Involvement of cytochrome c CymA in the anaerobic metabolism of RDX by Shewanella oneidensis MR-1

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitramine explosive commonly used for military applications that is responsible for severe soil and groundwater contamination. In this study, Shewanella oneidensis MR-1 was shown to efficiently degrade RDX anaerobically (3.5?μmol·h(-1)·(g protein)(-1)) via two initial routes: (1) sequential N-NO(2) reductions to the corresponding nitroso (N-NO) derivatives (94% of initial RDX degradation) and (2) denitration followed by ring cleavage. To identify genes involved in the anaerobic metabolism of RDX, a library of ~2500 mutants of MR-1 was constructed by random transposon mutagenesis and screened for mutants with a reduced ability to degrade RDX compared with the wild type. An RDX-defective mutant (C9) was isolated that had the transposon inserted in the c-type cytochrome gene cymA. C9 transformed RDX at ~10% of the wild-type rate, with degradation occurring mostly via early ring cleavage caused by initial denitration leading to the formation of methylenedinitramine, 4-nitro-2,4-diazabutanal, formaldehyde, nitrous oxide, and ammonia. Genetic complementation of mutant C9 restored the wild-type phenotype, providing evidence that electron transport components have a role in the anaerobic reduction of RDX by MR-1.     

A protein kinase A and Wnt-dependent network regulating an intermediate stage in epithelial tubulogenesis during kidney development

Genetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.     

Functional analysis of an indole-diterpene gene cluster for lolitrem B biosynthesis in the grass endosymbiont Epichloë festucae

Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole-diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub-telomeric locus in the genome of E. festucae. Here we resolve the genetic basis for the remarkable indole-diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.     

Quantile-based permutation thresholds for quantitative trait Loci hotspots

Quantitative trait loci (QTL) hotspots (genomic locations affecting many traits) are a common feature in genetical genomics studies and are biologically interesting since they may harbor critical regulators. Therefore, statistical procedures to assess the significance of hotspots are of key importance. One approach, randomly allocating observed QTL across the genomic locations separately by trait, implicitly assumes all traits are uncorrelated. Recently, an empirical test for QTL hotspots was proposed on the basis of the number of traits that exceed a predetermined LOD value, such as the standard permutation LOD threshold. The permutation null distribution of the maximum number of traits across all genomic locations preserves the correlation structure among the phenotypes, avoiding the detection of spurious hotspots due to nongenetic correlation induced by uncontrolled environmental factors and unmeasured variables. However, by considering only the number of traits above a threshold, without accounting for the magnitude of the LOD scores, relevant information is lost. In particular, biologically interesting hotspots composed of a moderate to small number of traits with strong LOD scores may be neglected as nonsignificant. In this article we propose a quantile-based permutation approach that simultaneously accounts for the number and the LOD scores of traits within the hotspots. By considering a sliding scale of mapping thresholds, our method can assess the statistical significance of both small and large hotspots. Although the proposed approach can be applied to any type of heritable high-volume "omic" data set, we restrict our attention to expression (e)QTL analysis. We assess and compare the performances of these three methods in simulations and we illustrate how our approach can effectively assess the significance of moderate and small hotspots with strong LOD scores in a yeast expression data set.     

Extreme population subdivision in the crown conch (Melongena corona): historical and contemporary influences

Organisms with crawl-away larvae are thought to experience highly restricted gene flow. Here, we assess the pattern and magnitude of population subdivision of the direct developing snails in the Melongena corona complex and assess the validity of species and subspecies designations in the genus. A total of 516 individuals from 15 locations were assayed at 8 microsatellite loci. Levels of genetic diversity were moderate and typical of gastropods. There were from 8 to 28 alleles per locus and the average observed per sample heterozygosity ranged from 0.16 to 0.79. Levels of genetic divergence were generally large with all sample pairwise F(ST) values statistically significant and ranging from 0.011 to 0.438 and Jost's D(EST) ranging from 0.028 to 0.731. A Bayesian analysis identified 7 clusters of, usually adjoining, samples. The population subdivision is likely derived from a complex mixture of life-history attributes, frequent short-distance dispersal via swimming larvae, rare short- and long-distance dispersal of rafting larvae and eggs, and a patchwork of adjacent and adjoining habitats. As with a previous study, the current taxonomy is not supported by the genetic results and the complex can be considered as M. corona, a single, albeit clearly geographically genetically structured, species.     

Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9?? resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.     

NITROGEN FIXATION,HYDROGEN CYCLING,AND ELECTRON TRANSPORT KINETICS IN TRICHODESMIUM ERYTHRAEUM (CYANOBACTERIA) STRAIN IMS1011

This study describes the relationships between dinitrogen (N₂) fixation, dihydrogen (H₂) production, and electron transport associated with photosynthesis and respiration in the marine cyanobacterium Trichodesmium erythraeum Ehrenb. strain IMS101. The ratio of H₂ produced:N₂ fixed (H₂:N₂) was controlled by the light intensity and by the light spectral composition and was affected by the growth irradiance level. For Trichodesmium cells grown at 50 μmol photons · m^?2 · s^?1, the rate of N₂ fixation, as measured by acetylene reduction, saturated at light intensities of 200 μmol photons · m^?2 · s^?1. In contrast, net H₂ production continued to increase with light levels up to 1,000 μmol photons · m^?2 · s^?1. The H₂:N₂ ratios increased monotonically with irradiance, and the variable fluorescence measured using a fast repetition rate fluorometer (FRRF) revealed that this increase was accompanied by a progressive reduction of the plastoquinone (PQ) pool. Additions of 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone (DBMIB), an inhibitor of electron transport from PQ pool to PSI, diminished both N₂ fixation and net H₂ production, while the H₂:N₂ ratio increased with increasing level of PQ pool reduction. In the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), nitrogenase activity declined but could be prolonged by increasing the light intensity and by removing the oxygen supply. These results on the coupling of N₂ fixation and H₂ cycling in Trichodesmium indicate how light intensity and light spectral quality of the open ocean can influence the H₂:N₂ ratio and modulate net H₂ production.     

Zoonotic and human parasites of inhabitants of Cueva de los Muertos Chiquitos, Rio Zape Valley, Durango, Mexico

We present the first reconstruction of the parasitoses among the people of the Loma San Gabriel culture, as represented by 36 coprolites excavated from the Cueva de los Muertos Chiquitos in Durango, Mexico. The coprolites date to approximately 1,400-yr-ago. Species identified based on eggs recovered include the trematode Echinostoma sp., the tapeworms Hymenolepis sp. and Dipylidium caninum , and the nematodes Ancylostoma duodenale, Enterobius vermicularis, and Trichuris trichiura. After rehydration and screening, 2 methods were used to recover eggs from these samples including spontaneous sedimentation and flotation. Samples were analyzed by 3 different laboratories for independent verification and comparison of methods. Spontaneous sedimentation resulted in the discovery of hymenolepidid eggs that were not found with flotation. Sedimentation was a more-sensitive indicator of prevalence as well. The modified method of flotation permitted estimation of egg concentration and resulted in the detection of a few specimens not found by sedimentation. The results of both methods showed that 19 (of 36) coprolites contained helminth eggs. Our results detected the presence of pathogenic helminths including hookworms and whipworms. The cestodes found do not cause severe pathology in humans. The early dates of hookworm and whipworm, relative to other findings in the southwest United States, indicate that these parasites arrived relatively late in prehistory in Arizona and New Mexico, probably moving into the area with travelers from Mesoamerica.