In vivo random mutagenesis of Bacillus subtilis by use of TnYLB-1, a mariner-based transposon

This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either sigmaA- or sigmaB-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan r cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm r gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10(-2)) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a "TA" dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo- clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.     

Exposure of mycobacteria to cell wall-inhibitory drugs decreases production of arabinoglycerolipid related to Mycolyl-arabinogalactan-peptidoglycan metabolism

The "cell wall core" consisting of a mycolyl-arabinogalactan-peptidoglycan (mAGP) complex represents the hallmark of the mycobacterial cell envelope. It has been the focus of intense research at both structural and biosynthetic levels during the past few decades. Because it is essential, mAGP is also regarded as a target for several antitubercular drugs. Herein, we demonstrate that exposure of Mycobacterium bovis Bacille Calmette-Guérin or Mycobacterium marinum to thiacetazone, a second line antitubercular drug, is associated with a severe decrease in the level of a major apolar glycolipid. This inhibition requires MmaA4, a methyltransferase reported to participate in the activation process of thiacetazone. Following purification, this glycolipid was subjected to detailed structural analyses, combining gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance. This allowed to identify it as a 5-O-mycolyl-β-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1)-Gro, designated dimycolyl diarabinoglycerol (DMAG). The presence of DMAG was subsequently confirmed in other slow growing pathogenic species, including Mycobacterium tuberculosis. DMAG production was stimulated in the presence of exogenous glycerol. Interestingly, DMAG appears structurally identical to the terminal portion of the mycolylated arabinosyl motif of mAGP, and the metabolic relationship between these two components was provided using antitubercular drugs such as ethambutol or isoniazid known to inhibit the biosynthesis of arabinogalactan or mycolic acid, respectively. Finally, DMAG was identified in the cell wall of M. tuberculosis. This opens the possibility of a potent biological function for DMAG that may be important to mycobacterial pathogenesis.     

Identification of gene encoding Plasmodium knowlesi phosphatidylserine decarboxylase by genetic complementation in yeast and characterization of in vitro maturation of encoded enzyme

The 23-megabase genome of Plasmodium falciparum, the causative agent of severe human malaria, contains ～5300 genes, most of unknown function or lacking homologs in other organisms. Identification of these gene functions will help in the discovery of novel targets for the development of antimalarial drugs and vaccines. The P. falciparum genome is unusually A+T-rich, which hampers cloning and expressing these genes in heterologous systems for functional analysis. The large repertoire of genetic tools available for Saccharomyces cerevisiae makes this yeast an ideal system for large scale functional complementation analyses of parasite genes. Here, we report the construction of a cDNA library from P. knowlesi, which has a lower A+T content compared with P. falciparum. This library was applied in a yeast complementation assay to identify malaria genes involved in the decarboxylation of phosphatidylserine. Transformation of a psd1Δpsd2Δdpl1Δ yeast strain, defective in phosphatidylethanolamine synthesis, with the P. knowlesi library led to identification of a new parasite phosphatidylserine decarboxylase (PkPSD). Unlike phosphatidylserine decarboxylase enzymes from other eukaryotes that are tightly associated with membranes, the PkPSD enzyme expressed in yeast was equally distributed between membrane and soluble fractions. In vitro studies reveal that truncated forms of PkPSD are soluble and undergo auto-endoproteolytic maturation in a phosphatidylserine-dependent reaction that is inhibited by other anionic phospholipids. This study defines a new system for probing the function of Plasmodium genes by library-based genetic complementation and its usefulness in revealing new biochemical properties of encoded proteins.     

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase

ΔN₁₂₃-glucan-binding domain-catalytic domain 2 (ΔN₁₂₃-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN₁₂₃-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN₁₂₃-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN₁₂₃-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN₁₂₃-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.     

Icy: an open bioimage informatics platform for extended reproducible research

Current research in biology uses evermore complex computational and imaging tools. Here we describe Icy, a collaborative bioimage informatics platform that combines a community website for contributing and sharing tools and material, and software with a high-end visual programming framework for seamless development of sophisticated imaging workflows. Icy extends the reproducible research principles, by encouraging and facilitating the reusability, modularity, standardization and management of algorithms and protocols. Icy is free, open-source and available at http://icy.bioimageanalysis.org/.     

Larval Dispersal Modeling of Pearl Oyster Pinctada margaritifera following Realistic Environmental and Biological Forcing in Ahe Atoll Lagoon

Studying the larval dispersal of bottom-dwelling species is necessary to understand their population dynamics and optimize their management. The black-lip pearl oyster (Pinctada margaritifera) is cultured extensively to produce black pearls, especially in French Polynesia''s atoll lagoons. This aquaculture relies on spat collection, a process that can be optimized by understanding which factors influence larval dispersal. Here, we investigate the sensitivity of P. margaritifera larval dispersal kernel to both physical and biological factors in the lagoon of Ahe atoll. Specifically, using a validated 3D larval dispersal model, the variability of lagoon-scale connectivity is investigated against wind forcing, depth and location of larval release, destination location, vertical swimming behavior and pelagic larval duration (PLD) factors. The potential connectivity was spatially weighted according to both the natural and cultivated broodstock densities to provide a realistic view of connectivity. We found that the mean pattern of potential connectivity was driven by the southwest and northeast main barotropic circulation structures, with high retention levels in both. Destination locations, spawning sites and PLD were the main drivers of potential connectivity, explaining respectively 26%, 59% and 5% of the variance. Differences between potential and realistic connectivity showed the significant contribution of the pearl oyster broodstock location to its own dynamics. Realistic connectivity showed larger larval supply in the western destination locations, which are preferentially used by farmers for spat collection. In addition, larval supply in the same sectors was enhanced during summer wind conditions. These results provide new cues to understanding the dynamics of bottom-dwelling populations in atoll lagoons, and show how to take advantage of numerical models for pearl oyster management.     

Trunk's Natural Inclination Influences Stance Limb Kinetics,but Not Body Kinematics,during Gait Initiation in Able Men

The imposing mass of the trunk in relation to the whole body has an important impact on human motion. The objective of this study is to determine the influence of trunk''s natural inclination - forward (FW) or backward (BW) with respect to the vertical - on body kinematics and stance limb kinetics during gait initiation.Twenty-five healthy males were divided based on their natural trunk inclination (FW or BW) during gait initiation. Instantaneous speed was calculated at the center of mass at the first heel strike. The antero-posterior impulse was calculated by integrating the antero-posterior ground reaction force in time. Ankle, knee, hip and thoraco-lumbar (L5) moments were calculated using inverse dynamics and only peaks of the joint moments were analyzed. Among all the investigated parameters, only joint moments present significant differences between the two groups. The knee extensor moment is 1.4 times higher (P<0.001) for the BW group, before the heel contact. At the hip, although the BW group displays a flexor moment 2.4 times higher (P<0.001) before the swing limb''s heel-off, the FW group displays an extensor moment 3.1 times higher (P<0.01) during the swing phase. The three L5 extensor peaks after the toe-off are respectively 1.7 (P<0.001), 1.4 (P<0.001) and 1.7 (P<0.01) times higher for the FW group. The main results support the idea that the patterns described during steady-state gait are already observable during gait initiation. This study also provides reference data to further investigate stance limb kinetics in specific or pathologic populations during gait initiation. It will be of particular interest for elderly people, knowing that this population displays atypical trunk postures and present a high risk of falling during this forward stepping.     

Phylogenetic signal from rearrangements in 18 Anopheles species by joint scaffolding extant and ancestral genomes

Background

Genomes rearrangements carry valuable information for phylogenetic inference or the elucidation of molecular mechanisms of adaptation. However, the detection of genome rearrangements is often hampered by current deficiencies in data and methods: Genomes obtained from short sequence reads have generally very fragmented assemblies, and comparing multiple gene orders generally leads to computationally intractable algorithmic questions.

Results

We present a computational method, ADseq, which, by combining ancestral gene order reconstruction, comparative scaffolding and de novo scaffolding methods, overcomes these two caveats. ADseq provides simultaneously improved assemblies and ancestral genomes, with statistical supports on all local features. Compared to previous comparative methods, it runs in polynomial time, it samples solutions in a probabilistic space, and it can handle a significantly larger gene complement from the considered extant genomes, with complex histories including gene duplications and losses. We use ADseq to provide improved assemblies and a genome history made of duplications, losses, gene translocations, rearrangements, of 18 complete Anopheles genomes, including several important malaria vectors. We also provide additional support for a differentiated mode of evolution of the sex chromosome and of the autosomes in these mosquito genomes.

Conclusions

We demonstrate the method’s ability to improve extant assemblies accurately through a procedure simulating realistic assembly fragmentation. We study a debated issue regarding the phylogeny of the Gambiae complex group of Anopheles genomes in the light of the evolution of chromosomal rearrangements, suggesting that the phylogenetic signal they carry can differ from the phylogenetic signal carried by gene sequences, more prone to introgression.

     

The role of the CroR response regulator in resistance of Enterococcus faecalis to D‐cycloserine is defined using an inducible receiver domain

Enterococcus faecalis is an opportunistic multidrug‐resistant human pathogen causing severe nosocomial infections. Previous investigations revealed that the CroRS two‐component regulatory pathway likely displays a pleiotropic role in E. faecalis, involved in virulence, macrophage survival, oxidative stress response as well as antibiotic resistance. Therefore, CroRS represents an attractive potential new target for antibiotherapy. In this report, we further explored CroRS cellular functions by characterizing the CroR regulon: the ‘domain swapping’ method was applied and a CroR chimera protein was generated by fusing the receiver domain from NisR to the output domain from CroR. After demonstrating that the chimera CroR complements a croR gene deletion in E. faecalis (stress response, virulence), we conducted a global gene expression analysis using RNA‐Seq and identified 50 potential CroR targets involved in multiple cellular functions such as cell envelope homeostasis, substrate transport, cell metabolism, gene expression regulation, stress response, virulence and antibiotic resistance. For validation, CroR direct binding to several candidate targets was demonstrated by EMSA. Further, this work identified alr, the gene encoding the alanine racemase enzyme involved in E. faecalis resistance to D‐cycloserine, a promising antimicrobial drug to treat enterococcal infections, as a member of the CroR regulon.