Differential Effects of Nitrogenous Fertilizers on Methane-Consuming Microbes in Rice Field and Forest Soils

The impact of environmental perturbation (e.g., nitrogenous fertilizers) on the dynamics of methane fluxes from soils and wetland systems is poorly understood. Results of fertilizer studies are often contradictory, even within similar ecosystems. In the present study the hypothesis of whether these contradictory results may be explained by the composition of the methane-consuming microbial community and hence whether methanotrophic diversity affects methane fluxes was investigated. To this end, rice field and forest soils were incubated in microcosms and supplemented with different nitrogenous fertilizers and methane concentrations. By labeling the methane with ¹³C, diversity and function could be coupled by analyses of phospholipid-derived fatty acids (PLFA) extracted from the soils at different time points during incubation. In both rice field and forest soils, the activity as well as the growth rate of methane-consuming bacteria was affected differentially. For type I methanotrophs, fertilizer application stimulated the consumption of methane and the subsequent growth, while type II methanotrophs were generally inhibited. Terminal restriction fragment length polymorphism analyses of the pmoA gene supported the PLFA results. Multivariate analyses of stable-isotope-probing PLFA profiles indicated that in forest and rice field soils, Methylocystis (type II) species were affected by fertilization. The type I methanotrophs active in forest soils (Methylomicrobium/Methylosarcina related) differed from the active species in rice field soils (Methylobacter/Methylomonas related). Our results provide a case example showing that microbial community structure indeed matters, especially when assessing and predicting the impact of environmental change on biodiversity loss and ecosystem functioning.     

Secreted proteins of Uromyces fabae: similarities and stage specificity

Uromyces fabae on Vicia faba is a model system for obligate biotrophic interactions. Searching for potential effector proteins we investigated the haustorial secretome of U. fabae (biotrophic stage) and compared it with the secretome of in vitro grown infection structures, which represent the pre-biotrophic stage. Using the yeast signal sequence trap method we identified 62 genes encoding proteins secreted from haustoria and 42 genes encoding proteins secreted from in vitro grown infection structures. Four of these genes were identical in both libraries, giving a total of 100 genes coding for secreted proteins. This finding indicates a strong stage-specific regulation of protein secretion. Similarity with previously identified proteins was found for 39 of the sequences analysed, 28 of which showed similarity to proteins identified among members of the order Uredinales only. This might be taken as an indication for possible roles in virulence and host specificity unique to the Uredinales.     

Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts

Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca²⁺ from thapsigargin-sensitive stores. Here, we have studied Ca²⁺ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca²⁺]_i was elevated and agonist-induced [Ca²⁺]_i increases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca²⁺. Importantly, [Ca²⁺]_i increases and Ca²⁺ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca²⁺ storage in S1P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca²⁺ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca²⁺]_i increase and the other with a slower and prolonged [Ca²⁺]_i elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca²⁺]_i increases, reflecting overall Ca²⁺ release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca²⁺ storage and elevated basal [Ca²⁺]_i. S1P metabolism thus plays a role not only in acute Ca²⁺ mobilization but also in long-term regulation of Ca²⁺ homeostasis.     

An integrated approach for environmental assessments

LCA is a system-wide assessment, and the LCIA phase is confronted with the difficulties of local and regional effects in a number of impact categories. We integrate three different environmental techniques to demonstrate how these effects can be addressed in an environmental assessment. The techniques are life cycle inventory, environmental fate models, and an ecological impact assessment using fuzzy expert systems. Results of the LCI are mass and energy flows. In the environmental fate modelling step these mass flows are transformed into concentration and immission values by dispersion-reaction models. A generalised fuzzy expert system for the environmental mechanisms compares calculated exposure with site specific buffering capacities and formulates a generalised dose-response relationship. This generalised fuzzy expert system is used as a template for the assessment of local and regional environmental impacts. An application of this integrated approach is shown for a practical problem: production of magnesium car components. The environmental fate of nitrogen oxides which are released due to the major combustion source within that production system is simulated. Fuzzy expert models for crop damage, soil acidification and eutrophication determine the possible environmental impact of the immited nitrogen oxides. The important methodological extension of this integrated approach is a regionalised impact assessment depending on the spatial distribution of environmental characteristics.     

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro³¹⁴–Trp³¹⁶ turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro³¹⁴ of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.     

Different Bacterial Populations Associated with the Roots and Rhizosphere of Rice Incorporate Plant-Derived Carbon

Microorganisms associated with the roots of plants have an important function in plant growth and in soil carbon sequestration. Rice cultivation is the second largest anthropogenic source of atmospheric CH₄, which is a significant greenhouse gas. Up to 60% of fixed carbon formed by photosynthesis in plants is transported below ground, much of it as root exudates that are consumed by microorganisms. A stable isotope probing (SIP) approach was used to identify microorganisms using plant carbon in association with the roots and rhizosphere of rice plants. Rice plants grown in Italian paddy soil were labeled with ¹³CO₂ for 10 days. RNA was extracted from root material and rhizosphere soil and subjected to cesium gradient centrifugation followed by 16S rRNA amplicon pyrosequencing to identify microorganisms enriched with ¹³C. Thirty operational taxonomic units (OTUs) were labeled and mostly corresponded to Proteobacteria (13 OTUs) and Verrucomicrobia (8 OTUs). These OTUs were affiliated with the Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria classes of Proteobacteria and the “Spartobacteria” and Opitutae classes of Verrucomicrobia. In general, different bacterial groups were labeled in the root and rhizosphere, reflecting different physicochemical characteristics of these locations. The labeled OTUs in the root compartment corresponded to a greater proportion of the 16S rRNA sequences (∼20%) than did those in the rhizosphere (∼4%), indicating that a proportion of the active microbial community on the roots greater than that in the rhizosphere incorporated plant-derived carbon within the time frame of the experiment.     

BIOCHEMICAL CHARACTERIZATION OF PARALYTIC SHELLFISH TOXIN BIOSYNTHESIS IN VITRO1

Saxitoxin (STX) and its analogs are voltage‐gated sodium‐channel blockers that cause paralytic shellfish poisoning (PSP) and negatively affect human health and seafood industries worldwide. Little is known about the molecular biology of PSP‐toxin synthesis. Saxitoxin precursors were identified 25 years ago, and a hypothetical biosynthesis pathway was proposed; however, the correct sequence of reactions and enzymes involved in their catalysis remains to be identified. This study describes the optimization of in vitro biosynthesis of PSP toxins by cellular lysates of the toxic cyanobacterium Cylindrospermopsis raciborskii (Wo?osz.) Seenaya et Subbaraju T3 and the characterization of its biochemical requirements. Enzymes involved in PSP‐toxin synthesis are located in the cytosol. The molecular components of in vitro biosynthesis reactions could not be completely defined because of the requirement of an unknown cofactor. Evidence is presented that supports the previous suggestion that STX biosynthesis involves a Claisen condensation between arginine and acetate. In addition, carbamoyl phosphate was identified as a likely precursor for carbamated PSP toxins. Predictions have been made regarding the enzymes that may be involved in the biosynthesis of PSP toxins. These included class II aminotransferase; nonheme iron oxygenase, containing flavin, and possibly ferredoxin, as the prosthetic groups; and an O‐carbamoyltransferase. On the other hand, the involvement of cytochrome P450 monooxygenase was excluded.     

Two protein-protein interaction sites on the spliceosome-associated human cyclophilin CypH

Cyclophilins are a family of proteins that share a common, highly conserved sequence motif. Cyclophilins bind transiently to other proteins and facilitate their folding. One member of the family, hCypH, is part of the human spliceosomal [U4/U6·U5] tri-snRNP complex; it associates specifically and stably with the U4/U6-specific protein 60K. Here, we demonstrate that recombinant hCypH exhibits peptidyl–prolyl isomerase (PPIase) activity, and describe mutagenesis studies demonstrating that it shares the catalytic pocket with other members of the cyclophilin family. However, neither the PPIase activity nor the catalytic pocket is required for binding of protein 60K. Rather, hCypH contains a small insertion in a loop of the otherwise conserved cyclophilin backbone, and this minor change creates a highly specific binding site that is responsible for the association of this cyclophilin, but not others, with protein 60K. hCypH is thus the first small cyclophilin shown to have a second protein–protein interaction site and the ability to bind stably to another protein. Since the catalytic pocket and the second binding site are located on opposite sides of the cyclophilin structure, this opens up the interesting possibility that hCypH may serve as a bridge mediating interactions between protein 60K of the U4/U6 snRNP and other as yet unknown factors.