Exoenzymes

The impact of acid rock drainage (ARD) in stream sediments on microbial communities above and below two abandoned mine sites in South Australia (Brukunga Mine) and in Australia' s Northern Territory (Rum Jungle Mine) was quantified by phospholipid profiling (PLFA). This technique has not previously been applied to environmental sites impacted by ARD. The study investigated the relationships between the community structure, biomass and a heavy metal-low pH contamination gradient. The ecosystems studied also suffered from additional environmental insults, including sewage and salinity. Physiochemical studies of the water and sediments were conducted in parallel to the PLFA analysis. The PLFA technique was also compared to additional growth and activity methods currently in use such as Biolog™, exoenzyme activities, plate counts and macroinvertebrate counts. Data was accumulated during the period 1998 to 2002. Both univariate and multivariate statistical techniques were applied to the data sets. Redundancy analysis (RDA) was used to identify the environmental parameters with the greatest bearing on the PLFA profiles. Analysis was conducted to compare spatial seasonal and temporal variations in the PLFA profiles. Multivariate analyses suggested that chemical and biological impacts of ARD downstream Brukunga Mine was limited to 14.5 km in the dry summer period, but extended as far as 22 km downstream in other seasons. In contrast, ARD impact downstream of the post-rehabilitated Rum Jungle Mine was limited to the first 3 km. The community structure of sediments downstream of both mines was dominated by prokaryotes. Aerobic prokaryotes and heterotrophic eukaryotes composed the largest group, followed by Gram-positive bacteria, with micro eukaryotes and SRB present only in minute proportions. Levels of Gram-positive bacteria were consistently suppressed downstream of both mines. Eukaryote levels, particularly fungal markers, were notably elevated just below Brukunga Mine. In contrast, the proportions of eukaryotes were markedly lower downstream Rum Jungle Mine. Principal component analysis (PCA) revealed a number of PLFA which were most influential in discriminating between ARD polluted sites and the rest of the sites. These PLFA included the Gram-negative markers: 20Hl2:0, 30Hl2:0, 30Hl4:0; the fungal marker: 18:2w6 and the Acidithiobacillus markers 10mel6:1 and 10mel8:1. The PLFA species 18:2w6, 20Hl2:0, 30Hl2:0 also characterised sites below Rum Jungle Mine. Elevated biomass at heavily ARD impacted sites below both mines was attributed to more favourable conditions for growth of acidophilic microorganisms. The study demonstrated the successful application of PLF A profiling technique to rapidly assess the toxicity of ARD affected waters and sediments and to differentiate this response from the effect of other pollutants, viz increased nutrients and salinity. This technique was sensitive enough to monitor even moderate levels of pollution, such as in Rum Jungle Mine. The technique was particularly useful when the PLFA absolute abundance data were converted to mol% of the total fatty acids and analysed by multivariate techniques.

Summary.  To characterize strains of Microsporum canis that infect dogs and cats in Sa˜o Paulo city, 30 isolates of this dermatophyte were tested for their ability to assimilate carbon and nitrogen sources, for proteinase and phospholipase secretion, for susceptibility to yeast killer toxins, and for susceptibility to the antifungals fluconazole, ketoconazole, itraconazole, 5-fluorocytosine and amphotericin B, in E test. All samples assimilated the nitrogen sources asparagine, ammonium sulphate, urea and sodium nitrate, as well as the carbon sources inulin, mannitol, trehalose, meso-erythritol, maltose, mannose, sorbitol, cellobiose, fructose and dextrin. Not all the samples assimilated adonitol, galactose, arabinose, rhamnose, raffinose, melibiose, ribose and sucrose, and none of them was capable of growing with dulcitol, lactose, or xylose as the only carbon source. Proteinase and phospholipase secretion was observed for most isolates. In the test of yeast killer toxin, 10 types could be identified, with four types exclusively observed in isolates from dogs and two types exclusively observed in isolates from cats. In the E test, all isolates were found to be resistant to the fluconazole and 5-fluorocytosine, while they were variably sensitive to amphotericin B, ketoconazole and itraconazole. When the data were submitted to the qualitative analysis in the matrix distance program Fitopac, the similarity of the isolates could be assessed.Zusammenfassung.  Dreißig Microsporum canis-Isolate, von Hunden und Katzen in Sao Paulo isoliert, wurden auf ihre Proteinase- und Phospholipase-Produktion und ihre Empfindlichkeit gegen Killer-Toxine geprüft. Die Sensibilität dieser Isolate gegenüber Fluconazol, Ketoconazol, Itraconazol, 5-Fluorocytosin und Amphotericin B wurde im E-test untersucht. Alle M. canis-Stämme metabolisierten Asparagin, Ammoniumsulfat, Harnstoff und Natriumnitrat, um aus diesen Stickstoff-Quellen Energie zu gewinnen. Inulin, Mannitol, Trehalose, Meso-Erythritol, Maltose, Mannose, Sorbitol, Cellobiose, Fructose und Dextrin wurden also Kohlenstoffquelle verstoffwechselt. Adonitol, Galaktose, Arabinose, Rhamnose, Raffinose, Melibiose, Ribose und Saccharose wurde von den M. canis-Stämmen unterschiedlich verwertet. Kein Isolat war bei Zusatz von Dulcitol, Lactose und Xylose gewachsen. Proteinase und Phospholipase konnte bei den meisten Isolaten nachgewiesen werden. Durch den Killer-Test wurden 10 verschiedene M. canis-Typen identifiziert, wobei vier Typen vorwiegend bei Hunden und zwei nur bei Katzen gefunden wurden. Alle M. canis-Isolate erwiesen sich als resistent gegen Fluconazol und 5-Fluorocytosin im E-test. Unterschiedliche Sensibilitäten wurden gegen Ketoconazol, Itraconazol und Amphotericin B beobachtet. Durch qualitative Auswertung der Daten mittels des Matrix-Distanzprogrammes Fitopac konnte die Ähnlichkeit der Isolate bestimmt werden.Zusammenfassung.  Dreißig Microsporum canis-Isolate, von Hunden und Katzen in Sao Paulo isoliert, wurden auf ihre Proteinase- und Phospholipase-Produktion und ihre Empfindlichkeit gegen Killer-Toxine geprüft. Die Sensibilität dieser Isolate gegenüber Fluconazol, Ketoconazol, Itraconazol, 5-Fluorocytosin und Amphotericin B wurde im E-test untersucht. Alle M. canis-Stämme metabolisierten Asparagin, Ammoniumsulfat, Harnstoff und Natriumnitrat, um aus diesen Stickstoff-Quellen Energie zu gewinnen. Inulin, Mannitol, Trehalose, Meso-Erythritol, Maltose, Mannose, Sorbitol, Cellobiose, Fructose und Dextrin wurden also Kohlenstoffquelle verstoffwechselt. Adonitol, Galaktose, Arabinose, Rhamnose, Raffinose, Melibiose, Ribose und Saccharose wurde von den M. canis-Stämmen unterschiedlich verwertet. Kein Isolat war bei Zusatz von Dulcitol, Lactose und Xylose gewachsen. Proteinase und Phospholipase konnte bei den meisten Isolaten nachgewiesen werden. Durch den Killer-Test wurden 10 verschiedene M. canis-Typen identifiziert, wobei vier Typen vorwiegend bei Hunden und zwei nur bei Katzen gefunden wurden. Alle M. canis-Isolate erwiesen sich als resistent gegen Fluconazol und 5-Fluorocytosin im E-test. Unterschiedliche Sensibilitäten wurden gegen Ketoconazol, Itraconazol und Amphotericin B beobachtet. Durch qualitative Auswertung der Daten mittels des Matrix-Distanzprogrammes Fitopac konnte die Ähnlichkeit der Isolate bestimmt werden.

Summary.  To characterize strains of Microsporum canis that infect dogs and cats in Sa˜o Paulo city, 30 isolates of this dermatophyte were tested for their ability to assimilate carbon and nitrogen sources, for proteinase and phospholipase secretion, for susceptibility to yeast killer toxins, and for susceptibility to the antifungals fluconazole, ketoconazole, itraconazole, 5-fluorocytosine and amphotericin B, in E test. All samples assimilated the nitrogen sources asparagine, ammonium sulphate, urea and sodium nitrate, as well as the carbon sources inulin, mannitol, trehalose, meso-erythritol, maltose, mannose, sorbitol, cellobiose, fructose and dextrin. Not all the samples assimilated adonitol, galactose, arabinose, rhamnose, raffinose, melibiose, ribose and sucrose, and none of them was capable of growing with dulcitol, lactose, or xylose as the only carbon source. Proteinase and phospholipase secretion was observed for most isolates. In the test of yeast killer toxin, 10 types could be identified, with four types exclusively observed in isolates from dogs and two types exclusively observed in isolates from cats. In the E test, all isolates were found to be resistant to the fluconazole and 5-fluorocytosine, while they were variably sensitive to amphotericin B, ketoconazole and itraconazole. When the data were submitted to the qualitative analysis in the matrix distance program Fitopac, the similarity of the isolates could be assessed.Zusammenfassung.  Dreißig Microsporum canis-Isolate, von Hunden und Katzen in Sao Paulo isoliert, wurden auf ihre Proteinase- und Phospholipase-Produktion und ihre Empfindlichkeit gegen Killer-Toxine geprüft. Die Sensibilität dieser Isolate gegenüber Fluconazol, Ketoconazol, Itraconazol, 5-Fluorocytosin und Amphotericin B wurde im E-test untersucht. Alle M. canis-Stämme metabolisierten Asparagin, Ammoniumsulfat, Harnstoff und Natriumnitrat, um aus diesen Stickstoff-Quellen Energie zu gewinnen. Inulin, Mannitol, Trehalose, Meso-Erythritol, Maltose, Mannose, Sorbitol, Cellobiose, Fructose und Dextrin wurden also Kohlenstoffquelle verstoffwechselt. Adonitol, Galaktose, Arabinose, Rhamnose, Raffinose, Melibiose, Ribose und Saccharose wurde von den M. canis-Stämmen unterschiedlich verwertet. Kein Isolat war bei Zusatz von Dulcitol, Lactose und Xylose gewachsen. Proteinase und Phospholipase konnte bei den meisten Isolaten nachgewiesen werden. Durch den Killer-Test wurden 10 verschiedene M. canis-Typen identifiziert, wobei vier Typen vorwiegend bei Hunden und zwei nur bei Katzen gefunden wurden. Alle M. canis-Isolate erwiesen sich als resistent gegen Fluconazol und 5-Fluorocytosin im E-test. Unterschiedliche Sensibilitäten wurden gegen Ketoconazol, Itraconazol und Amphotericin B beobachtet. Durch qualitative Auswertung der Daten mittels des Matrix-Distanzprogrammes Fitopac konnte die Ähnlichkeit der Isolate bestimmt werden.Zusammenfassung.  Dreißig Microsporum canis-Isolate, von Hunden und Katzen in Sao Paulo isoliert, wurden auf ihre Proteinase- und Phospholipase-Produktion und ihre Empfindlichkeit gegen Killer-Toxine geprüft. Die Sensibilität dieser Isolate gegenüber Fluconazol, Ketoconazol, Itraconazol, 5-Fluorocytosin und Amphotericin B wurde im E-test untersucht. Alle M. canis-Stämme metabolisierten Asparagin, Ammoniumsulfat, Harnstoff und Natriumnitrat, um aus diesen Stickstoff-Quellen Energie zu gewinnen. Inulin, Mannitol, Trehalose, Meso-Erythritol, Maltose, Mannose, Sorbitol, Cellobiose, Fructose und Dextrin wurden also Kohlenstoffquelle verstoffwechselt. Adonitol, Galaktose, Arabinose, Rhamnose, Raffinose, Melibiose, Ribose und Saccharose wurde von den M. canis-Stämmen unterschiedlich verwertet. Kein Isolat war bei Zusatz von Dulcitol, Lactose und Xylose gewachsen. Proteinase und Phospholipase konnte bei den meisten Isolaten nachgewiesen werden. Durch den Killer-Test wurden 10 verschiedene M. canis-Typen identifiziert, wobei vier Typen vorwiegend bei Hunden und zwei nur bei Katzen gefunden wurden. Alle M. canis-Isolate erwiesen sich als resistent gegen Fluconazol und 5-Fluorocytosin im E-test. Unterschiedliche Sensibilitäten wurden gegen Ketoconazol, Itraconazol und Amphotericin B beobachtet. Durch qualitative Auswertung der Daten mittels des Matrix-Distanzprogrammes Fitopac konnte die Ähnlichkeit der Isolate bestimmt werden.

A key challenge to understanding the effects of climate change and nutrient deposition on ecosystem functioning is our lack of knowledge about nutrient limitations of heterotrophic and phototrophic microbial communities. This is especially true in high elevation ecosystems where it has been shown that earlier melt-out of snow beds and glacial retreat is allowing photosynthetic microbes and plants to move into previously unvegetated areas. We used landscape-level analyses of microbial enzyme stoichiometries combined with soil microcosm fertilization studies to determine which nutrients are limiting to microbes in plant-free or sparsely vegetated, snow bed areas of the Colorado Front Range. Both of these independent approaches indicated that the ultimate limiting nutrient in unvegetated and sparsely vegetated soils is phosphorus (P) for phototrophic microbes, with co-limitation by carbon (C) for the entire microbial community. In contrast, vegetated soils in the same watersheds showed more balanced nitrogen (N), P and C co-limitation similar to patterns seen in other plant-dominated ecosystems. In microcosm experiments, P additions resulted in increased growth rates and percent cover by phototrophs, whereas N additions decreased the relative abundances of phototrophs. Taken together, our findings indicate that the colonization of high elevation ecosystems being impacted by N deposition and climate warming will likely be constrained by P limitation of both heterotrophic and phototrophic microbes and by negative impacts of N on microbial phototrophs. These effects may in turn limit the ability of these fragile ecosystems to immobilize inputs of atmospheric N causing increased runoff of excess N to downstream ecosystems.

The importance of crustal fluid chemical composition in driving the marine deep subseafloor biosphere was examined in northeast Pacific ridge-flank sediments. At IODP Site U1301, sulfate from crustal fluids diffuses into overlying sediments, forming a transition zone where sulfate meets in situ-produced methane. Enhanced cell counts and metabolic activity suggest that sulfate stimulates microbial respiration, specifically anaerobic methane oxidation coupled to sulfate reduction. Cell counts and activity are also elevated in basement-near layers. Owing to the worldwide expansion of the crustal aquifer, we postulate that crustal fluids may fuel the marine deep subseafloor biosphere on a global scale.