Where is the unseen fungal diversity hidden? A study of Mortierella reveals a large contribution of reference collections to the identification of fungal environmental sequences

? Estimation of the proportion of undescribed fungal taxa is an issue that has remained unresolved for many decades. Several very different estimates have been published, and the relative contributions of traditional taxonomic and next-generation sequencing (NGS) techniques to species discovery have also been called into question recently. ? Here, we addressed the question of what proportion of hitherto unidentifiable molecular operational taxonomic units (MOTUs) have already been described but not sequenced, and how many of them represent truly undescribed lineages. We accomplished this by modeling the effects of increasing type strain sequencing effort on the number of identifiable MOTUs of the widespread soil fungus Mortierella. ? We found a nearly linear relationship between the number of type strains sequenced and the number of identifiable MOTUs. Using this relationship, we made predictions about the total number of Mortierella species and found that it was very close to the number of described species in Mortierella. ? These results suggest that the unusually high number of unidentifiable MOTUs in environmental sequencing projects can be, at least in some fungal groups, ascribed to a lag in type strain and specimen sequencing rather than to a high number of undescribed species.     

Late Quaternary paleoenvironmental records from the western Lena Delta,Arctic Siberia

The three main Lena Delta terraces were formed during different stages of the late Quaternary. While only the first floodplain terrace is connected with active deltaic processes, the second and third terraces, which dominate the western part of the delta, are erosional remnants of arctic paleolandscapes affected by periglacial processes. The landscape dynamics of the second and the third terraces, and their relationship to each other, are of particular importance in any effort to elucidate the late Quaternary paleoenvironment of western Beringia.Multidisciplinary studies of permafrost deposits on the second terrace were carried out at several sites of the Arga Complex, named after the largest delta island, Arga–Muora–Sise. The frozen sediments predominantly consist of fluvial sands several tens of meters thick, radiocarbon-dated from > 52 to 16 kyr BP. These sands were deposited under changing fluvial conditions in a dynamic system of shifting river channels, and have been additionally modified by synsedimentary and postsedimentary cryogenesis. Later thermokarst processes affected this late Pleistocene fluvial landscape during the Lateglacial and the Holocene. In addition, eolian activity reworked the fluvial sands on exposed surfaces at least since the Lateglacial, resulting in dune formation in some areas. Contrary to the Arga Complex, the third terrace is mainly composed of polygenetic alluvial and proluvial ice-rich permafrost sequences (Ice Complex deposits) radiocarbon-dated from 50 to 17 kyr BP which cover older fluvial sand units luminescence-dated to about 100–50 kyr BP. Paleoecological records reflect tundra-steppe conditions that varied locally, depending on landscape dynamics, during the Marine Isotope Stage (MIS) 4 and 3 periods, and a persistent change to shrub and arctic tundra during Lateglacial and Holocene periods.The study results indicate a continuous fluvial sedimentation environment for the Laptev Sea shelf in the region of the second Lena Delta terrace during the late Pleistocene, and confirm the presence of a dynamic channel system of the paleo-Lena River that flowed at the same time as the nearby subaerial Ice Complex deposits were being formed.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains

The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.     

Entianin, a novel subtilin-like lantibiotic from Bacillus subtilis subsp. spizizenii DSM 15029T with high antimicrobial activity

Lantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and subtilin autoinduction are available. In this report, we used the previously reported subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029(T). Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated subtilin, B. subtilis DSM 15029(T) secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated subtilin (S-subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.     

Versatile metabolic adaptations of Ralstonia eutropha H16 to a loss of PdhL, the E3 component of the pyruvate dehydrogenase complex

A previous study reported that the Tn5-induced poly(3-hydroxybutyric acid) (PHB)-leaky mutant Ralstonia eutropha H1482 showed a reduced PHB synthesis rate and significantly lower dihydrolipoamide dehydrogenase (DHLDH) activity than the wild-type R. eutropha H16 but similar growth behavior. Insertion of Tn5 was localized in the pdhL gene encoding the DHLDH (E3 component) of the pyruvate dehydrogenase complex (PDHC). Taking advantage of the available genome sequence of R. eutropha H16, observations were verified and further detailed analyses and experiments were done. In silico genome analysis revealed that R. eutropha possesses all five known types of 2-oxoacid multienzyme complexes and five DHLDH-coding genes. Of these DHLDHs, only PdhL harbors an amino-terminal lipoyl domain. Furthermore, insertion of Tn5 in pdhL of mutant H1482 disrupted the carboxy-terminal dimerization domain, thereby causing synthesis of a truncated PdhL lacking this essential region, obviously leading to an inactive enzyme. The defined ΔpdhL deletion mutant of R. eutropha exhibited the same phenotype as the Tn5 mutant H1482; this excludes polar effects as the cause of the phenotype of the Tn5 mutant H1482. However, insertion of Tn5 or deletion of pdhL decreases DHLDH activity, probably negatively affecting PDHC activity, causing the mutant phenotype. Moreover, complementation experiments showed that different plasmid-encoded E3 components of R. eutropha H16 or of other bacteria, like Burkholderia cepacia, were able to restore the wild-type phenotype at least partially. Interestingly, the E3 component of B. cepacia possesses an amino-terminal lipoyl domain, like the wild-type H16. A comparison of the proteomes of the wild-type H16 and of the mutant H1482 revealed striking differences and allowed us to reconstruct at least partially the impressive adaptations of R. eutropha H1482 to the loss of PdhL on the cellular level.     

Theoretical Analysis of Time-to-Peak Responses in Biological Reaction Networks

Processing of information by signaling networks is characterized by properties of the induced kinetics of the activated pathway components. The maximal extent of pathway activation (maximum amplitude) and the time-to-peak-response (position) are key determinants of biological responses that have been linked to specific outcomes. We investigate how the maximum amplitude of pathway activation and its position depend on the input and wiring of a signaling network. For this purpose, we consider a simple reaction A→B that is regulated by a transient input and extended this to include back-reaction and additional partners. In particular, we show that a unique maximum of B(t) exists. Moreover, we prove that the position of the maximum is independent of the applied input but regulated by degradation reactions of B. Indeed, the time-to-peak-response decreases with increasing degradation rate, which we prove for small models and show in simulations for more complex ones. The identified dependencies provide insights into design principles that facilitate the realization dynamical characteristics like constant position of maximal pathway activation and thereby guide the characterization of unknown kinetics within larger protein networks.     

OpenCMISS: A multi-physics & multi-scale computational infrastructure for the VPH/Physiome project

The VPH/Physiome Project is developing the model encoding standards CellML (cellml.org) and FieldML (fieldml.org) as well as web-accessible model repositories based on these standards (models.physiome.org). Freely available open source computational modelling software is also being developed to solve the partial differential equations described by the models and to visualise results. The OpenCMISS code (opencmiss.org), described here, has been developed by the authors over the last six years to replace the CMISS code that has supported a number of organ system Physiome projects.OpenCMISS is designed to encompass multiple sets of physical equations and to link subcellular and tissue-level biophysical processes into organ-level processes. In the Heart Physiome project, for example, the large deformation mechanics of the myocardial wall need to be coupled to both ventricular flow and embedded coronary flow, and the reaction-diffusion equations that govern the propagation of electrical waves through myocardial tissue need to be coupled with equations that describe the ion channel currents that flow through the cardiac cell membranes.In this paper we discuss the design principles and distributed memory architecture behind the OpenCMISS code. We also discuss the design of the interfaces that link the sets of physical equations across common boundaries (such as fluid-structure coupling), or between spatial fields over the same domain (such as coupled electromechanics), and the concepts behind CellML and FieldML that are embodied in the OpenCMISS data structures. We show how all of these provide a flexible infrastructure for combining models developed across the VPH/Physiome community.     

Heat shock protein expression and change of cytochrome c oxidase activity: presence of two phylogenic old systems to protect tissues in ischemia and reperfusion

Induction of heat shock proteins (hsp) has been shown to protect cells from ischemia by providing transient tolerance against myocardial injury and improving postischemic functional recovery. Attenuation of ATP depletion and earlier restoration of ATP content on reperfusion are thought to play a role in this scenario. Hsp induction is accompanied by altered enzyme activity of the respiratory chain, the major generator of ATP under physiological conditions. This report addresses the question whether processing and final assembly of the active holoenzyme cytochrome c oxidase (CcO, complex IV), member of the respiratory chain, is compromised under hypoxic conditions unless protected by stress proteins. Special focus is laid on function of the enzyme’s subunits and importance of cellular energy availability and maintenance.     

Do global change experiments overestimate impacts on terrestrial ecosystems?

In recent decades, many climate manipulation experiments have investigated biosphere responses to global change. These experiments typically examined effects of elevated atmospheric CO(2), warming or drought (driver variables) on ecosystem processes such as the carbon and water cycle (response variables). Because experiments are inevitably constrained in the number of driver variables tested simultaneously, as well as in time and space, a key question is how results are scaled up to predict net ecosystem responses. In this review, we argue that there might be a general trend for the magnitude of the responses to decline with higher-order interactions, longer time periods and larger spatial scales. This means that on average, both positive and negative global change impacts on the biosphere might be dampened more than previously assumed.