Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. β-, γ-, δ-, and -proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating δ-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous γ-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.     

Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake

Abstract Three membrane-bound acid-stable cytochromes c with molecular masses of 46, 30 and 21 kDa were characterized from a new Thiobacillus ferrooxidans strain. They were solubilized with high concentrations of dodecylmaltoside at pH 8. The 30 kDa cytochrome c was purified to a homogeneous state as established by SDS-PAGE analysis. It showed an absorption peak at 410 nm in the oxidized form and at 418, 523 and 552 nm in the reduced form. The 46 kDa cytochrome c co-purified with a non-heme protein of 36 kDa. The amino acid composition and the N-terminal amino acid sequence of the 46 kDa cytochrome c were determined and compared with those of the soluble 14 kDa and the membrane-bound 21, 22.3 and 68 kDa cytochromes c isolated from two different strains. The results clearly show that this cytochrome is distinct from both the 22.3, 21 and 14 kDa cytochrome species, and exhibits some similarities with the 68 kDa cytochrome c as regards its amino acid composition.     

Dominant microbial populations in limestone-corroding stream biofilms, Frasassi cave system, Italy

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. beta-, gamma-, delta-, and epsilon-proteobacteria in sulfur-cycling clades accounted for > or = 75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating delta-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous gamma-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of epsilon-proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.     

Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy

The sulfide-rich Frasassi cave system hosts an aphotic, subsurface microbial ecosystem including extremely acidic (pH 0-1), viscous biofilms (snottites) hanging from the cave walls. We investigated the diversity and population structure of snottites from three locations in the cave system using full cycle rRNA methods and culturing. The snottites were composed primarily of bacteria related to Acidithiobacillus species. Other populations present in the snottites included Thermoplasmata group archaea, bacteria related to Sulfobacillus, Acidimicrobium, and the proposed bacterial lineage TM6, protists, and filamentous fungi. Based on fluorescence in situ hybridization population counts, Acidithiobacillus are key members of the snottite communities, accompanied in some cases by smaller numbers of archaea related to Ferroplasma and other Thermoplasmata. Diversity estimates show that the Frasassi snottites are among the lowest-diversity natural microbial communities known, with one to six prokaryotic phylotypes observed depending on the sample. This study represents the first in-depth molecular survey of cave snottite microbial diversity and population structure, and contributes to understanding of rapid limestone dissolution and cave formation by microbially mediated sulfuric acid speleogenesis.     

Community Structure of Subsurface Biofilms in the Thermal Sulfidic Caves of Acquasanta Terme,Italy

We performed a microbial community analysis of biofilms inhabiting thermal (35 to 50°C) waters more than 60 m below the ground surface near Acquasanta Terme, Italy. The groundwater hosting the biofilms has 400 to 830 μM sulfide, <10 μM O₂, pH of 6.3 to 6.7, and specific conductivity of 8,500 to 10,500 μS/cm. Based on the results of 16S rRNA gene cloning and fluorescent in situ hybridization (FISH), the biofilms have low species richness, and lithoautotrophic (or possibly mixotrophic) Gamma- and Epsilonproteobacteria are the principle biofilm architects. Deltaproteobacteria sequences retrieved from the biofilms have <90% 16S rRNA similarity to their closest relatives in public databases and may represent novel sulfate-reducing bacteria. The Acquasanta biofilms share few species in common with Frasassi cave biofilms (13°C, 80 km distant) but have a similar community structure, with representatives in the same major clades. The ecological success of Sulfurovumales-group Epsilonproteobacteria in the Acquasanta biofilms is consistent with previous observations of their dominance in sulfidic cave waters with turbulent water flow and high dissolved sulfide/oxygen ratios.Despite rapid progress in the past decade, the deep subsurface remains one of the least explored microbial habitats on earth. Recent studies illustrate the presence of significant spatial heterogeneity (13, 53) and the strong influence of mineralogy and fluid flow on subsurface microbial biodiversity (9, 16, 31, 61). Data obtained by drilling are complemented by an increasing number of studies that exploit subsurface passages navigable by humans (17). These subsurface passages include caves (14, 46) and mines (22, 32, 47, 52, 57). Approximately 10% of known caves (49) and perhaps more (33) are formed where reduced, sulfidic groundwaters interact with oxidized water descending from surface environments. Limestone dissolution in these groundwater mixing zones results in deep caves that receive few organic inputs from the surface. Due to the presence of both sulfide and oxidants where the groundwaters mix, lithoautotrophic microorganisms thrive and supply the primary productivity for food chains that may include invertebrate and vertebrate animals (11, 23). Interest in these isolated terrestrial chemosynthetic microbial communities is fueled by their potential as model systems for microbial biogeography and as analogs for oxygen-poor, sulfur-rich environments prevalent early in Earth history or on other planets.The sulfidic caves studied by microbiologists to date include Lower Kane Cave in Wyoming (15), Cueva de Villa Luz in Mexico (5, 23), Movile Cave in Romania (7, 28), Parker Cave in Kentucky (4), and the Frasassi caves in Italy (38, 39). The average water temperatures of previously studied sulfidic caves range from 12 to 28°C. In contrast, the groundwater in the Grotta Nuova di Rio Garrafo and associated caves near Acquasanta Terme (Italy) reaches temperatures up to 50°C (M. Mainiero, unpublished results). The Acquasanta caves host conspicuous microbial biofilms that have not been previously investigated, presenting an opportunity to compare subsurface environments with similar energy resources but large differences in temperature.A reconnaissance of the Acquasanta caves (Fig. ​(Fig.1)1) showed that they contain biofilm types also reported in other sulfidic caves, including viscous snottites on walls above the water table (see Fig. S1a, b, and e in the supplemental material), reddish clay-rich deposits (“ragu”) similar to those on walls in Cueva de Villa Luz (see Fig. S1e in the supplemental material), and white biofilms covering sediments below the water table (see Fig. S1d and f in the supplemental material). As in Cueva de Villa Luz, subaerial surfaces in areas with high sulfur gas concentrations are covered with elemental sulfur deposits (see Fig. S1c in the supplemental material). Here, we describe the diversity and community structure of biofilms in the thermal Acquasanta groundwater. In addition, we investigate whether the Acquasanta stream biofilms have a structure consistent with a simple ecological niche model developed for sulfur-oxidizing clades inhabiting nonthermal sulfidic caves (37). The niche model considers aqueous sulfide and oxygen concentrations and hydrodynamic shear and suggests that in turbulently flowing (high shear) waters with high sulfide/oxygen ratios, Epsilonproteobacteria should outcompete filamentous Gammaproteobacteria, such as Beggiatoa and Thiothrix. We find that Acquasanta cave stream biofilms share very few phylotypes in common with other sulfidic caves studied to date and are dominated by Epsilonproteobacteria as predicted by the niche model.Open in a separate windowFIG. 1.(A) Plan view map of the Rio Garrafo caves. (Modified from reference 21 with kind permission from Springer Science+Business Media.) Contour lines indicate surface elevation in meters. (B) Cross section of Grotta Nuova del Rio Garrafo. (Modified from reference 18 with permission of the National Speleological Society.) Black circles indicate sampling locations for biofilms depicted in Fig. S1 in the supplemental material. Arrows indicate flow directions for water (solid) and gas (dashed). Samples analyzed in the current study were collected exclusively at site AS1.     

Unsuspected diversity of <Emphasis Type="Italic">Niphargus</Emphasis> amphipods in the chemoautotrophic cave ecosystem of Frasassi,central Italy

Background  

The sulfide-rich Frasassi caves in central Italy contain a rare example of a freshwater ecosystem supported entirely by chemoautotrophy. Niphargus ictus, the sole amphipod species previously reported from this locality, was recently shown to host the first known case of a freshwater chemoautotrophic symbiosis. Since the habitat of N. ictus is highly fragmented and is comprised of streams and lakes with various sulfide concentrations, we conducted a detailed study to examine the potential genetic diversity of this species within Frasassi.     

Mineral Precipitation by Epilithic Biofilms in the Speed River, Ontario, Canada

Epilithic microbial communities, ubiquitously found in biofilms on submerged granite, limestone, and sandstone, as well as on the concrete support pillars of bridges, were examined in the Speed River, Ontario, Canada. Transmission electron microscopy showed that attached bacteria (on all substrata) were highly mineralized, ranging from Fe-rich capsular material to fine-grained (<1 μm) authigenic (primary) mineral precipitates. The authigenic grains exhibited a wide range of morphologies, from amorphous gel-like phases to crystalline structures. Energy-dispersive X-ray spectroscopy indicated that the most abundant mineral associated with epilithic bacteria was a complex (Fe, Al) silicate of variable composition. The gel-like phases were similar in composition to a chamositic clay, whereas the crystalline structures were more siliceous and had compositions between those of glauconite and kaolinite. The consistent formation of (Fe, Al) silicates by all bacterial populations, regardless of substratum lithology, implies that biomineralization was a surface process associated with the anionic nature of the cell wall. The adsorption of dissolved constituents from the aqueous environment contributed significantly to the mineral formation process. In this regard, it appears that epilithic microbial biofilms dominate the reactivity of the rock-water interface and may determine the type of minerals formed, which will ultimately become part of the riverbed sediment. Because rivers typically contain high concentrations of dissolved iron, silicon, and aluminum, these findings provide a unique insight into biogeochemical activities that are potentially widespread in natural waters.     

Acidophilic granular cells in the epidermis of the brown trout,Salmo trutta L.

Summary Acidophilic cells occur in the epidermis of several species of salmonid fish, although their abundance fluctuates considerably between individuals within the same population and at different times during the life cycle. The histology, histochemistry and ultrastructure of an acidophilic, granular celltype in the epidermis of the brown trout, Salmo trutta L., is described. At the light microscope level this cell type is easily distinguished from the large, mucus-secreting, epidermal goblet cells by its acidophilic, proteinaceous secretion. At the ultrastructural level this secretion consists of membrane-bound granules formed by the very active Golgi region. It is argued that the acidophilic, granular cell is not a transformed blood cell but constitutes a normal epidermal component of the brown trout. Possible roles of this cell in the function(s) of the epidermis are discussed.     

Influence of sulfur-oxidizing bacteria on the budget of sulfate in Yugama crater lake, Kusatsu-Shirane volcano, Japan

Sulfur-oxidizing bacteria, Thiobacillus thiooxidans, werefound in a highly acidic (pH = 11.5) crater lake, Yugama,seasonally flowing streams and soil in the catchment area of thecrater. Thiobacillus ferrooxidans was also found in some ofthe streams but not in the lake itself. The lake water containsaqueous carbon dioxide, hydrogen sulfide, polythionates andelemental sulfur in suspension which are the substrates for thegrowth of the sulfur-oxidizing bacteria as no organic compoundsexcept for the microorganisms themselves were detected. Thebacteria isolated from the Yugama water preferred polythionatesin the following order: S₄O₆ ²> S₅O₆ ²> S₆O₆ ²On the other hand,H₂S was more rapidly consumed by the bacteria thanpolythionates which were followed by elemental sulfur. In thecase of test-tube incubation, the optimum pH of the solution forgrowth of the bacteria was between 1.0 and 1.5, and forcultivation in growth medium plates between 2.5 and 3.5. Thebacteria hardly proliferated at pH 0.5 or below. In accordancewith these characteristics of the bacteria, numbers of thebacteria in the surface Yugama crater lake water were at minimum(< a few cells/mL) in February and at maximum (10⁶ cell/mL)in August. The bacterial activity changed in accordance with thesurface lake water temperature, but not necessarily with thevariations in H₂S and polythionates concentrations of the lakewater. Based on the variation in sulfur isotope ratios of sulfateand experimentally determined rate of oxidation of H₂S in thelake water, the sulfate production rate by the bacteria in thecatchment area and the lake were estimated to 9.5 and 8.4g/m²/day, respectively, during the period from 1988 to 1990when the volcanic activity at Yugama was at minimum. Also stream,hydrothermal, H₂S-oxidated SO² ₄-inputs and outputs byseepage and precipitation have been calculated as 4.1, 32, 0.56,36, and 1.2 ton/day, respectively.     

Design,synthesis and biological evaluation of novel hamamelitannin analogues as potentiators for vancomycin in the treatment of biofilm related Staphylococcus aureus infections

Staphylococcus aureus is a frequent cause of biofilm-related infections. Bacterial cells within a biofilm are protected from attack by the immune system and conventional antibiotics often fail to penetrate the biofilm matrix. The discovery of hamamelitannin as a potentiator for antibiotics, recently led to the design of a more drug-like lead. In the present study, we want to gain further insight into the structure–activity relationship (S.A.R.) of the 5-position of the molecule, by preparing a library of 21 hamamelitannin analogues.     

Occurrence,structure and function of intracellular polyglucose in the obligate chemolithotroph Thiobacillus neapolitanus

Nitrogen-limited cells of the obligate chemolithotroph Thiobacillus neapolitanus formed an intracellular polymer during growth in the chemostat. This polymer was isolated and characterized as a branched polyglucose composed of units joined by -14 and -16 linkages. Polyglucose in T. neapolitanus can be considered a storage compound since formation of this compound took place during excess of energy and CO₂ whilst shortage of CO₂ resulted in rapid breakdown of polyglucose. Moreover the breakdown of polyglucose generated metabolically useful energy as could be demonstrated by polyglucose-dependent protein synthesis. Possession of polyglucose did not influence the viability of T. neapolitanus during prolonged periods of energy starvation. Activities of key enzymes of the oxidative pentose phosphate cycle, glucose-6-phosphatedehydrogenase and 6-phospho-gluconate-dehydrogenase, were demonstrated in cell free extracts of T. neapolitanus and appeared to increase 5- and 3-fold, respectively, during growth on NO ₃ ^- instead of NH ₄ ⁺ as a nitrogen source.     

Sacciform cells in the epidermis of the brown trout,Salmo trutta,and the Arctic char,Salvetinus alpinus

Summary Sacciform cells containing an acidophilic, proteinaceous secretion, were identified in the epidermis of the brown trout and Arctic char. This cell type increased in number during the chronic stages of infestation by the ectoparasitic flagellate, Ichthyobodo sp., in immature brown trout, and decreased during sexual maturation in male brown trout and char. It is suggested that the salmonid sacciform cell produces a secretion which protects the fish against infestation or damage by skin parasites.     

Temporal changes in phytoplankton structure and composition at the Turkwel Gorge Reservoir,Kenya

Temporal changes in phytoplankton chlorophyll a, composition, diversity, biomass (density and fresh weight) and primary production were investigated at the Turkwel Gorge Reservoir (Kenya) over a two year period (1994 and 1995). The phytoplankton properties investigated revealed a seasonal pattern that was very distinct in 1994 and muted in 1995. The wet season was characterized by higher levels of chlorophyll a, biomass and primary production and a lower diversity. A prominent seasonality in 1994 was found to be the result of a higher river inflow volume as compared to 1995. Chlorophyll a changes showed some positive correlation to changes in total nitrogen and total phosphorus. Diversity changes were inversely correlated to changes in total counts (R = −0.84 and −0.96 for 1994 and 1995 respectively). Individual species density changes varied from a distinct seasonal pattern to a nearly uniform density. While the diatom Achnanthes dominated the wet season in 1994, coccoid blue green algae were dominant during most of 1995. Throughout the study period, most biomass was due to the diatoms but with a lower percentage of total biomass in 1995 (40%) as compared to 1994 (88%). The wet season biomass in each year was dominated by the diatoms. Dominance of the intervening period changed irregularly between diatoms, dinoflagellates, green algae and blue green algae. The range of variation in chlorophyll a, total biomass and primary production were; 4.9 to 36.8 μg l^-1, 440.14 to 11172.70 mg m^-3 and 1.85 to 9.67 g O₂ m^-2 d^-1 in 1994 and 4.9 to 11.5 μg l^-1, 486.46 to 1351.39 mg l^-1 and 3.08 to 5.41 g O₂ m^-2 d^-1 in 1995 in the same order. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biofilms as food for decapods (Atyidae,Palaemonidae) in the River Murray,South Australia

Stable water levels and turbidity associated with flow regulation in the River Murray have promoted the growth of filamentous green algae and Cyanobacteria in biofilms on submerged wood. We investigated the assimilation of biofilm algae by two dominant consumers, the decapod crustaceans Macrobrachium australiense (Palaemonidae) and Paratya australiensis (Atyidae), in two river reaches differing in the extent of floodplain development, hence wetland connectivity. Filamentous Cyanobacteria, a major part of the biofilms assimilated in combination with other foods, were up to 83% of the algal component of the gut content volume of P. australiensis and 44% that of M. australiense. Cyanobacteria have not previously been reported as a major source of nutrition for adult decapods. There was little difference between the stable isotopic signatures (¹³C/¹²C, ¹⁵N/¹⁴N) of the two decapod species, or between decapods in the two reaches. Coarse and fine particulate organic matter from the gorge had similar isotopic signatures to those from upstream and so were likely derived from macrophyte detritus rather than local willows. Red gum leaves and wood were too depleted in both ¹³C and ¹⁵N to register in the diets of either decapod in gorge or floodplain reaches. The most likely food sources for the decapods are littoral plants in the gorge reach and fine particulate organic matter material processed upstream. This is consistent with current hypotheses of organic matter flux in large river systems.     

Examining time trends in the Oldowan technology at Beds I and II,Olduvai Gorge

The lithic analysis of the Bed I and II assemblages from Olduvai Gorge reveals both static and dynamic time trends in early hominids' technology from 1.8 to 1.2 m.y.a. The Bed I Oldowan (1.87-1.75 m.y.a.) is characterized by the least effort strategy in terms of raw material exploitation and tool production. The inclusion of new raw material, chert, for toolmaking in the following Developed Oldowan A (DOA, 1.65-1.53 m.y.a.) facilitated more distinctive and variable flaking strategies depending on the kind of raw materials. The unique characters of DOA are explainable by this raw material factor, rather than technological development of hominids. The disappearance of chert in the subsequent Developed Oldowan B and Acheulian (1.53-1.2 m.y.a.) necessitated a shift in tool production strategy more similar to that of Bed I Oldowan than DOA. However, the evidence suggests that Bed II hominids might have been more skillful toolmakers, intensive tool-users, and engaged in more active transport of stone tools than the Bed I predecessors. Koobi Fora hominids maintained a more static tool-using behavior than their Olduvai counterparts due mainly to a stable supply of raw materials. They differed from Olduvai hominids in terms of less battering of cores, consistent transport behavior, and few productions of side-struck flakes, indicating a regional variation of toolmaking and using practice. However, they shared with Olduvai hominids a temporal trend toward the production of larger flakes from larger cores after 1.6 m.y.a. Increased intake of animal resources and the expansion of ranging area of Homo ergaster would have led to the development of technological organization. Technological changes in the Oldowan industry are attested at Olduvai Gorge, Koobi Fora, and Sterkfontein, suggesting that it was a pan-African synchronous phenomenon, beginning at 1.5 m.y.a.     

Changes in experimental gillnet catches from the Kafue Floodplain, Zambia, since construction of the Kafue Gorge Dam

The Kafue Floodplain fishery produces an annual fish yield of about 5000 tonnes. The 1971 closure of the Kafue Gorge Dam downstream from the floodplain modified flooding patterns. Pre-impoundment studies predicted this modification would increase fish stocks. Experimental gillnets set in 1975 and 1976 caught 18 species on a regular basis, nine of which were abundant enough for comparison with catches in similar gillnet samples taken prior to completion of the Kafue Gorge Dam. Of these nine species, experimental gillnet catches of three predator species were smaller in 1975–76 than in 1969–70. Catches of only one species increased significantly. Whether this change is due to the modified flooding pattern is unclear; unusually dry flood conditions in 1973 and increased fishing effort may have contributed.     

河北省邢台西部山区种子植物科的区系分析

邢台西部山区位于河北太行山脉的南段,区内植物种类丰富,共有野生种子植物93科,393属,823种,其中含23种以上的优势科有12科,反映区系地方特征的表征科有10科,区域单种科、单属科共37科,植物生活型以草本为主;本区系具明显的温带性质,同时,与热带又有一定的历史渊源;区系成分复杂,与其它地区植物区系联系广泛,具有过渡性,在起源上又具有古老性。研究结果表明,本区系是河北省植物区系的重要组成部分,在太行山区具有较强的代表性。     

Methylamine,an inhibitor of ammonium oxidation and chemoautotrophic growth in the marine nitrifying bacteriumNitrosococcus oceanus

Methylamine (CH₃NH ₃ ⁺ ) appeared to utilize the same transport mechanism as ammonium (NH ₄ ⁺ ) to enter cells ofNitrosococcus oceanus. Methylamine uptake did not show clear evidence of saturable kinetics and was not fully saturated at 20 mM. Assimilated CH₃NH ₃ ⁺ was not incorporated into macromolecular constituents, but inhibited rates of nitrification, chemoautotrophic CO₂ fixation and growth. The degree of inhibition was dependent on the relative concentrations of NH ₄ ⁺ and CH₃NH ₃ ⁺ . Rates of CO₂ fixation and growth were inhibited four times more than the rate of nitrification.