E. coli multidrug transporter MdfA is a monomer

MdfA is a 410-residue-long secondary multidrug transporter from E. coli. Cells expressing MdfA from a multicopy plasmid exhibit resistance against a diverse group of toxic compounds, including neutral and cationic ones, because of active multidrug export. As a prerequisite for high-resolution structural studies and a better understanding of the mechanism of substrate recognition and translocation by MdfA, we investigated its biochemical properties and overall structural characteristics. To this end, we purified the beta-dodecyl maltopyranoside (DDM)-solubilized protein using a 6-His tag and metal affinity chromatography, and size exclusion chromatography (SE-HPLC). Purified MdfA was analyzed for its DDM and phospholipid (PL) content, and tetraphenylphosphonium (TPP+)-binding activity. The results are consistent with MdfA being an active monomer in DDM solution. Furthermore, an investigation of two-dimensional crystals by electron crystallography and 3D reconstruction lent support to the notion that MdfA may also be monomeric in reconstituted proteoliposomes.     

High-resolution species-distribution model based on systematic sampling and indirect observations

Species distribution models (SDMs) are often limited by the use of coarse-resolution environmental variables and by the number of observations required for their calibration. This is particularly true in the case of elusive animals. Here, we developed a SDM by combining three elements: a database of explanatory variables, mapped at a fine resolution; a systematic sampling scheme; and an intensive survey of indirect observations. Using MaxEnt, we developed the SDM for the population of the Asiatic wild ass (Equus hemionus), a rare and elusive species, at three spatial scales: 10, 100, and 1000 m per pixel. We used indirect observations of feces mounds. We constructed 14 layers of explanatory variables, in five categories: water, topography, biotic conditions, climatic variables and anthropogenic variables. Woody vegetation cover and slopes were found to have the strongest effect on the distribution of wild ass and were included as the main predictors in the SDM. Model validation revealed that an intensive survey of feces mounds and high-resolution predictor layers resulted in a highly accurate and informative SDM. Fine-grain (10 and 100 m) SDMs can be utilized to: (1) characterize the variables influencing species distribution at high resolution and local scale, including anthropogenic effects and geomorphologic features; (2) detect potential population activity centers; (3) locate potential corridors of movement and possible isolated habitat patches. Such information may be useful for the conservation efforts of the Asiatic wild ass. This approach could be applied to other elusive species, particularly large mammals.     

Novel Primers Reveal Wider Diversity among Marine Aerobic Anoxygenic Phototrophs

Aerobic anoxygenic phototrophic bacteria (AAnPs) were previously proposed to account for up to 11% of marine bacterioplankton and to potentially have great ecological importance in the world's oceans. Our data show that previously used primers based on the M subunit of anoxygenic photosynthetic reaction center genes (pufM) do not comprehensively identify the diversity of AAnPs in the ocean. We have designed and tested a new set of pufM-specific primers and revealed several new AAnP variants in environmental DNA samples and genomic libraries.     

Roseobacter-Like Bacteria in Red and Mediterranean Sea Aerobic Anoxygenic Photosynthetic Populations

Bacteriochlorophyll a-containing aerobic anoxygenic phototrophs (AAnP) have been proposed to account for up to 11% of the total surface water microbial community and to potentially have great ecological importance in the world's oceans. Recently, environmental and genomic data based on analysis of the pufM gene identified the existence of α-proteobacteria as well as possible γ-like proteobacteria among AAnP in the Pacific Ocean. Here we report on analyses of environmental samples from the Red and Mediterranean Seas by using pufM as well as the bchX and bchL genes as molecular markers. The majority of photosynthesis genes retrieved from these seas were related to Roseobacter-like AAnP sequences. Furthermore, the sequence of a novel photosynthetic operon organization from an uncultured Roseobacter-like bacterial artificial chromosome retrieved from the Red Sea is described. The data show the presence of Roseobacter-like bacteria in Red and Mediterranean Sea AAnP populations in the seasons analyzed.     

Differential protein expression in Colletotrichum acutatum: changes associated with reactive oxygen species and nitrogen starvation implicated in pathogenicity on strawberry

The cellular outcome of changes in nitrogen availability in the context of development and early stages of pathogenicity was studied by quantitative analysis of two-dimensional gel electrophoresis of Colletotrichum acutatum infecting strawberry. Significant alterations occurred in the abundance of proteins synthesized during appressorium formation under nitrogen-limiting conditions compared with a complete nutrient supply. Proteins that were up- or down-regulated were involved in energy metabolism, nitrogen and amino acid metabolism, protein synthesis and degradation, response to stress and reactive oxygen scavenging. Members belonging to the reactive oxygen species (ROS) scavenger machinery, superoxide dismutase and glutathione peroxidase, were up-regulated at the appressorium formation stage, as well as under nitrogen-limiting conditions relative to growth with a complete nutrient supply, whereas abundance of bifunctional catalase was up-regulated predominantly at the appressorium formation stage. Fungal ROS were detected within germinating conidia during host pre-penetration, penetration and colonization stages, accompanied by plant ROS, which were abundant in the apoplastic space. Application of exogenous antioxidants quenched ROS production and reduced the frequency of appressorium formation. Up-regulation in metabolic activity was detected during appressorium formation and nutrient deficiency compared with growth under complete nutrient supply. Enhanced levels of proteins related to the glyoxylate cycle and lipid metabolism (malate dehydrogenase, formate dehydrogenase and acetyl-CoA acetyltransferase) were observed at the appressorium formation stage, in contrast to down-regulation of isocitrate dehydrogenase. The present study demonstrates that appressoria formation processes, occurring under nutritional deprivation, are accompanied by metabolic shifts, and that ROS production is an early fungal response that may modulate initial stages of pathogen development.     

The TSC-mTOR Pathway Mediates Translational Activation of TOP mRNAs by Insulin Largely in a Raptor- or Rictor-Independent Manner

The stimulatory effect of insulin on protein synthesis is due to its ability to activate various translation factors. We now show that insulin can increase protein synthesis capacity also by translational activation of TOP mRNAs encoding various components of the translation machinery. This translational activation involves the tuberous sclerosis complex (TSC), as the knockout of TSC1 or TSC2 rescues TOP mRNAs from translational repression in mitotically arrested cells. Similar results were obtained upon overexpression of Rheb, an immediate TSC1-TSC2 target. The role of mTOR, a downstream effector of Rheb, in translational control of TOP mRNAs has been extensively studied, albeit with conflicting results. Even though rapamycin fully blocks mTOR complex 1 (mTORC1) kinase activity, the response of TOP mRNAs to this drug varies from complete resistance to high sensitivity. Here we show that mTOR knockdown blunts the translation efficiency of TOP mRNAs in insulin-treated cells, thus unequivocally establishing a role for mTOR in this mode of regulation. However, knockout of the raptor or rictor gene has only a slight effect on the translation efficiency of these mRNAs, implying that mTOR exerts its effect on TOP mRNAs through a novel pathway with a minor, if any, contribution of the canonical mTOR complexes mTORC1 and mTORC2. This conclusion is further supported by the observation that raptor knockout renders the translation of TOP mRNAs rapamycin hypersensitive.     

BchY-Based Degenerate Primers Target All Types of Anoxygenic Photosynthetic Bacteria in a Single PCR

To detect anoxygenic bacteria containing either type 1 or type 2 photosynthetic reaction centers in a single PCR, we designed a degenerate primer set based on the bchY gene. The new primers were validated in silico using the GenBank nucleotide database as well as by PCR on pure strains and environmental DNA.Anoxygenic photosynthetic bacteria are diverse and important members of microbial communities (11, 13, 17, 20). There are five bacterial phyla containing anoxygenic phototrophs: Proteobacteria (purple bacteria), Chlorobi (green sulfur bacteria), Chloroflexi (green nonsulfur bacteria), Acidobacteria (“Candidatus Chloracidobacterium thermophilum” [7]), and Firmicutes (heliobacteria). While Heliobacterium modesticaldum, Chlorobi, and “Ca. Chloracidobacterium thermophilum” have a type 1 reaction center (RC1) similar to photosystem I in Cyanobacteria and higher plants, Chloroflexi and Proteobacteria possess a type 2 reaction center (RC2) similar to photosystem II of oxygenic phototrophs (7, 16).Primers based on pufM, the gene encoding the M subunit of RC2, have been widely used to detect phototrophic purple bacteria (1, 4, 12, 19). However, phototrophic bacteria that do not possess RC2 are not retrieved when pufM is used as the target. Achenbach and coworkers (1) developed primers targeting rRNA genes of Chlorobi, Chloroflexi, and heliobacteria, while Alexander and coworkers (2) have developed primers to specifically detect green sulfur bacteria (Chlorobi) by using 16S rRNA and fmoA as gene targets and applied these primers in environmental studies (3). No currently available primer set can simultaneously target phototrophs containing either RC1 or RC2.Since it is well established that both RC1- and RC2-containing anoxygenic phototrophs synthesize bacteriochlorophylls (BChls), we searched for a universal anoxygenic photosynthesis gene marker among all enzymes involved in BChl biosynthetic pathways. All known pathways for chlorophyll and BChl biosynthesis branch from the heme biosynthesis pathway at protoporphyrin IX and continue to chlorophyllide a (Chlide a) through the same intermediates (9). Chlide a is the branching point that separates chlorophyll and BChl biosynthetic pathways. Moreover, pathways for the synthesis of different BChls are also split at this stage: chlorophyllide oxidoreductase converts Chlide a to 3-vinyl-bacteriophyllide a, which is the precursor for BChls a, b, and g, while a yet unknown enzyme reduces Chlide a to 3-vinyl-bacteriophyllide d, a precursor for antenna BChls c, d, and e in Chlorobium spp. (9). Since 3-vinyl-bacteriophyllide a is the last common intermediate in the synthesis of BChl a and BChl g, and the latter is the only BChl in heliobacteria (14, 15), chlorophyllide oxidoreductase is the only enzyme that is (i) present in anoxygenic phototrophic bacteria and not in oxygenic phototrophs and (ii) common to all known anoxygenic phototrophic bacterial species (with the exception of “Ca. Chloracidobacterium thermophilum,” where the pathway for BChl synthesis is not yet known). Analyzing multiple alignments of the subunits of chlorophyllide oxidoreductase, we found that only the Y subunit (encoded by the BchY gene) had two conserved regions distinguishing this protein from its closest homologs; therefore, the bchY gene was chosen as a universal marker for anoxygenic photosynthesis.Due to likely codon variations coding identical amino acid sequences in different genomes (19), degenerate BchY primers were designed by reverse translation of two conserved regions of the BchY alignment (Fig. ​(Fig.1):1): bchY_fwd (5′-CCNCARACNATGTGYCCNGCNTTYGG-3′ [26 bases; 2,048 variants; corresponding amino acid sequence, PQTMCPAFG]) and bchY_rev (5′-GGRTCNRCNGGRAANATYTCNCC-3′ [23 bases; 4,096 variants; corresponding amino acid sequence, GE{I/M}FP{A/ V}DP]). Each primer had no more than two bases deviating from known bchY sequences in the GenBank nr database (except for H. modesticaldum) as well as to environmental BchY variants in the GenBank env_nr database. None of these deviations were located in the 3′ ends of the primers (see Tables S2 and S3 in the supplemental material). These primers, therefore, were predicted to amplify a wide diversity of bchY genes under nonstringent PCR conditions (50 to 52°C annealing temperature). The lengths of the expected PCR products were either 480 bp (for green sulfur, green nonsulfur bacteria, and heliobacteria) or 510 bp (for purple bacteria).Open in a separate windowFIG. 1.Multiple-amino-acid alignment of BchY proteins. Sequence abbreviations: R.den, Roseobacter denitrificans (gi|110677524); R.gel, Rubrivivax gelatinosus (gi|29893484); R.cap, Rhodobacter capsulatus (gi|114868); C.lit, Congregibacter litoralis KT 71 (gi|88706663); H.hal, Halorhodospira halophila (gi|121998388); C.aur, Chloroflexus aurantiacus (gi|163849328); C.tep, Chlorobium tepidum (gi|66576270); and H.mod, Heliobacterium modesticaldum (gi|167629410).In order to check primer specificity in silico, a screening procedure was developed. Putative primer sites (tags) for both the bchY_fwd and the bchY_rev primers were gathered from the GenBank nucleotide collection (nt) by BLAST with relaxed search conditions; the tags having mismatches at the 3′ end or more than five overall mismatches from their primer were filtered out, and the remaining tags were mapped to their sequences mimicking PCR primer annealing. Fragments ranging from 300 to 700 bp (virtual “PCR products”) were retrieved from GenBank and annotated (see Table S4 in the supplemental material). All bchY genes present in the GenBank nt database were virtually “amplified,” pointing to the robustness of the primers and our in silico PCR analysis. On the other hand, all nonspecific “amplicons” have major deviations from the primer sequences and would likely not be amplified by a real PCR. The same screening procedure was performed against the GenBank environmental nucleotide collection (env_nt) (see Table S5 in the supplemental material), and as in the case with the nt database, only bchY fragments were virtually “amplified.”The BchY primer set was validated using five key control organisms, including the RC2-containing the purple sulfur bacterium Allochromatium vinosum and the purple nonsulfur bacterium Rhodobacter capsulatus as well as the RC1-containing green sulfur bacterium Chlorobium limicola, green nonsulfur bacterium Chloroflexus aurantiacus, and the heliobacterium H. modesticaldum. Amplifications yielded the predicted products of 510 bp from the purple bacteria and 480 bp from the green sulfur and nonsulfur bacteria and H. modesticaldum. Negative-control Escherichia coli and Synechocystis sp. strain PCC 6803 did not yield amplification products when the bchY primers were used.The designed BchY primer set successfully amplified bchY genes from DNA obtained from both marine (East Mediterranean Sea) and freshwater (Lake Kinneret) environments (see Table S6 in the supplemental material for best BLASTX hits for selected sequenced fragments). These habitats were chosen for testing due to the previously reported wide diversity of their anoxygenic phototrophs (8, 10, 18, 19). A phylogenetic tree of bchY gene fragments amplified from both freshwater and marine DNA samples is shown in Fig. ​Fig.22.Open in a separate windowFIG. 2.BchY phylogenetic tree based on a maximum likelihood tree to which short sequences were added by ARB parsimony. The branches that appeared on the original maximum likelihood tree are shown with thicker lines. Bootstrap values greater than 50% are indicated next to the branches. Sequences obtained in this study are shown in bold. For reasons of clarity, not all BchY sequences retrieved are shown in the tree. For cases in which a BchY fragment was found in more than three clones, the numbers of clones are given in parentheses. Clones m21_2 and m21_3 are identical to the bchY gene of Hoeflea phototrophica strain DFL-43 (6); the m20_2 clone was identical to the bchY gene of Dinoroseobacter shibae (5).Our study underlines the utility of the bchY gene as a molecular marker for revealing genetic heterogeneity in phototrophic microbial populations. Using both wide-scale bioinformatic analysis and PCR on control strains and naturally occurring microbial community DNA, we have confirmed the specificity and coverage of the proposed degenerate BchY primers.     

Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies

Background  

In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction.