Contents Volume 69, 2004

Volume Contents

Contents Volume 69, 2004     

Sporocarp δ15N and use of inorganic and organic nitrogen in vitro differ among host-specific suilloid fungi associated with high elevation five-needle pines

Widespread decline of whitebark and limber pines in the northern Rocky Mountains (USA) has created an imperative to understand functional diversity in their ectomycorrhizal associates. Because suilloid fungi are likely important in successful reestablishment of pines the nitrogen-related functional traits of 28 high-elevation suilloid isolates were examined. Radial growth, mass accumulation and mycelial density were measured for isolates on six different nitrogen sources. The δ¹⁵N values of suilloid sporocarps used as sources for pure cultures were compared against growth parameters to investigate a possible link between these N-related functional traits. Isolates grew poorly on nitrate and BSA and grew well on glutamine, alanyl-glutamine and ammonium phosphate, with somewhat slower growth on alanine. Isolates and species varied considerably in their growth response to different nitrogen sources. Effective use of nitrate and BSA was uncommon and associated with isolates with high inherent growth rates. Sporocarp δ¹⁵N was negatively correlated with relative growth on alanine of the corresponding isolates. Our results suggest strong similarities in N source use patterns of suilloid fungi of whitebark pine origin and those of another high-elevation five-needle stone pine, the Swiss stone pine.     

Response of tundra CH4 and CO2 flux tomanipulation of temperature and vegetation

We conducted plant species removals, air temperaturemanipulations, and vegetation and soil transplants inAlaskan wet-meadow and tussock tundra communities todetermine the relative importance of vegetation typeand environmental variables in controlling ecosystemmethane (CH₄) and carbon dioxide (CO₂) flux. Plastic greenhouses placed over wet-meadow tundraincreased air temperature, soil temperature, and soilmoisture, but did not affect CH₄ or CO₂ flux(measured in the dark). By contrast, removal ofsedges in the wet meadow significantly decreased fluxof CH₄, while moss removal tended to increaseCH₄ emissions. At 15 cm depth, pore-waterCH₄ concentrations were higher in sedge-removalthan in control plots, suggesting that sedgescontribute to CH₄ emissions by transportingCH₄ from anaerobic soil to the atmosphere, ratherthan by promoting methanogenesis. Inreciprocal-ecosystem transplants between thewet-meadow and tussock tundra communities, CH₄and CO₂ emissions were higher overall in thewet-meadow site, but were unrelated to transplantorigin. Methane flux was correlated with localvariation in soil temperature, thaw depth, andwater-table depth, but the relative importance ofthese factors varied through the season. Our resultssuggest that future changes in CH₄ and CO₂flux in response to climatic change will be morestrongly mediated by large-scale changes in vegetationand soil parameters than by direct temperature effects.     

Tracing metabolic pathways of lipid biosynthesis in ectomycorrhizal fungi from position‐specific 13C‐labelling in glucose

Six position‐specific ¹³C‐labelled isotopomers of glucose were supplied to the ectomycorrhizal fungi Suillus pungens and Tricholoma flavovirens. From the resulting distribution of ¹³C among fungal PLFAs, the overall order and contribution of each glucose atom to fatty acid ¹³C enrichment was: C6 (～31%) > C5 (～25%) > C1 (～18%) > C2 (～18%) > C3 (～8%) > C4 (～1%). These data were used to parameterize a metabolic model of the relative fluxes from glucose degradation to lipid synthesis. Our data revealed that a higher amount of carbon is directed to glycolysis than to the oxidative pentose phosphate pathway (60% and 40% respectively) and that a significant part flows through these pathways more than once (73%) due to the reversibility of some glycolysis reactions. Surprisingly, 95% of carbon cycled through glyoxylate prior to incorporation into lipids, possibly to consume the excess of acetyl‐CoA produced during fatty acid turnover. Our approach provides a rigorous framework for analysing lipid biosynthesis in fungi. In addition, this approach could ultimately improve the interpretation of isotopic patterns at natural abundance in field studies.     

In vitro activation of the Serratia marcescens hemolysin through modification and complementation.

The hemolytic activity of Serratia marcescens is determined by two polypeptides, termed ShlA and ShlB. ShlA is synthesized as an inactive precursor (ShlA*) and secreted with the help of ShlB, which is located in the outer membrane. In this study, it is shown that a cell lysate containing ShlB as well as partially purified ShlB converted ShlA* to the active ShlA hemolysin. ShlA remained active after removal of ShlB by column chromatography. In contrast to the stable modification of ShlA* by ShlB, a reversible activation was achieved by adding to ShlA* an N-terminal fragment of ShlA (ShlA16), consisting of 269 amino acid residues of ShlA and 18 residues of the vector. The nonhemolytic ShlA16 complemented ShlA* only when it was synthesized in an ShlB-producing cell. A deletion derivative of ShlA*, lacking residues 4 to 117, was complemented by ShlA16 but not activated by ShlB. Activation of ShlA* by ShlB at 4 degrees C proceeded at a much slower rate than complementation by ShlA16. It is concluded that ShlA* is modified by ShlB. ShlA16 modified by ShlB complements the missing modification of ShlA* in trans. Modification by ShlB occurs in the N-terminal part of ShlA*, which is also the reaction in vivo which results in active ShlA hemolysin in the culture supernatant. The HpmA hemolysin of Proteus mirabilis, which is very similar to ShlA, was also activated in vitro by ShlB and complemented by ShlA16.     

Convergent Surface Water Distributions in U.S. Cities

Earth’s surface is rapidly urbanizing, resulting in dramatic changes in the abundance, distribution and character of surface water features in urban landscapes. However, the scope and consequences of surface water redistribution at broad spatial scales are not well understood. We hypothesized that urbanization would lead to convergent surface water abundance and distribution: in other words, cities will gain or lose water such that they become more similar to each other than are their surrounding natural landscapes. Using a database of more than 1 million water bodies and 1 million km of streams, we compared the surface water of 100 US cities with their surrounding undeveloped land. We evaluated differences in areal (A _WB) and numeric densities (N _WB) of water bodies (lakes, wetlands, and so on), the morphological characteristics of water bodies (size), and the density (D _C) of surface flow channels (that is, streams and rivers). The variance of urban A _WB, N _WB, and D _C across the 100 MSAs decreased, by 89, 25, and 71%, respectively, compared to undeveloped land. These data show that many cities are surface water poor relative to undeveloped land; however, in drier landscapes urbanization increases the occurrence of surface water. This convergence pattern strengthened with development intensity, such that high intensity urban development had an areal water body density 98% less than undeveloped lands. Urbanization appears to drive the convergence of hydrological features across the US, such that surface water distributions of cities are more similar to each other than to their surrounding landscapes.     

Toward a molecular equivalent dose: use of the medaka model in comparative risk assessment

Recent changes in the risk assessment landscape underscore the need to be able to compare the results of toxicity and dose-response testing between a growing list of animal models and, quite possibly, an array of in vitro screening assays. How do we compare test results for a given compound between vastly different species? For example, what dose level in the ambient water of a small fish model would be equivalent to 10 ppm of a given compound in the rat's drinking water? Where do we begin? To initially address these questions, and in order to compare dose-response tests in a standard rodent model with a fish model, we used the concept of molecular dose. Assays that quantify types of DNA damage that are directly relevant to carcinogenesis integrate the factors such as chemical exposure, uptake, distribution, metabolism, etc. that tend to vary so widely between different phyletic levels. We performed parallel exposures in F344 rats and Japanese medaka (Oryzias latipes) to the alkylating hepatocarcinogen, dimethylnitrosamine (DMN). In both models, we measured the DNA adducts 8-hydroxyguanine, N(7)-methylguanine and O(6)-methylguanine in the liver; mutation frequency using lambda cII transgenic medaka and lambda cII transgenic (Big Blue(R)) rats; and early morphological changes in the livers of both models using histopathology and immunohistochemistry. Pulse dose levels in fish were 0, 10, 25, 50, or 100 ppm DMN in the ambient water for 14 days. Since rats are reported to be especially sensitive to DMN, they received 0, 0.1, 1, 5, 10, or 25 ppm DMN in the drinking water for the same time period. While liver DNA adduct concentrations were similar in magnitude, mutant frequencies in the DMN-exposed medaka were up to 20 times higher than in the Big Blue rats. Future work with other compounds will generate a more complete picture of comparative dose response between different phyletic levels and will help guide risk assessors using "alternative" models.