Environmental niches and metabolic diversity in Neoarchean lakes.

The diversification of macro-organisms over the last 500 million years often coincided with the development of new environmental niches. Microbial diversification over the last 4 billion years likely followed similar patterns. However, linkages between environmental settings and microbial ecology have so far not been described from the ancient rock record. In this study, we investigated carbon, nitrogen, and molybdenum isotopes, and iron speciation in five non-marine stratigraphic units of the Neoarchean Fortescue Group, Western Australia, that are similar in age (2.78-2.72 Ga) but differ in their hydro-geologic setting. Our data suggest that the felsic-dominated and hydrologically open lakes of the Bellary and Hardey formations were probably dominated by methanogenesis (δ13 Corg  = -38.7 ± 4.2‰) and biologic N2 fixation (δ15 Nbulk  =-0.6 ± 1.0‰), whereas the Mt. Roe, Tumbiana and Kylena Formations, with more mafic siliciclastic sediments, preserve evidence of methanotrophy (δ13 Corg as low as -57.4‰, δ13 Ccarb as low as -9.2‰) and NH3 loss under alkaline conditions. Evidence of oxygenic photosynthesis is recorded only in the closed evaporitic Tumbiana lakes marked by abundant stromatolites, limited evidence of Fe and S cycling, fractionated Mo isotopes (δ98/95 Mo = +0.4 ± 0.4‰), and the widest range in δ13 Corg (-57‰ to -15‰), suggesting oxidative processes and multiple carbon fixation pathways. Methanotrophy in the three mafic settings was probably coupled to a combination of oxidants, including O2 and SO42- . Overall, our results may indicate that early microbial evolution on the Precambrian Earth was in part influenced by geological parameters. We speculate that expanding habitats, such as those linked to continental growth, may have been an important factor in the evolution of life.

Linkages between mineralogy, fluid chemistry, and microbial communities within hydrothermal chimneys from the Endeavour Segment, Juan de Fuca Ridge.

Rock and fluid samples were collected from three hydrothermal chimneys at the Endeavour Segment, Juan de Fuca Ridge to evaluate linkages among mineralogy, fluid chemistry, and microbial community composition within the chimneys. Mössbauer, midinfrared thermal emission, and visible-near infrared spectroscopies were utilized for the first time to characterize vent mineralogy, in addition to thin-section petrography, X-ray diffraction, and elemental analyses. A 282°C venting chimney from the Bastille edifice was composed primarily of sulfide minerals such as chalcopyrite, marcasite, and sphalerite. In contrast, samples from a 300°C venting chimney from the Dante edifice and a 321°C venting chimney from the Hot Harold edifice contained a high abundance of the sulfate mineral anhydrite. Geochemical modeling of mixed vent fluids suggested the oxic-anoxic transition zone was above 100°C at all three vents, and that the thermodynamic energy available for autotrophic microbial redox reactions favored aerobic sulfide and methane oxidation. As predicted, microbes within the Dante and Hot Harold chimneys were most closely related to mesophilic and thermophilic aerobes of the Betaproteobacteria and Gammaproteobacteria and sulfide-oxidizing autotrophic Epsilonproteobacteria. However, most of the microbes within the Bastille chimney were most closely related to mesophilic and thermophilic anaerobes of the Deltaproteobacteria, especially sulfate reducers, and anaerobic hyperthermophilic archaea. The predominance of anaerobes in the Bastille chimney indicated that other environmental factors promote anoxic conditions. Possibilities include the maturity or fluid flow characteristics of the chimney, abiotic Fe2+ and S2- oxidation in the vent fluids, or O2 depletion by aerobic respiration on the chimney outer wall.

Did life originate from a global chemical reactor?

Many decades of experimental and theoretical research on the origin of life have yielded important discoveries regarding the chemical and physical conditions under which organic compounds can be synthesized and polymerized. However, such conditions often seem mutually exclusive, because they are rarely encountered in a single environmental setting. As such, no convincing models explain how living cells formed from abiotic constituents. Here, we propose a new approach that considers the origin of life within the global context of the Hadean Earth. We review previous ideas and synthesize them in four central hypotheses: (i) Multiple microenvironments contributed to the building blocks of life, and these niches were not necessarily inhabitable by the first organisms; (ii) Mineral catalysts were the backbone of prebiotic reaction networks that led to modern metabolism; (iii) Multiple local and global transport processes were essential for linking reactions occurring in separate locations; (iv) Global diversity and local selection of reactants and products provided mechanisms for the generation of most of the diverse building blocks necessary for life. We conclude that no single environmental setting can offer enough chemical and physical diversity for life to originate. Instead, any plausible model for the origin of life must acknowledge the geological complexity and diversity of the Hadean Earth. Future research may therefore benefit from identifying further linkages between organic precursors, minerals, and fluids in various environmental contexts.

Diversity among three novel groups of hyperthermophilic deep-sea Thermococcus species from three sites in the northeastern Pacific Ocean.

Eight new strains of deep-sea hyperthermophilic sulfur reducers were isolated from hydrothermal vent fields at 9 degrees 50'N East Pacific Rise (EPR) and at the Cleft and CoAxial segments along the Juan de Fuca Ridge (JdFR). 16S rRNA gene sequence analysis showed that each strain belongs to the genus Thermococcus. Restriction fragment length polymorphism patterns of the 16S/23S rRNA intergenic spacer region revealed that these isolates fell into three groups: those from the EPR, those from fluid and rock sources on the JdFR, and those isolated from Paralvinella spp. polychaete vent worms from the JdFR. The optimum-temperature specific growth rates and the temperature ranges for growth were significantly higher and broader for those strains isolated from worms relative to those isolated from low-temperature diffuse hydrothermal fluids. Furthermore, the worm-derived isolates generally produced a larger array of proteases and amylases based on zymogram analyses. The zymogram patterns also changed with growth temperature suggesting that these organisms alter their lytic protein suites in response to changes in temperature. This study suggests that there is significant phenotypic diversity in Thermococcus that is not apparent from their highly conserved 16S rRNA nucleotide sequences.

A novel microbial habitat in the mid-ocean ridge subseafloor.

The subseafloor at the mid-ocean ridge is predicted to be an excellent microbial habitat, because there is abundant space, fluid flow, and geochemical energy in the porous, hydrothermally influenced oceanic crust. These characteristics also make it a good analog for potential subsurface extraterrestrial habitats. Subseafloor environments created by the mixing of hot hydrothermal fluids and seawater are predicted to be particularly energy-rich, and hyperthermophilic microorganisms that broadly reflect such predictions are ejected from these systems in low-temperature ( approximately 15 degrees C), basalt-hosted diffuse effluents. Seven hyperthermophilic heterotrophs isolated from low-temperature diffuse fluids exiting the basaltic crust in and near two hydrothermal vent fields on the Endeavour Segment, Juan de Fuca Ridge, were compared phylogenetically and physiologically to six similarly enriched hyperthermophiles from samples associated with seafloor metal sulfide structures. The 13 organisms fell into four distinct groups: one group of two organisms corresponding to the genus Pyrococcus and three groups corresponding to the genus Thermococcus. Of these three groups, one was composed solely of sulfide-derived organisms, and the other two related groups were composed of subseafloor organisms. There was no evidence of restricted exchange of organisms between sulfide and subseafloor habitats, and therefore this phylogenetic distinction indicates a selective force operating between the two habitats. Hypotheses regarding the habitat differences were generated through comparison of the physiology of the two groups of hyperthermophiles; some potential differences between these habitats include fluid flow stability, metal ion concentrations, and sources of complex organic matter.

Phylogenetic analysis of particle-attached and free-living bacterial communities in the Columbia river, its estuary, and the adjacent coastal ocean.

The Columbia River estuary is a dynamic system in which estuarine turbidity maxima trap and extend the residence time of particles and particle-attached bacteria over those of the water and free-living bacteria. Particle-attached bacteria dominate bacterial activity in the estuary and are an important part of the estuarine food web. PCR-amplified 16S rRNA genes from particle-attached and free-living bacteria in the Columbia River, its estuary, and the adjacent coastal ocean were cloned, and 239 partial sequences were determined. A wide diversity was observed at the species level within at least six different bacterial phyla, including most subphyla of the class Proteobacteria. In the estuary, most particle-attached bacterial clones (75%) were related to members of the genus Cytophaga or of the alpha, gamma, or delta subclass of the class Proteobacteria. These same clones, however, were rare in or absent from either the particle-attached or the free-living bacterial communities of the river and the coastal ocean. In contrast, about half (48%) of the free-living estuarine bacterial clones were similar to clones from the river or the coastal ocean. These free-living bacteria were related to groups of cosmopolitan freshwater bacteria (beta-proteobacteria, gram-positive bacteria, and Verrucomicrobium spp.) and groups of marine organisms (gram-positive bacteria and alpha-proteobacteria [SAR11 and Rhodobacter spp.]). These results suggest that rapidly growing particle-attached bacteria develop into a uniquely adapted estuarine community and that free-living estuarine bacteria are similar to members of the river and the coastal ocean microbial communities. The high degree of diversity in the estuary is the result of the mixing of bacterial communities from the river, estuary, and coastal ocean.

The quantum event of oceanic crustal accretion: impacts of diking at mid-ocean ridges.

Seafloor diking-eruptive events represent the irreducible, quantum events of upper oceanic crustal accretion. They record events by which a large portion of the oceanic crust has formed through geological history. Since 1993, the U.S. Navy's real-time Sound Surveillance System has allowed location of ongoing acoustic signatures of dike emplacement and basalt eruptions at ridge crests in the northeast Pacific. These diking-eruptive events trigger a sequence of related, rapidly evolving physical, chemical, and biological processes. Magmatic volatiles released during these events may provide nutrients for communities of subsea-floor microorganisms, some of which thrive in high-temperature anaerobic environments. Many of the organisms identified from these systems are Archaea. If microorganisms can thrive in the water-saturated pores and cracks within deep, volcanically active portions of our planet, other hydrothermally active planets may harbor similar life forms.

Cadmium removal by a new strain of Pseudomonas aeruginosa in aerobic culture.

A fluorescent pseudomonad (strain CW-96-1) isolated from a deep-sea vent sample grew at 30 degrees C under aerobic conditions in an artificial seawater medium and tolerated cadmium concentrations up to 5 mM. After 140 h, strain CW-96-1 removed > 99% of the cadmium from solution. Energy dispersive microanalysis revealed that the cadmium was removed by precipitation on the cell wall; sulfide production was confirmed by growth on Kligler's agar. Based on 16S ribosomal DNA sequencing and fatty acid analysis, the microorganism is closely related to Pseudomonas aeruginosa.

Deep-sea smokers: windows to a subsurface biosphere?

Since the discovery of hyperthermophilic microbial activity in hydrothermal fluids recovered from "smoker" vents on the East Pacific Rise, the widely accepted upper temperature limit for life (based on pure culture data) has risen from below the boiling point of water at atmospheric pressure to approximately 115 degrees C. Many microbiologists seem willing to speculate that the maximum may be closer to 150 degrees C. We have postulated not only higher temperatures than these (under deep-sea hydrostatic pressures), but also the existence of a biosphere subsurface to accessible seafloor vents. New geochemical information from the Endeavour Segment of the Juan de Fuca Ridge indicative of subsurface organic material caused us to re-examine both the literature on hyperthermophilic microorganisms cultured from deep-sea smoker environments and recent results of microbial sampling efforts at actively discharging smokers on the Endeavour Segment. Here we offer the case for a subsurface biosphere based on an interdisciplinary view of microbial and geochemical analyses of Endeavour smoker fluids, a case in keeping with rapidly evolving geophysical understanding of organic stability under deep-sea hydrothermal conditions.

Enhanced thermotolerance and temperature-induced changes in protein composition in the hyperthermophilic archaeon ES4.

The hyperthermophilic archaeon ES4, a heterotrophic sulfur reducer isolated from a deep-sea hydrothermal vent, is capable of protecting itself from thermal stress at temperatures above its optimum for growth. The thermotolerance of ES4 was determined by exposing log-phase cells to various lethal high temperatures. When ES4 was shifted from 95 to 102 degrees C, it displayed recovery from an exponential rate of death, followed by transient thermotolerance. When ES4 was shifted directly from 95 to either 105 or 108 degrees C, only exponential death occurred. However, a shift from 95 to 105 degrees C with an intermediate incubation at 102 degrees C also gave ES4 transient thermotolerance to 105 degrees C. The protein composition of ES4 was examined at temperatures ranging from 75 to 102 degrees C by one-dimensional electrophoresis. Two proteins with molecular masses of approximately 90 and 150 kDa significantly decreased in abundance with increasing growth temperature, while a 98-kDa protein, present at very low levels at normal growth temperatures (76 to 99 degrees C), was more abundant at higher temperatures. The enhanced tolerance to hyperthermal conditions after a mild hyperthermal exposure and the increased abundance of the 98-kDa protein at above-optimal temperatures imply that ES4 is capable of a heat shock-like response previously unseen in hyperthermophilic archaea.

Particulate DNA in smoker fluids: evidence for existence of microbial populations in hot hydrothermal systems.

As part of an interdisciplinary study of hydrothermal vents on the Endeavour Segment of the Juan de Fuca Ridge, we used the submersible ALVIN to collect 57 fluid samples in titanium syringes and Go Flo Niskin bottles from 17 different hot vents (smokers and flanges) and their environs for the purpose of extracting particulate DNA. The relative purity of the vent fluids collected was determined by Mg content as an indicator of seawater entrainment. Particulate material concentrated from these samples was lysed enzymatically (enz) and by a combination of enzyme and French press treatment (fp). Concentrations of partially purified DNA recovered from these lysates were determined spectrofluorometrically by using the dye Hoechst 33258. Ambient seawater surrounding the vents was found to contain low DNA concentrations, 0.18 to 0.32 ng of DNA per ml (n = 4; mean(enz) = 0.23 +/- 0.05; mean(fp) = 0.26 +/- 0.05), while low-temperature vent samples yielded significantly higher concentrations of 0.37 to 2.12 ng of DNA per ml (n = 4; mean(enz) = 0.97 +/- 0.68; mean(fp) = 1.05 +/- 0.54). Although DNA recovery values from superheated (210 to 345 degrees C) flange samples (mean(enz) = 0.14 +/- 0.10; mean(fp) = 0.12 +/- 0.14) were not significantly different from ambient seawater values, most of the superheated (174 to 357 degrees C) smoker fluid samples contained particulate DNA in concentrations too high to be attributable to entrained seawater. Detailed sampling at one smoker site demonstrated not only the existence of significant levels of particulate DNA in the superheated smoker fluids but also the presence of an elevated microbial population in the buoyant plume 20 to 100 m above the smoker. These results underscore the heterogeneity of smoker environments within a given hydrothermal vent field and indicate that microorganisms exist in some superheated fluids.

Rapid bacterial growth in the hindgut of a marine deposit feeder.

Antibiotic-insensitive mutants of natural sedimentary bacteria from an intertidal site were selected on gradient plates. Two of these strains, anAeromonas sp. andVibrio alginolyticus, were mixed with natural sediments from the field and fed toAbarenicola vagabunda, an intertidal lugworm characteristic of sandy beaches in the Pacific Northwest. Digestive removal was apparent in the midgut, 97% efficiency being seen forAeromonas sp. Both strains showed rapid growth in the hindgut, increasing between 2 and 3 orders of magnitude in abundance between the midgut and rectum of the polychaete, corresponding with a doubling time of about 50 min for each strain. Direct epifluorescence counts of natural bacteria in guts of animals freshly collected from the field suggest a mean doubling time that is only slightly greater (66 min) for all ingested bacteria that survive midgut digestion. These bacterial growth rates exceed by orders of magnitude the greatest rates reported for ambient marine sediments and suggest that hindgut bacterial growth, though of little immediate importance in the energetics of the animals, may strongly influence both population dynamics of marine bacteria and diagenesis of sedimentary organic matter.

Solid Medium for Culturing Black Smoker Bacteria at Temperatures to 120 degrees C.

A solid, highly thermostable medium, based on the new gelling agent GELRITE, was devised to facilitate the culturing of extremely thermophilic microorganisms from submarine hydrothermal vents. The medium remained solid at temperatures to 120 degrees C at vapor pressures and hydrostatic pressures to 265 atm. It proved useful to its maximum tested limits in isolating colonies of black smoker bacteria from hydrothermal fluids recently collected at the Juan de Fuca Ridge in the Pacific Ocean.

Initial effects of the mount st. Helens eruption on nitrogen cycle and related chemical processes in ryan lake.

Ryan Lake, a 1.6-hectare basin lake near the periphery of the tree blowdown area in the blast zone 19 km north of Mount St. Helens, was studied from August to October 1980 to determine the microbial and chemical response of the lake to the eruption. Nutrient enrichment through the addition of fresh volcanic material and the organic debris from the surrounding conifer forest stimulated intense microbial activity. Concentrations of such nutrients as phosphorus, sulfur, manganese, iron, and dissolved organic carbon were markedly elevated. Nitrogen cycle activity was especially important to the lake ecosystem in regulating biogeochemical cycling owing to the limiting abundance of nitrogen compounds. Nitrogen fixation, both aerobic and anaerobic, was active from aerobic benthic and planktonic cyanobacteria with rates up to 210 nmol of N(2) cm h and 667 nmol of N(2) liter h, respectively, and from anaerobic bacteria with rates reaching 220 nmol of N(2) liter h. Nitrification was limited to the aerobic epilimnion and littoral zones where rates were 43 and 261 nmol of NO(2) liter day, respectively. Potential denitrification rates were as high as 30 mumol of N(2)O liter day in the anaerobic hypolimnion. Total bacterial numbers ranged from 1 x 10 to 3 x 10 ml with the number of viable sulfur-metal-oxidizing bacteria reaching 2 x 10 ml in the hypolimnion. A general scenario for the microbial cycling of nitrogen, carbon, sulfur, and metals is presented for volcanically impacted lakes. The important role of nitrogen as these lakes recover from the cataclysmic eruption and proceed back towards their prior status as oligotrophic alpine lakes is emphasized.

Partial purification and characterization of hemolysin from a psychrotrophic kanagawa-positive marine Vibrio.

Psychrotrophic Kanagawa-positive marine vibrios were isolated from soft-shelled clams (Mya arenaria) collected in Yaquina Bay, Oreg. The 235 vibrio isolates obtained were screened for Gram reaction and morphology, Kanagawa reaction on Wagastsuma agar, and response to selected biochemical tests. The vibrio selected for further study was grown in broth, and the hemolysin was precipitated from a cleared supernatant with solid ammonium sulfate. The hemolytic substance was partially purified by DEAE-cellulose and Sephadex G-100 column chromatography. The hemolysin contained protein essential for activity, was thermolabile, and was more active against rabbit erythrocytes at 37 degrees C than at lower temperatures. The molecular weight was estimated at 55,000 by using a Sephadex G-100 column. Hemolytic activity was partially inactivated by gangliosides and lowered against horse erythrocytes. The hemolysin did not react with antibody prepared against vibriolysin from Vibrio parahaemolyticus WP-1 by the Ouchterlony method. The hemolysin was high in aspartic and glutamic acids and low in arginine and histidine. Electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel gave three major bands. The hemolysin from a psychrotrophic vibrio and the hemolytic exotoxin of V. parahaemolyticus had some similar and dissimilar characteristics. The possibility that a Vibrio sp. other than V. parahaemolyticus might serve as the reservoir for the Kanagawa phenotype is discussed.

Incidence of Vibrio parahaemolyticus bacteriophages and other Vibrio bacteriophages in marine samples.

Vibrio bacteriophages were isolated by enrichment from 177 of 643 samples of marine molluscan shellfish, crustaceans, seawater, and sediments. The predominant bacteriophage types isolated were specific for some strains of Vibrio parahaemolyticus. A high frequency of phage isolations was also observed with strains of agar-digesting vibrios (21 of 56) and psychrophilic vibrios (14 of 72) that were originally isolated from non-shellfish growing areas. No bacteriophages were isolated against V. alginolyticus and only rarely for V. anguillarum even though these were the two most abundant species found in near-shore environments. No V. cholerae phages were isolated. It was also determined from quantitative studies on the Pacific oyster (Crassostrea gigas) obtained from two environments in Washington and Oregon that the titers of V. parahaemolyticus bacteriophages increased with increasing seasonal water temperatures and that this was proportional to the increase in numbers of mesophilic vibrios and not with the incidence of V. parahaemolyticus. Titers of V. parahaemolyticus bacteriophages occasionally exceeded 10(6) per g of oyster during the summer months. Specific V. parahaemolyticus bacteriophages were also isolated from market seafoods and other marine samples that originated in cold environments where no mesophilic vibrios are expected to be found. The possibility that V. parahaemolyticus bacteriophages originate from Vibrio spp. other than V. parahaemolyticus and the role of these bacteriophages in the ecology of marine vibrios are discussed.

Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by bacteriophage susceptibility patterns.

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the O and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter-and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agar-digesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.

Survival of human enteric and other sewage microorganisms under simulated deep-sea conditions.

The survival of pure cultures of Escherichia coli, Streptococcus faecalis, Clostridium perfringens, and Vibrio parahaemolyticus under simulated deep-sea conditions of low temperature (4 C), seawater, and hydrostatic pressures ranging from 1 to 1,000 atm was determined over a period exceeding 300 h. The viability of E. coli and total aerobic bacteria in seawater-diluted raw sewage subjected to these deep-sea conditions was also measured. There was a greater survival of both E. coli and S. faecalis at 250 and 500 atm than at 1 atm at 4 C. S. faecalis was quite insensitive to 1,000 atm, whereas with E. coli there was a 10-fold die-off per 50-h exposure to 1,000 atm. In contrast, V. parahaemolyticus and C. perfringens were quite sensitive to pressures exceeding 250 atm, and with both of these species there was a total loss of viability of approximately 10(8) cells per ml within 100 h at 1,000 arm and within 200 h at 500 atm. The viability of the naturally occurring fecal coliforms in sewage exposed to moderate pressures at 4 C was found to be similar to the survival patterns demonstrated with pure cultures of E. coli. The total numbers of aerobic bacteria in these sewage samples, however, stabilized at 500 and 1,000 atm after 100 h, and at 1 and 250 atm there was significant growth of sewage-associated bacteria, which apparently utilized the organic compounds in the seawater-diluted sewage samples. A preliminary classification of some of these bacteria indicated that approximately 90% (160 isolates) of the organisms that survived over a 400-h exposure to 500 and 1,000 atm were Arthrobacter/Corynebacterium species, and the representative organisms capable of growing at 1 and 250 atm in seawater at 4 C were gram-positive cellulose digesters and an unidentified gram-negative coccus. The significance of these results with respect to the contamination of the deep ocean with human pathogens and the possibility of sewage-associated microorganisms growing and competing with indigenous marine microbial flora in situ is discussed.