolution, complexed with a methionine analogue: l-2-amino-4-methoxy-cis-but-3-enoic acid (l-cisAMB). The enzyme consists of four identical subunits arranged in two tight dimers that are related by crystallographic 2-fold symmetry. The crystal structure shows the positions of the relevant cysteine residues in the chain, and that Cys35 and Cys61 are perfectly oriented for forming a disulphide link. This result leads us to propose a hypothesis to explain the control of MAT I/III exchange and hence, the effects observed on activity. We have identified the methionine-binding site into the active-site cavity, for the first time. The l-cisAMB inhibitor is stacked against Phe251 aromatic ring in a rather planar conformation, and its carboxylate group coordinates a Mg(2+), which, in turn, is linked to Asp180. The essential role of the involved residues in MAT activity has been confirmed by site-directed mutagenesis. Phe251 is exposed to solvent and is located in the beginning of the flexible loop Phe251-Ala260 that is connecting the N-terminal domain to the central domain. We postulate that a conformational change may take place during the enzymatic reaction and this is possibly the reason of the unusual two-step mechanism involving tripolyphosphate hydrolysis. Other important mechanistic implications are discussed on the light of the results. Moreover, the critical role that certain residues identified in this study may have in methionine recognition opens further possibilities for rational drug design.