Life in hot springs and hydrothermal vents

Hot springs and hydrothermal systems occurring within volcanic areas are inhabited by hyperthermophilic microorganisms, some of which grow at temperatures up to 110 °C. Hyperthermophiles grow anaerobically or aerobically by diverse metabolic types. Within the high temperature ecocystems, primary production is independent from solar energy.Presented at the Session Water in the Solar System and Its Role in Exobiology during the 26th General Assembly of the European Geophysical Society, 22–26 april 1991 in Wiesbaden, Germany.     

Influence of electron donor/acceptor concentrations on hydrous ferric oxide (HFO) bioreduction

Dissimilatory metal-reducing bacteria (DMRB) facilitate the reduction of Feand Mn oxides in anoxic soils and sediments and play an important role inthe cycling of these metals and other elements such as carbon in aqueousenvironments. Previous studies investigating the reduction of Fe(III) oxidesby DMRB focused on reactions under constant initial electron donor (lactate)and electron acceptor (Fe oxide) concentrations. Because the concentrationsof these reactants can vary greatly in the environment and would be expectedto influence the rate and extent of oxide reduction, the influence of variableelectron acceptor and donor concentrations on hydrous ferric oxide (HFO)bioreduction was investigated. Batch experiments were conducted in pH 7 HCO₃– buffered media using Shewanella putrefaciens strain CN32. In general, the rate of Fe(III) reduction decreased with increasing HFO:lactateratios, resulting in a relatively greater proportion of crystalline Fe(III) oxidesof relatively low availability for DMRB. HFO was transformed to a variety ofcrystalline minerals including goethite, lepidocrocite, and siderite but was almostcompletely dissolved at high lactate to HFO ratios. These results indicate thatelectron donor and acceptor concentrations can greatly impact the bioreductionof HFO and the suite of Fe minerals formed as a result of reduction. The respirationdriven rate of Fe(II) formation from HFO is believed to be a primary factor governingthe array of ferrous and ferric iron phases formed during reduction.     

Biomineralization at hot springs and mineral springs, and their significance in relation to the Earth's history]

Recently, there is strong interest on microbe-mineral interactions. This is related also to recent expanded knowledges on extremely severe environments in which microbes live. Interaction between microbes and minerals contains biomineralization processes. Varieties of biomineralization products are found not only in various geologic materials and processes in the earth's history but also in present surface environments. Some hot springs represent such environments similar to those of unique and extremely severe environments for life. In this short review, the author briefly shows some examples of biomineralizations at some hot springs and mineral springs, Japan. In such environments, iron ore was formed and some varieties of growing stromatolites were found. The varieties of stromatolite are siliceous, calcic and manganese types. Cyanobacteria and the other bacteria are related to form the stromatolite structure. In the Gunma iron ore, sedimentary iron ores were mineralogically described in order to evaluate the role of microorganisms and plants in ore formation. The iron ore is composed of nanocrystalline goethite. Algal fossils are clearly preserved in some ores. Various products of biomineralization are found in the present pH 2-3, Fe2(+)- and SO4(2-)-rich streams. Bacterial precipitation had variations from amorphous Fe-P-(S) precipitates near the outlet of mineral spring, to Fe-P-S precipitates and to Fe-S-(P) precipitates. Mosses and green algae are also collecting Fe precipitates in and around the living and dead cells. The Gunma Iron Ore can be said as Biologically Induced Iron Ore. At Onikobe and Akakura hot springs, growing stromatolites of siliceous and calcareous types, were found, respectively. At Onikobe, The stromatolites grow especially near the geyser. Cyanobacterial filaments in stromatolite were well preserved in the siliceous and calcic stromatolites. The filaments oriented in two directions which form the layered structures were found. At Yunokoya hot spring, black and brittle stromatolitic structures which were composed of amorphous Mn minerals are growing. The form of these structures are hemispherical. Many bacteria that were coated with amorphous Mn minerals were found on these structures. Furthermore, Precambrian (Proterozoic : Wittenoom-Chichester region, western Australia) manganese stromatolite was briefly shown in comparison. The black stromatolite has been clarified to be composed of todorokite. Small spotty and donuts-like shaped todorokite aggregates which are very similar to biologically induced Mn-precipitates were found in massive dolomite layers.     

In search of the microbe/mineral interface: quantitative analysis of bacteria on metal surfaces using vertical scanning interferometry

To understand the development of biofilms on metal surfaces, analysis of initial bacterial attachment to surfaces is crucial. Here we present the results of a study, using Shewanella oneidensis MR-1 as a model organism, in which vertical scanning interferometry (VSI) was used to investigate the initial stages of cell attachment to glass, steel and aluminium surfaces. It was found that while VSI gave unambiguous results with opaque surfaces, when reflective surfaces were used, an artifact sometimes appeared, with the bacteria appearing as rod-shaped pits rather than as cells on the surface. When the bacteria were altered to increase opacity, this artifact disappeared, and upon further investigation, it was found that the observational artifact was the result of a conflict between light reflected from the bacteria and the light reflected from the bacteria–metal interface. These results suggest that not only can bacteria be measured on surfaces using VSI, but with some modifications to the analytical software, there may be a unique window for studying the bacterial/substrate interface that can be used for quantitative observations. Imaging and characterization of the bacteria–substrate interface in vivo (previously invisible) will provide new insights into the interactions that occur at this important juncture.     

Chemical composition of surface antigens in the pseudotubercular microbe]

Superficially-active complexes, having a toxic effect and promoting generalization of the infestation process in the animal organism have been isolated from the pseudotubercular microbe. The chemical composition of these biopolymers has been studied.     

Heterotrophic bacterial populations in the mineral waters of thermal springs in Spain

The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 10³ cfu ml^-1 and the number of sporulated bacteria less than 10² cfu ml^-1. No significant differences were foundin the counts obtained with Plate Count Agar (PCA) and PCA diluted 1 : 10 and incubated at 22°, 37° and 45°C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85·4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.     

Chemical reactivity and microbicidal action of bethoxazin

Bethoxazin is a new broad spectrum industrial microbicide with applications in material and coating preservation. However, little is known of its reactivity profile and mechanism of action. In this study, we examined the reactivity of bethoxazin toward biologically important nucleophilic groups using UV-vis spectroscopy and LC-MS/MS techniques and found the molecule to be highly electrophilic. Bethoxazin reacted with molecules containing free sulfhydryl groups such as GSH and human serum albumin to form covalent adducts that were detectable by MS, but did not react with amino, carboxylic, phenolic, amino oxo, alcoholic, and phosphate functional groups. Bethoxazin potently inhibited the catalytic activity of yeast DNA topoisomerase II and the growth of yeast BY4742 cells at low micromolar concentrations. However, the reduced form of bethoxazin and GSH-treated bethoxazin were both inactive in these assays. The experimentally determined relative reactivity of bethoxazin and its reduced form analog correlated with their biological activities as well as their quantum-mechanically calculated electrophilicity properties. Taken together, the results suggest that bethoxazin may exert its microbicidal action by reacting with sensitive endogenous sulfhydryl biomolecules of microbial cells. Consistent with this view, the inhibitory activity of bethoxazin on topoisomerase II may be due to its ability to react with critical free cysteine sulfhydryl groups on the enzyme. Our studies have provided for the first time a better understanding of the reactivity of bethoxazin, as well as some insights into the mechanism by which the compound exerts its microbicidal action.     

Chemical markers of prebiotic chemistry in hydrothermal systems.

The goal of this chapter is to suggest some organic compounds which may be indicative of prebiotic processes in hydrothermal systems or laboratory simulations of them. While the exact processes which led to the origins of life are not known, studies of life's origins of the past forty years have uncovered a plethora of potential precursor molecules. Some of these same molecules were probably present in hydrothermal systems if chemical processes there had a role in the origins of life. The types of molecules formed in primitive Earth simulation experiments and observed in the interstellar medium, on comets and meteorites will be reviewed in Section 2 of this chapter. Some reactions involving these molecules which may have been important in prebiotic syntheses will be outlined. Since near- to supercritical water is found in hydrothermal systems, its properties and aspects of organic chemistry in supercritical water at high temperature and pressure will be discussed in Section 3. Fischer-Tropsch type (FTT) reactions, which are a potential source of the building blocks of biological molecules in hydrothermal systems, are discussed in Section 4. In the concluding section, Section 5, the possible formation in hydrothermal systems of organic molecules that are believed to have been important for the origins of life is discussed.     

Chemical reactivity and antimicrobial activity of N-substituted maleimides

Several N-substituted maleimides containing substituents of varying bulkiness and polarity were synthesised and tested for antimicrobial and cytostatic activity. Neutral maleimides displayed relatively strong antifungal effect minimum inhibitory concentrations (MICs in the 0.5-4 μg ml(-1) range); their antibacterial activity was structure dependent and all were highly cytostatic, with IC(50) values below 0.1 μg ml(-1). Low antimicrobial but high cytostatic activity was noted for basic maleimides containing tertiary aminoalkyl substituents. Chemical reactivity and lipophilicity influenced antibacterial activity of neutral maleimides but had little if any effect on their antifungal and cytostatic action. N-substituted maleimides affected biosynthesis of chitin and β(1,3)glucan, components of the fungal cell wall. The membrane enzyme, β(1,3)glucan synthase has been proposed as a putative primary target of N-ethylmaleimide and some of its analogues in Candida albicans cells.     

A new technique of depositing phospholipid bilayers on quartz surfaces: its use in membrane spin-label studies

We have developed a new improved technique termed the parallel-beam spattering (PBS) method for depositing phospholipid bilayers on quartz surfaces. This technique involves atomizing the phospholipid mixture with a stream of nitrogen gas and passing this atomized mixture through two orifices separated by a distance to achieve a parallel beam of atomized particles before deposition on the quartz plate. A static electric field can easily be applied to the quartz surface. Also a goniometer of new design has been constructed to allow precise positioning of the deposited phospholipid bilayers with reference to the magnetic field. We have utilized the PBS method to deposit phosphatidylcholine/nitroxyl labeled cholestane mixtures on quartz plates and have found that hydrated bilayers of these mixtures yield ESR spectra with essentially the same characteristics as those obtained using more conventional techniques. The distinct advantage of the new technique for depositing bilayers is that there is no spectral anomaly present which usually is present when the more conventional method of depositing bilayers is used. The spectral anomaly is apparently caused by a portion of the bilayers aligned in directions not directly parallel to the quartz surface. For precision work the spectral anomaly is unacceptable. It is not observed with the new PBS method which has yielded highly reproducible results.     

Correlation of the abundance of betaproteobacteria on mineral surfaces with mineral weathering in forest soils

Pyrosequencing-based analysis of 16S rRNA gene sequences revealed a significant correlation between apatite dissolution and the abundance of betaproteobacteria on apatite surfaces, suggesting a role for the bacteria belonging to this phylum in mineral weathering. Notably, the cultivation-dependent approach demonstrated that the most efficient mineral-weathering bacteria belonged to the betaproteobacterial genus Burhkolderia.     

The role of mineral surfaces in the origin of life

Adsorption of nucleoside phosphates on the surfaces of volcanic rocks has been studied. Differences in the adsorption of some nucleoside phosphates on the surface of basalt cinder have been found. Differences in the adsorption of similarmolecules on different mineral surfaces have also been shown. Different adsorptive capacities may have served as a mechanism for the selection of organic molecules during prebiotic evolution.     

A possible energetic role of mineral surfaces in chemical evolution

The postulated roles of clays and other minerals in chemical evolution and the origin of life are reconsidered in terms of the interaction of these minerals with penetrating sources of energy such as ionizing radiation and mechanical stress. This interaction, including such facets as excitation, degradation, storage, and transfer, is considered here with regard to its profound potential for altering the capabilities of minerals to serve both as substrates for prebiological chemistry and as inorganic prototypic life forms. The interaction of minerals and energy in relationship to surface chemistry is discussed in terms of the spectroscopic properties of minerals, the interaction of energy with condensed phases, some commonly accepted concepts of heterogeneous catalysis in the absence of electronic energy inputs, and some commonly accepted and novel means by which surface activity might be enhanced in the presence of energy inputs.An estimation is made of the potential contribution of two poorly characterized prebiotic energy sources, natural radioactive decay and triboelectric energy. These estimates place a conservative lower limit on their prebiotic abundance. Also some special properties of these energy sources, relative to solar energy, are pointed out which might give them particular suitability for driving reactions occurring under geological conditions.Skeletal support for this broadly defined framework of demonstrated and potential relationships between minerals, electronic excitation, and surface reactivity, as applied to chemical evolution, is provided from the results of our studies on 1/1 clays. We have discovered and partially characterized a number of novel luminescent properties of these clays, that indicate energy storage and transfer processes in clays. These luminescent properties are interpreted in relationship to the electron spin resonance phenomena, to provide a basis for estimating the potential significance of energy storage and transduction in monitoring or driving clay surface chemistry.Consideration of the electronic structure of abundant minerals in terms of band theory and localized defect centers provides a predictive theoretical framework from which to rationalize the capacity of these materials to store and transduce energy. The bulk crystal is seen as a collecting antenna for electronic energy, with the defect centers serving as storage sites.The clay properties produced by isomorphic substitution appear to be intimately associated with all of the life-mimetic chemical processes that have been attributed to clays. It appears sensible to postulate that the energetic properties of these substitutional defect centers may also be influential in these biomimetic processes: the promotion of surface reactions, storage of information, replication with transfer of information, and asymmetric separation of electrical charges, as well as their more recently hypothesized roles in energy storage and transduction. The identity of the sites implicated in all of the biomimetic functions of clays as well as their capacity for energy storage is seen to offer significant potential for coupling these functions to an environmental energy source. A yet more specific and experimentally testable hypothesis is offered for a new biomimetic process performed by clays. This hypothesis is that energy stored near isomorphically substituted sites provides the energetic basis for the coupled transport of electrical charge and/or electronic energy through the clay layer, which operates via environmental activation of electron/hole mobility. This is to say that mobility of charge/electronic excitation between defect centers serves as the basis for a primordial inorganic electron transport chain.     

Interactions between larvae of the hoverfly Eristalinus sepulchralis and microorganisms in the hydrothermal springs of the Uzon caldera, Kamchatka

The physicochemical and microbial characteristics of some medium-temperature hydrotherms of Kamchatka Peninsula (Uzon caldera), habitats of the hoverfly Eristalinus sepulchralis larvae, were studied. In these hydrothermal vents, the larvae were found to use various prokaryotic and eukaryotic microorganisms as a nutrient substrate. The rates of chemo- and photosynthetic activity of the suspended microbial communities inhabiting the hydrotherms and supporting the existence of larvae were measured. By light and electron microscopy, exo- and endosymbiotic prokaryotic microorganisms were revealed in the digestive and respiratory systems of larvae.     

The role of mineral surfaces in the origin of life.

Adsorption of nucleoside phosphates on the surfaces of volcanic rocks has been studied. Differences in the absorption of some nucleoside phosphates on the surface of basalt cinder have been found. Differences in the adsorption of similar molecules on different mineral surfaces have also been shown. Different adsorptive capacities may have served as a mechanism for the selection of organic molecules during prebiotic evolution.     

Nitric oxide and vascular reactivity in developing zebrafish, Danio rerio

We used a newly developed digital motion analysis video technique to study the effects of nitric oxide (NO) and epinephrine on the early larval arterial and venous vasculature of zebrafish. Application of the NO donor sodium nitroprusside resulted in a significant increase in both the venous and arterial vessel diameters, whereas N(G)-nitro-L-arginine methyl ester caused a significant decrease in the same diameters. Thus our results show that both the venous and arterial vasculature of the 5- and 6-day-old zebrafish larvae are influenced by endogenously produced NO. By use of immunohistochemistry, NO synthase immunoreactivity was demonstrated in endothelial cells of the dorsal vein. Local application of epinephrine onto the dorsal artery had no effect on vessel diameter. However, if the embryos were preincubated with N(omega)-nitro-L-arginine methyl ester, addition of epinephrine resulted in a significant reduction in both arterial and venous vessel diameters. Thus this study provides increasing evidence that before a functional autonomic innervation of the peripheral vascular system, vascular tone in larval tissue is regulated by a complex interaction of vasoactive substances that are produced locally by vascular endothelial cells.     

Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter

By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.