Macrocytic Anemia and Mitochondriopathy Resulting from a Defect in Sideroflexin 4

We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case. In vitro and in vivo complementation studies with fibroblasts from the affected individuals and zebrafish demonstrated the requirement of SFXN4 for mitochondrial respiratory homeostasis and erythropoiesis. Our findings establish mutations in SFXN4 as a cause of mitochondriopathy and macrocytic anemia.     

Structural Basis for Biofilm Formation via the Vibrio cholerae Matrix Protein RbmA

During the transition from a free-swimming, single-cell lifestyle to a sessile, multicellular state called a biofilm, bacteria produce and secrete an extracellular matrix comprised of nucleic acids, exopolysaccharides, and adhesion proteins. The Vibrio cholerae biofilm matrix contains three major protein components, RbmA, Bap1, and RbmC, which are unique to Vibrio cholerae and appear to support biofilm formation at particular steps in the process. Here, we focus on RbmA, a structural protein with an unknown fold. RbmA participates in the early cell-cell adhesion events and is found throughout the biofilm where it localizes to cell-cell contact sites. We determined crystal structures of RbmA and revealed that the protein folds into tandem fibronectin type III (FnIII) folds. The protein is dimeric in solution and in crystals, with the dimer interface displaying a surface groove that is lined with several positively charged residues. Structure-guided mutagenesis studies establish a crucial role for this surface patch for RbmA function. On the basis of the structure, we hypothesize that RbmA serves as a tether by maintaining flexible linkages between cells and the extracellular matrix.     

Evaluation of suppressive and pro-resolving effects of EPA and DHA in human primary monocytes and T-helper cells

Despite their beneficial anti-inflammatory properties, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may increase the infection risk at high doses, likely by generating an immune-depressed state. To assess the contribution of different immune cell populations to the immunomodulatory fatty acid effect, we comparatively investigated several aspects of inflammation in human T-helper (Th) cells and monocytes. Both fatty acids, but DHA to a lesser extent compared with EPA, selectively and dose-dependently reduced the percentage of cytokine-expressing Th cells in a peroxisome proliferator-activated receptor (PPAR)γ-dependent fashion, whereas the expression of the cell surface marker CD69 was unaltered on activated T cells. In monocytes, both EPA and DHA increased interleukin (IL)-10 without affecting tumor necrosis factor (TNF)-α and IL-6. Cellular incorporation of EPA and DHA occurred mainly at the expense of arachidonic acid. Concomitantly, thromboxane B (TXB)2 and leukotriene B (LTB)4 in supernatants decreased, while levels of TXB3 and LTB5 increased. This increase was independent of activation and in accordance with cyclooxygenase expression patterns in monocytes. Moreover, EPA and DHA gave rise to a variety of mono- and trihydroxy derivatives of highly anti-inflammatory potential, such as resolvins and their precursors. Our results suggest that EPA and DHA do not generally affect immune cell functions in an inhibitory manner but rather promote pro-resolving responses.     

Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expression as fusion with maltose binding protein was not successful but cytoplasmic expression as His-tagged TRX fusion proteins with a TEV recognition sequence resulted in proteins of correct size. Use of the SHuffle strain C3030 further improved the expression. Fusion proteins inhibited growth of Escherichia coli. They could be cleaved by TEV protease, releasing authentic proproteins without any additional amino acid at the N-terminus.     

Structure and Community Composition of Sprout-Like Bacterial Aggregates in a Dinaric Karst Subterranean Stream

The Vjetrenica cave in the Dinaric Karst hosts a worldwide extraordinarily high cave biodiversity. Beside a diverse and specialized cave fauna, sprout-like formations attached to the bed of the cave stream were observed and described, but not further characterized, almost a century ago. Here we investigated these sprout-like microbial aggregates by the rRNA approach and detailed microscopy. Based on fluorescence in situ hybridization and ultrastructural analysis, the sprout-like formations are morphologically highly organized, and their core consists of a member of a novel deep-branching lineage in the bacterial phylum Nitrospirae. This organism displays an interesting cellular ultrastructure with different kinds of cytoplasmic inclusions and is embedded in a thick extracellular matrix, which contributes to the stability and shape of the aggregates. This novel bacterium has been provisionally classified as “Candidatus Troglogloea absoloni.” The surface of the sprout-like aggregates is more diverse than the core. It is colonized by a bacterial biofilm consisting primarily of filamentous Betaproteobacteria, whereas other microbial populations present in the crust include members of the Bacteriodetes, Gammaproteobacteria, Actinombacteria, Alphaproteobacteria, and Planctomycetes, which are intermingled with mineral inclusions. This study represents the first thorough molecular and ultrastructural characterization of the elusive sprout-like bacterial aggregates, which are also found in other cave systems of the Dinaric Karst. The discovery of Ca. Troglogloea absoloni contributes to the known biodiversity of subterranean ecosystems and especially of macroscopic structures formed in caves by microorganisms, whose composition and ecological function often remain enigmatic.     

Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

Clostridium beijerinckii is a well-known solvent-producing microorganism with great potential for biofuel and biochemical production. To better understand and improve the biochemical pathway to solvents, the development of genetic tools for engineering C. beijerinckii is highly desired. Based on mobile group II intron technology, a targetron gene knockout system was developed for C. beijerinckii in this study. This system was successfully employed to disrupt acid production pathways in C. beijerinckii, leading to pta (encoding phosphotransacetylase)- and buk (encoding butyrate kinase)-negative mutants. In addition to experimental characterization, the mutant phenotypes were analyzed in the context of our C. beijerinckii genome-scale model. Compared to those of the parental strain (C. beijerinckii 8052), acetate production in the pta mutant was substantially reduced and butyrate production was remarkably increased, while solvent production was dependent on the growth medium. The pta mutant also produced much higher levels of lactate, suggesting that disrupting pta influenced the energy generation and electron flow pathways. In contrast, acetate and butyrate production in the buk mutant was generally similar to that of the wild type, but solvent production was consistently 20 to 30% higher and glucose consumption was more rapid and complete. Our results suggest that the acid and solvent production of C. beijerinckii can be effectively altered by disrupting the acid production pathways. As the gene disruption method developed in this study does not leave any antibiotic marker in a disrupted allele, multiple and high-throughput gene disruption is feasible for elucidating genotype and phenotype relationships in C. beijerinckii.     

Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.