Anaerobic Carbon Monoxide Dehydrogenase Diversity in the Homoacetogenic Hindgut Microbial Communities of Lower Termites and the Wood Roach

Anaerobic carbon monoxide dehydrogenase (CODH) is a key enzyme in the Wood-Ljungdahl (acetyl-CoA) pathway for acetogenesis performed by homoacetogenic bacteria. Acetate generated by gut bacteria via the acetyl-CoA pathway provides considerable nutrition to wood-feeding dictyopteran insects making CODH important to the obligate mutualism occurring between termites and their hindgut microbiota. To investigate CODH diversity in insect gut communities, we developed the first degenerate primers designed to amplify cooS genes, which encode the catalytic (β) subunit of anaerobic CODH enzyme complexes. These primers target over 68 million combinations of potential forward and reverse cooS primer-binding sequences. We used the primers to identify cooS genes in bacterial isolates from the hindgut of a phylogenetically lower termite and to sample cooS diversity present in a variety of insect hindgut microbial communities including those of three phylogenetically-lower termites, Zootermopsis nevadensis, Reticulitermes hesperus, and Incisitermes minor, a wood-feeding cockroach, Cryptocercus punctulatus, and an omnivorous cockroach, Periplaneta americana. In total, we sequenced and analyzed 151 different cooS genes. These genes encode proteins that group within one of three highly divergent CODH phylogenetic clades. Each insect gut community contained CODH variants from all three of these clades. The patterns of CODH diversity in these communities likely reflect differences in enzyme or physiological function, and suggest that a diversity of microbial species participate in homoacetogenesis in these communities

Identification of a second Xenopus Twisted Gastrulation gene

Twisted Gastrulation (Tsg) is a secreted molecule which regulates BMP signalling in the extracellular space as part of an evolutionarily conserved network of interacting proteins. In Xenopus, maternal xTsg mRNA can be found throughout the early embryo. After gastrulation, xTsg is expressed as part of the BMP4 synexpression group until late tadpole stages. Here we report the identification of a second Xenopus Tsg gene (xTsg-2). Xenopus Tsg-2 is highly homologous to xTsg. In particular, amino acid residues which have been shown to be required for the binding of xTsg to BMP and to Chordin are conserved. The expression of Xenopus Tsg-2 mRNA was restricted to late stages of embryonic development; it was detected at tadpole stages in lateral plate mesoderm, neural crest, branchial arches and head mesenchyme. In microinjection experiments, the activity of xTsg-2 mRNA was similar to that of xTsg. We conclude that two Tsg genes act in distinct temporal and spatial territories in the course of Xenopus embryonic development

Genetic analysis of MA4079, an aldehyde dehydrogenase homolog, in Methanosarcina acetivorans

When Methanosarcina acetivorans grows on carbon monoxide (CO), it synthesizes high levels of a protein, MA4079, homologous to aldehyde dehydrogenases. To investigate the role of MA4079 in M. acetivorans, mutants lacking the encoding gene were generated and phenotypically analyzed. Loss of MA4079 had no eVect on methylotrophic growth but led to complete abrogation of methylotrophic growth in the presence of even small amounts of CO, which indicated the mutant’s inability to acclimate to the presence of this toxic gas. Prolonged incubation with CO allowed the isolation of a strain in which the eVect of MA4079 deletion is suppressed. The strain, designated Mu3, tolerated the presence of high CO partial pressures even better than the wild type. Immunological analysis using antisera against MA4079 suggested that it is not abundant in M. acetivorans. Comparison of proteins diVerentially abundant in Mu3 and the wild type revealed an elevated level of methyl-coenzyme M reductase and a decreased level of one isoform of carbon monoxide dehydrogenase/acetyl-coenzyme A synthase, which suggests that pleiotropic mutation(s) compensating for the loss of MA4079 aVected catabolism. The data presented point toward a role of MA4079 to enable M. acetivorans to properly acclimate to CO

MicroRNA Profiling as Tool for <i>In Vitro</i> Developmental Neurotoxicity Testing: The Case of Sodium Valproate

<div><p>Studying chemical disturbances during neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative <i>in vitro</i> testing approach for the identification of developmental neurotoxicants. miRNAs represent a class of small non-coding RNA molecules involved in the regulation of neural development and ESC differentiation and specification. Thus, neural differentiation of mESCs <i>in vitro</i> allows investigating the role of miRNAs in chemical-mediated developmental toxicity. We analyzed changes in miRNome and transcriptome during neural differentiation of mESCs exposed to the developmental neurotoxicant sodium valproate (VPA). A total of 110 miRNAs and 377 mRNAs were identified differently expressed in neurally differentiating mESCs upon VPA treatment. Based on miRNA profiling we observed that VPA shifts the lineage specification from neural to myogenic differentiation (upregulation of muscle-abundant miRNAs, <i>mir-206, mir-133a</i> and <i>mir-10a</i>, and downregulation of neural-specific <i>mir-124a, mir-128</i> and <i>mir-137</i>). These findings were confirmed on the mRNA level and via immunochemistry. Particularly, the expression of myogenic regulatory factors (MRFs) as well as muscle-specific genes (<i>Actc1, calponin</i>, <i>myosin light chain, asporin, decorin</i>) were found elevated, while genes involved in neurogenesis (e.g. <i>Otx1</i>, <i>2, and Zic3, 4, 5</i>) were repressed. These results were specific for valproate treatment and―based on the following two observations―most likely due to the inhibition of histone deacetylase (HDAC) activity: (i) we did not observe any induction of muscle-specific miRNAs in neurally differentiating mESCs exposed to the unrelated developmental neurotoxicant sodium arsenite; and (ii) the expression of muscle-abundant <i>mir-206</i> and <i>mir-10a</i> was similarly increased in cells exposed to the structurally different HDAC inhibitor trichostatin A (TSA). Based on our results we conclude that miRNA expression profiling is a suitable molecular endpoint for developmental neurotoxicity. The observed lineage shift into myogenesis, where miRNAs may play an important role, could be one of the developmental neurotoxic mechanisms of VPA.</p></div

Valproate effects on viability and expression of β-III-Tubulin.

<p>The cells were induced to differentiate into neurons for 16 days under continuous substance exposure. Cell viability was estimated using CellTiterBlue assay and is shown as a percentage of solvent control (A), expression of β-III-tubulin was analyzed by flow cytometry and is shown as a percentage of solvent control for each concentration tested (B). Results represent a mean of three independent differentiation experiments ± SEM.</p

Gene expression under VPA treatment.

<p>A. RT-PCR verification of Affymetrix whole genome array data. The graph demonstrates mean of log₂ fold change in three independent differentiation processes (VPA vs. solvent control) ± SEM for nine upregulated and four downregulated mRNAs. (n = 3 independent biological replicates, t-test, *p<0.05, **p<0.01, ***p<0.001). B. Induction of expression of myogenic regulation factors (MRFs) by VPA in neural-differentiated ES cells. The graph demonstrates mean of log₂ fold change (VPA vs. solvent control) ± SEM. (n = 3 independent biological replicates, t-test, *p<0.05, **p<0.01, ***p<0.001).</p

Expression of neuron- and myocyte-markers under VPA exposure.

<p>Neural-differentiated ES cells were immuno-stained with neuron specific marker β-III-tubulin (red) and muscle specific marker α-actinin (green) after VPA (A) or PBS (B) treatment.</p