A role for polyproline motifs in the spinal muscular atrophy protein SMN. Profilins bind to and colocalize with smn in nuclear gems

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the loss of alpha-motoneurons in the spinal cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have been identified on human chromosome 5q. The corresponding SMN protein is expressed ubiquitously. It is coded by seven exons and contains conspicuous proline-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to bind to profilins (PFNs), small proteins engaged in the control of actin dynamics. We tested whether profilins interact with SMN via its polyproline stretches. Using the yeast two-hybrid system we show that profilins bind to SMN and that this binding depends on its proline-rich motifs. These results were confirmed by coimmunoprecipitation and by in vitro binding studies. Two PFN isoforms, I and II, are known, of which II is characteristic for central nervous system tissue. We show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons. In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN in the cytoplasm of the cell body and in nuclear gems. Likewise, SMN and PFN I colocalize in gems of HeLa cells. Although SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the SMN genes are expressed ubiquitously, our findings suggest that the interaction of PFN II with SMN may be involved in neuron-specific effects of SMN mutations.     

Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis

The large cytotoxins of Clostridia species glycosylate and thereby inactivate small GTPases of the Rho family. Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin use UDP-glucose as the donor for glucosylation of Rho/Ras GTPases. In contrast, alpha-toxin from Clostridium novyi N-acetylglucosaminylates Rho GTPases by using UDP-N-acetylglucosamine as a donor substrate. Based on the crystal structure of C. difficile toxin B, we studied the sugar donor specificity of the toxins by site-directed mutagenesis. The changing of Ile-383 and Gln-385 in toxin B to serine and alanine, respectively, largely increased the acceptance of UDP-N-acetylglucosamine as a sugar donor for modification of RhoA. The K(m) value was reduced from 960 to 26 mum for the double mutant. Accordingly, the potential of the double mutant of toxin B to hydrolyze UDP-N-acetylglucosamine was higher than that for UDP-glucose. The changing of Ile-383 and Gln-385 in the lethal toxin of C. sordellii allowed modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduced the acceptance of UDP-glucose as a donor for glycosylation. Vice versa, the changing of the equivalent residues in C. novyi alpha-toxin from Ser-385 and Ala-387 to isoleucine and glutamine, respectively, reversed the donor specificity of the toxin from UDP-N-acetylglucosamine to UDP-glucose. These data demonstrate that two amino acid residues are crucial for the co-substrate specificity of clostridial glycosylating toxins.     

Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.     

Soil respiration under elevated CO2 and its partitioning into recently assimilated and older carbon sources

Plant and Soil - Efflux of soil CO2 (soil respiration) plays a crucial role in the global carbon cycle and may be strongly altered by global change. In this study, we measured soil respiration in...     

Isotopic signatures of N2O produced by ammonia-oxidizing archaea from soils

N₂O gas is involved in global warming and ozone depletion. The major sources of N₂O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N₂O produced by soil AOA and associated N₂O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N₂O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), δ¹⁵N^bulk and δ¹⁸O -N₂O of soil AOA strains were 13–30%, −13 to −35% and 22–36%, respectively, and strains MY1–3 and other soil AOA strains had distinct isotopic signatures. A ¹⁵N-NH₄⁺-labeling experiment indicated that N₂O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N₂O from AOA may be attributable to the relative contributions of these two processes. The highest N₂O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N₂O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N₂O emissions from soil nitrification may be attributable to AOA.     

Elevated air carbon dioxide concentrations increase dissolved carbon leaching from a cropland soil

Terrestrial sources of nitrogen (N), particularly N-fixing alder, may be important for sustaining production in headwater streams that typically lack substantial subsidies of marine-derived nutrients from spawning salmon yet support upstream-dispersing juvenile salmonids. However, other physiographic characteristics, such as watershed slope and topographic wetness, also control transport of nutrients to streams and may confound apparent linkages between alder and stream N. Seasonal patterns in precipitation and temperature may interact with watershed characteristics to modulate stream N availability. We empirically modeled the effect of alder cover and other watershed physiographic variables on stream N and contrasted these relationships over the growing season among 25 first-order streams from the lower Kenai Peninsula, Alaska. For each date, percent alder cover, mean topographic wetness, and mean slope were used as watershed predictors of NO _x –N concentration (nitrate?+?nitrite) and daily NO _x –N yield using Generalized Additive Models (GAM) and compared using Akaike’s Information Criterion (AIC_c). Alder cover was the only probable model and explained 75–96% of the variation in NO _x –N concentration and 83–89% of the variation in daily NO _x –N yield. The relationship between alder and both NO _x –N concentration and daily NO _x –N yield changed from constant inputs in May across the range of alder cover (linear fit) to increasing inputs in July and September (non-linear fits) implying that high-alder watersheds were N-saturated. The strong linkage between alder and stream N coupled with the concurrent timing of maximum stream N from alder in the spring to salmon fry emergence indicates the potential importance of this subsidy to headwater stream ecosystems.     

Clostridium difficile glucosyltransferase toxin B-essential amino acids for substrate binding

Recently the crystal structure of the catalytic domain of Clostridium difficile toxin B was solved ( Reinert, D. J., Jank, T., Aktories, K., and Schulz, G. E. (2005) J. Mol. Biol. 351, 973-981 ). On the basis of this structure, we studied the functional role of several amino acids located in the catalytic center of toxin B. Besides the (286)DXD(288) motif and Trp(102), which were shown to be necessary for Mn(2+) and UDP binding, respectively, we identified by alanine scanning Asp(270), Arg(273), Tyr(284), Asn(384), and Trp(520) as being important for enzyme activity. The amino acids Arg(455), Asp(461), Lys(463), and Glu(472) and residues of helix alpha17 (e.g. Glu(449)) of toxin B are essential for enzyme-protein substrate recognition. Introduction of helix alpha17 of toxin B into Clostridium sordellii lethal toxin inhibited modification of Ras subfamily proteins but enabled glucosylation of RhoA, indicating that helix alpha17 is involved in RhoA recognition by toxin B. The data allow the design of a model of the interaction of the glucosyltransferase domain of toxin B with its protein substrate RhoA.     

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co‐expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive‐like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co‐expressed constitutively active Rop4, blocking the hypersensitive response caused by over‐expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop‐dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop‐dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.     

Cyanogenesis inhibits active defense reactions in plants

In the course of fungal attack on the cyanogenic rubber tree (Hevea brasiliensis Muell.-Arg.) HCN is liberated from infected tissue. The HCN interferes with plant host and fungal pathogen. It becomes inhibitory to active defense responses which are dependent on biosynthetic processes as far as a threshold concentration is transgressed.     

Interpretation of sulfur cycling in two catchments in the Black Forest (Germany) using stable sulfur and oxygen isotope data

The isotopic composition of SO ₄ ^2- in bulk precipitation, canopy throughfall, seepage water at three different soil depths, stream water, and groundwater was monitored in two forested catchments in the Black Forest (Germany) between November 1989 and February 1992. Isotope measurements on aqueous sulfate were complemented by ³⁴S-analyses on SO₂ in the air, total sulfur and inorganic sulfate in the soil, and bedrock sulfur, in order to identify sources and biogeochemical processes affecting S cycling in catchments with base poor, siliceous bedrock. Stable S isotope data indicated that atmospheric deposition and not mineral weathering is the major source of S in both catchments since ³⁴S-values for sulfate in the soil, in seepage water, and in stream water were generally found to be similar to the mean ³⁴S-values of precipitation SO ₄ ^2- (+2.1. However, ¹⁸O-values of seepage water SO ₄ ^2- at 30 cm and especially at 80 cm depth were depleted by several per mil with respect to those of the atmospheric deposition (+7.5 to +13.5. This indicates that in both catchments a considerable proportion of the seepage water SO ₄ ^2- is derived from mineralization of carbon-bonded soil S and must therefore have cycled through the organic soil S pool. ³⁴S-values for different S compounds in the solid soil were found to differ markedly depending on S fraction and soil depth. Since atmospheric S deposition with rather constant ³⁴S-values was identified as the dominant S source in both catchments, this is interpreted as a result ofin situ isotope fractionation rather than admixture of isotopically different S. The differences between the ³⁴S-values of seepage water and soil sulfate and those of organic soil S compounds are consistent with a model in which SO ₄ ^2- uptake by vegetation and soil microorganisms favours³⁴SO ₄ ^2- slightly, whereas during mineralization of organic soil S to aqueous SOSO ₄ ^2- ,³²S reacts preferentially. However, the data provide evidence for negligible isotope fractionation during physico-chemical S transformations such as adsorption/desorption in aerated forest soils.