Annelid phylogeny and the status of Sipuncula and Echiura

Background  

Annelida comprises an ancient and ecologically important animal phylum with over 16,500 described species and members are the dominant macrofauna of the deep sea. Traditionally, two major groups are distinguished: Clitellata (including earthworms, leeches) and "Polychaeta" (mostly marine worms). Recent analyses of molecular data suggest that Annelida may include other taxa once considered separate phyla (i.e., Echiura, and Sipuncula) and that Clitellata are derived annelids, thus rendering "Polychaeta" paraphyletic; however, this contradicts classification schemes of annelids developed from recent analyses of morphological characters. Given that deep-level evolutionary relationships of Annelida are poorly understood, we have analyzed comprehensive datasets based on nuclear and mitochondrial genes, and have applied rigorous testing of alternative hypotheses so that we can move towards the robust reconstruction of annelid history needed to interpret animal body plan evolution.     

Triplex-forming ability of modified oligonucleotides

We present our studies on the ability of several different nucleotide analogs as triplex-forming oligonucleotides. The modifications tested include 4'-C-hydroxymethyl, LNA, 2'-amino-LNA and N2'-functionalized 2'-amino-LNA. Triplexes containing monomers of N2'-glycyl-functionalized 2'-amino-LNA are particularly stable.     

Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks

Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, we establish that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. We propose that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals.     

Formation of atroviridin by Hypocrea atroviridis is conidiation associated and positively regulated by blue light and the G protein GNA3

Species of the mycoparasitic fungal genus Hypocrea/Trichoderma are prominent producers of peptaibols, a class of small linear peptides of fungal origin. Some of these peptaibols have been shown to act synergistically with cell-wall-degrading enzymes in the inhibition of the growth of other fungi in vitro and in vivo. Here we present the structure of the Hypocrea atroviridis peptaibol synthetase gene (pbs1), deduced from the genome sequence of H. atroviridis. It consists of 19 typical peptide synthetase modules with the required additional modifying domains at the N and C termini. Phylogenetic and similarity analyses of the individual amino acid-activating modules is consistent with its ability to synthesize atroviridins. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of surface-grown cultures of H. atroviridis showed that no peptaibols were formed during vegetative growth, but a microheterogenous mixture of atroviridins accumulated when the colonies started to sporulate. This correlation between sporulation and atroviridin formation was shown to be independent of the pathway inducing sporulation (i.e., light, mechanical injury and carbon starvation, respectively). Atroviridin formation was dependent on the function of the two blue light regulators, BLR1 and BLR2, under some but not all conditions of sporulation and was repressed in a pkr1 (regulatory subunit of protein kinase A) antisense strain with constitutively active protein kinase A. Conversely, however, loss of function of the Galpha-protein GNA3, which is a negative regulator of sporulation and leads to a hypersporulating phenotype, fully impairs atroviridin formation. Our data show that formation of atroviridin by H. atroviridis occurs in a sporulation-associated manner but is uncoupled from it at the stage of GNA3.     

Structural characterization of the pore forming protein TatAd of the twin-arginine translocase in membranes by solid-state N-NMR

The transmembrane protein TatA is the pore forming unit of the twin-arginine translocase (Tat), which has the unique ability of transporting folded proteins across the cell membrane. This ATP-independent protein export pathway is a recently discovered alternative to the general secretory (Sec) system of bacteria. To obtain insight in the translocation mechanism, the structure and alignment in the membrane of the well-folded segments 2-45 of TatA_d from Bacillus subtilis was studied here. Using solid-state NMR in bicelles containing anionic lipids, the topology and orientation of TatA_d was determined in an environment mimicking the bacterial membrane. A wheel-like pattern, characteristic for a tilted transmembrane helix, was observed in ¹⁵N chemical shift /¹⁵N-¹H dipolar coupling correlation NMR spectra. Analysis of this PISA wheel revealed a 14-16 residue long N-terminal membrane-spanning helix which is tilted by 17° with respect to the membrane normal. In addition, comparison of uniformly and selectively ¹⁵N-labeled TatA_2-45 samples allowed determination of the helix polarity angle.     

Structure-function relationships of the lanthionine cyclase SpaC involved in biosynthesis of the Bacillus subtilis peptide antibiotic subtilin

Biosynthesis of the lantibiotic subtilin in Bacillus subtilis is accomplished by a synthetase complex consisting of the dehydratase SpaB, cyclase SpaC, and transporter SpaT. Genetically engineered subtilin cyclases SpaC and related NisC and EriC proteins involved in biosynthesis of the lantibiotics nisin and ericin A/S, respectively, were analyzed to functionally substitute native SpaC in vivo. We could show for the first time posttranslational modification of a lantibiotic precursor peptide (subtilin) by a hybrid lantibiotic synthetase (SpaBT/EriC). Genetically engineered SpaC alanine replacement mutants revealed the essentiality of residues His231, Trp302, Cys303, Tyr304, Gly305, Cys349, and His350, as well as the conserved C-terminal motif Lys437-Ala438-Leu439-Leu440-Ile441 for subtilin biosynthesis. Assignment of these strictly conserved lantibiotic cyclase residues to the NisC structure [Li, B., Yu, J. B., Brunzelle, J. S., Moll, G. N., van der Donk, W. A., and Nair, S. K. (2006) Science, 311, 1464-1467] revealed the first experimental evidence for structure-function relationships in catalytic centers of lantibiotic cyclases. SpaC residues His231, Cys303, and Cys349 are involved in coordination of the central zinc ion. The pair His231/Tyr304 is discussed to act as general acid/base catalysts in lanthionine formation. Furthermore, pull-down experiments revealed that functional inactive SpaC mutants were still able to interact with the hexahistidine-tagged subtilin precursor peptide in vitro. Our results suggest that Trp302 and the C-terminal residues of SpaC are constituents of a hydrophobic cluster which is involved in stabilization of the catalytic center and binding of the subtilin precursor peptide.     

Enantiomer separation of fungicidal triazolyl alcohols by normal phase HPLC on polysaccharide‐based chiral stationary phases

Three fungicidal triazolyl alcohols (triadimenol, hexaconazole, and cis/trans‐1‐4‐chlorophenyl‐2‐1H‐1,2,4‐triazol‐1‐yl‐cycloheptanol) were completely separated into enantiomers by chiral HPLC using polysaccharide‐based chiral stationary phases. A better separation was achieved on cellulose and amylose carbamate phases compared with a cellulose ester phase. Peak shapes were almost symmetrical except for two cases, where tailing of the first eluted enantiomer and unusual symmetric peak broadening were observed. The effect of eluents on enantioseparation was also investigated. Chirality 11:195–200, 1999. © 1999 Wiley‐Liss, Inc.     

Critical summer foraging tradeoffs in a subarctic ungulate

Summer diets are crucial for large herbivores in the subarctic and are affected by weather, harassment from insects and a variety of environmental changes linked to climate. Yet, understanding foraging behavior and diet of large herbivores is challenging in the subarctic because of their remote ranges. We used GPS video‐camera collars to observe behaviors and summer diets of the migratory Fortymile Caribou Herd (Rangifer tarandus granti) across Alaska, USA and the Yukon, Canada. First, we characterized caribou behavior. Second, we tested if videos could be used to quantify changes in the probability of eating events. Third, we estimated summer diets at the finest taxonomic resolution possible through videos. Finally, we compared summer diet estimates from video collars to microhistological analysis of fecal pellets. We classified 18,134 videos from 30 female caribou over two summers (2018 and 2019). Caribou behaviors included eating (mean = 43.5%), ruminating (25.6%), travelling (14.0%), stationary awake (11.3%) and napping (5.1%). Eating was restricted by insect harassment. We classified forage(s) consumed in 5,549 videos where diet composition (monthly) highlighted a strong tradeoff between lichens and shrubs; shrubs dominated diets in June and July when lichen use declined. We identified 63 species, 70 genus and 33 family groups of summer forages from videos. After adjusting for digestibility, monthly estimates of diet composition were strongly correlated at the scale of the forage functional type (i.e., forage groups composed of forbs, graminoids, mosses, shrubs and lichens; r = 0.79, p < .01). Using video collars, we identified (1) a pronounced tradeoff in summer foraging between lichens and shrubs and (2) the costs of insect harassment on eating. Understanding caribou foraging ecology is needed to plan for their long‐term conservation across the circumpolar north, and video collars can provide a powerful approach across remote regions.     

Identification of ςB-Dependent Genes in Bacillus subtilis Using a Promoter Consensus-Directed Search and Oligonucleotide Hybridization

A consensus-directed search for sigma(B) promoters was used to locate potential candidates for new sigma(B)-dependent genes in Bacillus subtilis. Screening of those candidates by oligonucleotide hybridizations with total RNA from exponentially growing or ethanol-stressed cells of the wild type as well as a sigB mutant revealed 22 genes that required sigma(B) for induction by ethanol. Although almost 50% of the proteins encoded by the newly discovered sigma(B)-dependent stress genes seem to be membrane localized, biochemical functions have so far not been defined for any of the gene products. Allocation of the genes to the sigma(B)-dependent stress regulon may indicate a potential function in the establishment of a multiple stress resistance. AldY and YhdF show similarities to NAD(P)-dependent dehydrogenases and YdbP to thioredoxins, supporting our suggestion that sigma(B)-dependent proteins may be involved in the maintenance of the intracellular redox balance after stress.