Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis

The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.Thermophilic and hyperthermophilic organisms, like the vast majority of other microorganisms, accumulate compatible solutes in response to water stress imposed by salt. In fact, many of the (hyper)thermophiles known were isolated from geothermal areas venting seawater (36). However, the compatible solutes of thermophilic and hyperthermophilic prokaryotes are generally different from those of their mesophilic counterparts and some, namely, di-myo-inositol-phosphate (DIP), mannosyl-di-myo-inositol-phosphate (MDIP), diglycerol phosphate, and mannosylglyceramide, are confined to organisms that grow at extremely high temperatures (19, 22, 34, 38). Mannosylglycerate (2-α-d-mannosylglycerate; MG), for example, is a common compatible solute of thermophiles and hyperhermophiles (23, 27, 38) but has also been found in mesophilic organisms, such as red algae, where it was first identified (6). It should also be noted that there is a growing awareness that compatible solutes are involved in other types of stress; trehalose, for example, plays a role in osmotic stress, heat stress, desiccation, and freezing (9). Some compatible solutes of thermophilic organisms are extremely rare and have been encountered in only one or two, generally closely related, species. Among them are mannosylglyceramide in Rhodothermus marinus, diglycerol phosphate in Archaeoglobus fulgidus, and, more recently, mannosylglucosylglycerate (α-d-1→2-mannopyranosyl-α-d-1→2-glucopyranosylglycerate; MGG) identified in Petrotoga miotherma (16, 19, 38).The species of the genus Petrotoga represent slightly thermophilic members of the generally hyperthermophilic and deep-branching bacteria of the order Thermotogales (2, 3, 31). Organisms of this genus have all been isolated from hot oilfield water (21, 25), and have an optimum temperature for growth of 55 to 60°C in medium containing NaCl in the range of 0.5 to 10% (16). In Ptg. miotherma, the levels of MGG increased during low-level osmotic adaptation, whereas glutamate and proline were used for protection against hyperosmotic stress (16). The hyperthermophilic Thermotoga spp. accumulate primarily di-myo-inositol-phosphate and mannosyl-di-myo-inositol-phosphate during osmotic adjustment or during growth at temperatures above the optimum for growth (37).The novel compatible solute MGG is a derivative of glucosylglycerate (2-α-d-glucosylglycerate; GG) identified in the free form in Erwinia chrysanthemi, in the marine cyanobacteria Prochlorococcus marinus and Synechococcus sp. PCC7002, and in the thermophilic bacterium Persephonella marina, the latter of which possesses two alternative pathways for its synthesis (8, 13, 14, 18, 37). Glucosylglycerate has also been detected in trace amounts in Mycobacterium smegmatis, where it probably is the precursor of a polysaccharide involved in the regulation of fatty acid synthesis, as well as in the polar head group of a glycolipid from Nocardia otitidiscaviarum (17, 30).Two alternative pathways for the synthesis of GG have been identified and characterized. In the two-step reaction scheme, the synthesis of GG involves the condensation of nucleoside diphosphate (NDP)-glucose and d-3-phosphoglycerate (3-PGA) into glucosyl-3-phosphoglycerate (GPG), which in turn is dephosphorylated to yield GG. Yet, in a single-step pathway, the synthesis of GG occurs via the condensation of ADP-glucose with d-glycerate (13). Similar routes to those described above also lead to the synthesis of mannosylglycerate in Rhodothermus marinus (4).Two functionally connected genes encoding an “actinobacterial”-type glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase were detected in the genome of Petrotoga mobilis (12). In this study, we examine the synthesis of MGG through a phosphorylating pathway (with a phosphorylated intermediate) from 3-phosphoglycerate and UDP-glucose to the final compatible solute, in cell extracts and by functional characterization of recombinant enzymes. We also examine a second nonphosphorylating pathway (no phosphorylated intermediates) that could represent an alternative route for the synthesis of MGG in Ptg. mobilis that could lead to the direct conversion of GG and GDP-mannose to MGG. Pathway multiplicity likely reflects a crucial role for MGG in the physiology of Ptg. mobilis during stress adaptation.     

Dehydration of ribonucleotides catalyzed by ribonucleotide reductase: the role of the enzyme

This article focuses on the second step of the catalytic mechanism for the reduction of ribonucleotides catalyzed by the enzyme Ribonucleotide Reductase (RNR). This step corresponds to the protonation/elimination of the substrate's C-2' hydroxyl group. Protonation is accomplished by the neighbor Cys-225, leading to the formation of one water molecule. This is a very relevant step since most of the known inhibitors of this enzyme, which are already used in the fight against certain forms of cancer, are 2'-substituted substrate analogs. Even though some theoretical studies have been performed in the past, they have modeled the enzyme with minimal gas-phase models, basically represented by a part of the side chain of the relevant amino acids, disconnected from the protein backbone. This procedure resulted in a limited accuracy in the position and/or orientation of the participating residues, which can result in erroneous energetics and even mistakes in the choice of the correct mechanism for this step. To overcome these limitations we have used a very large model, including a whole R1 model with 733 residues plus the substrate and 10 A thick shell of water molecules, instead of the minimal gas-phase models used in previous works. The ONIOM method was employed to deal with such a large system. This model can efficiently account for the restrained mobility of the reactive residues, as well as the long-range enzyme-substrate interactions. The results gave additional information about this step, which previous small models could not provide, allowing a much clearer evaluation of the role of the enzyme. The interaction energy between the enzyme and the substrate along the reaction coordinate and the substrate steric strain energy have been obtained. The conclusion was that the barrier obtained with the present model was very similar to the one previously determined with minimal gas-phase models. Therefore, the role of the enzyme in this step was concluded to be mainly entropic, rather than energetic, by placing the substrate and the two reactive residues in a position that allows for the highly favorable concerted trimolecular reaction, and to protect the enzyme radical from the solvent.     

The Na+/H+ antiporter of the thermohalophilic bacterium Rhodothermus marinus

In the thermohalophilic bacterium Rhodothermus marinus, the NADH:quinone oxidoreductase (complex I) is encoded by two single genes and two operons, one of which contains the genes for five complex I subunits, nqo10-nqo14, a pterin carbinolamine dehydratase, and a putative single subunit Na+/H+ antiporter. Here we report that the latter encodes indeed a functional Na+/H+ antiporter, which is able to confer resistance to Na+, but not to Li+ to an Escherichia coli strain defective in Na+/H+ antiporters. In addition, an extensive amino acid sequence comparison with several single subunit Na+/H+ antiporters from different groups, namely NhaA, NhaB, NhaC, and NhaD, suggests that this might be the first member of a new type of Na+/H+ antiporters, which we propose to call NhaE.     

Biogeographic Patterns of Intertidal Macroinvertebrates and their Association with Macroalgae Distribution along the Portuguese Coast

Geographical patterns in the distribution of epifaunal crustaceans (Amphipoda, Isopoda and Tanaidacea) occurring with dominant macroalgal species were investigated along the Portuguese rocky coast. Three regions, each encompassing six shores, were studied. Algal species were selected according to their geographical distribution: Mastocarpusstellatus and Chondrus crispus (north); Bifurcariabifurcata (north-centre); Plocamiumcartilagineum and Cystoseiratamariscifolia (centre-south); Corallina spp. and Codiumtomentosum (entire coast). Multivariate techniques were used to test for differences in crustacean assemblage composition between sub-regions and host algal species. A clear gradient of species substitution was observed from north to south. Differences in abundance and diversity of epifaunal crustaceans were observed between southern locations and the remaining sites. Four species were recorded for the first time in the Portuguese coast. Among the 57 taxa identified, southern distribution limits were observed for three species and northern distribution limits were observed for four species. Interestingly, the observed geographical patterns in epifaunal abundance and diversity were not related with geographical changes in the indentity of the dominant algal species.     

Variability in infection efficiency in vitro of different strains of the microsporidian Encephalitozoon hellem

The infection efficiency of different strains of Encephalitozoon hellem of human origin was tested in Vero E6 cell cultures, scoring the number of infection foci (NIF) after 9, 14, 20, and 24 days of inoculation. The results revealed a strong interaction of the strain type with time: different strains showed different proliferative dynamics. Number of infection foci was lower on the first sampling day for CDC: V257, EHVS-96, and PV6-96, with a subsequent increase at a higher rate for the first strain and lower for the latter. In contrast, PV7-96 showed the highest NIF at the first sampling, followed by a slight decrease. Since these strains were selected by their genotype for the polar tube protein (PTP)-1A, 1B, 1C, and 2C, respectively, it is tempting to suggest a major role of this protein in the differences detected, although the influence of other genes that hypothetically may also differ among the strains employed cannot be discarded. The different in vitro infection efficiencies raise the possibility that some strains of E. hellem will also produce more aggressive features in infected patients.     

Molecular modeling of hair keratin/peptide complex: Using MM-PBSA calculations to describe experimental binding results

Molecular dynamics simulations of a keratin/peptide complex have been conducted to predict the binding affinity of four different peptides toward human hair. Free energy calculations on the peptides' interaction with the keratin model demonstrated that electrostatic interactions are believed to be the main driving force stabilizing the complex. The molecular mechanics-Poisson-Boltzmann surface area methodology used for the free energy calculations demonstrated that the dielectric constant in the protein's interior plays a major role in the free energy calculations, and the only way to obtain accordance between the free energy calculations and the experimental binding results was to use the average dielectric constant.     

Evaluation of the membrane lipid selectivity of the pea defensin Psd1

Psd1, a 46 amino acid residues defensin isolated from the pea Pisum sativum seeds, exhibits anti-fungal activity by a poorly understood mechanism of action. In this work, the interaction of Psd1 with biomembrane model systems of different lipid compositions was assessed by fluorescence spectroscopy. Partition studies showed a marked lipid selectivity of this antimicrobial peptide (AMP) toward lipid membranes containing ergosterol (the main sterol in fungal membranes) or specific glycosphingolipid components, with partition coefficients (K(p)) reaching uncommonly high values of 10(6). By the opposite, Psd1 does not partition to cholesterol-enriched lipid bilayers, such as mammalian cell membranes. The Psd1 mutants His36Lys and Gly12Glu present a membrane affinity loss relative to the wild type. Fluorescence quenching data obtained using acrylamide and membrane probes further clarify the mechanism of action of this peptide at the molecular level, pointing out the potential therapeutic use of Psd1 as a natural antimycotic agent.     

One or two cameras per station? Monitoring jaguars and other mammals in the Amazon

Camera trapping has become a popular technique to monitor carnivore populations due to its usefulness in estimating abundance. Nevertheless, there are a number of problems associated with study design which are motivating researchers to search for a compromise that ensures improvement of precision while being cost-effective. We have used data from a capture?Crecapture study in a forested area in central Brazil to evaluate the effectiveness of using one versus two cameras per trapping station for determining jaguar (Panthera onca) density and capture rates of several other mammals. The capture rate for the jaguar and other species recorded with only one camera was lower than that with two cameras. The number of jaguars identified using photos from one camera ranged between six and seven animals, but reached ten individuals when two-camera sets were used where pictures of both flanks could be positively individualized. These differences, combined with different estimates of effective sampled area size, resulted in jaguar densities estimates ranging from 2.18 to 5.40 and 3.99?individuals/100?km² when one and two cameras were used per station, respectively (using the half-MMDM and Heterogeneity model). Based on our results, we recommend the use of two cameras per station for jaguar density monitoring to ensure reasonable levels of reliability and accuracy of estimates despite a small sample size.     

Application of the Apn2/MAT locus to improve the systematics of the Colletotrichum gloeosporioides complex: an example from coffee (Coffea spp.) hosts

To improve phylogenetic resolution of the Colletotrichum gloeosporioides species complex we developed and tested the performance of a new set of primers for the Apn2/MAT locus with a case study of 22 isolates. These were isolated mainly from coffee plants and represent six divergent and well characterized species within the C. gloeosporioides complex. Following previous studies on this locus, we have generated sequence data from an expanded region (> 4600 bp), revealing increased phylogenetic informativeness when compared to other commonly used markers such as ITS, β-tub2 and GS. Within the Apn2/MAT locus the ApMAT marker alone was almost as informative in terms of phylogenetic resolution as a seven-gene concatenated dataset. Our results further revealed that gene-tree discordance may come to be a common issue in resolving evolutionary relationships in the C. gloeosporioides complex, highlighting the importance of multilocus approaches. The use of state-of-the-art data analysis techniques and a highly informative dataset as employed here may abate this issue and hopefully assist in disentangling the C. gloeosporioides complex.