Microbial biosignatures in ancient deep-sea hydrothermal sulfides

Deep-sea hydrothermal systems provide ideal conditions for prebiotic reactions and ancient metabolic pathways and, therefore, might have played a pivotal role in the emergence of life. To understand this role better, it is paramount to examine fundamental interactions between hydrothermal processes, non-living matter, and microbial life in deep time. However, the distribution and diversity of microbial communities in ancient deep-sea hydrothermal systems are still poorly constrained, so evolutionary, and ecological relationships remain unclear. One important reason is an insufficient understanding of the formation of diagnostic microbial biosignatures in such settings and their preservation through geological time. This contribution centers around microbial biosignatures in Precambrian deep-sea hydrothermal sulfide deposits. Intending to provide a valuable resource for scientists from across the natural sciences whose research is concerned with the origins of life, we first introduce different types of biosignatures that can be preserved over geological timescales (rock fabrics and textures, microfossils, mineral precipitates, carbonaceous matter, trace metal, and isotope geochemical signatures). We then review selected reports of biosignatures from Precambrian deep-sea hydrothermal sulfide deposits and discuss their geobiological significance. Our survey highlights that Precambrian hydrothermal sulfide deposits potentially encode valuable information on environmental conditions, the presence and nature of microbial life, and the complex interactions between fluids, micro-organisms, and minerals. It further emphasizes that the geobiological interpretation of these records is challenging and requires the concerted application of analytical and experimental methods from various fields, including geology, mineralogy, geochemistry, and microbiology. Well-orchestrated multidisciplinary studies allow us to understand the formation and preservation of microbial biosignatures in deep-sea hydrothermal sulfide systems and thus help unravel the fundamental geobiology of ancient settings. This, in turn, is critical for reconstructing life's emergence and early evolution on Earth and the search for life elsewhere in the universe.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Antigen-specific and nonspecific mediators of T-cell/B-cell cooperation. VI. Distinct patterns of cross-reactivity by two human gamma-globulin-primed helper T-cell subpopulations.

We have previously characterized the activities, in vitro, of two different helper T-cell subpopulations, primed with human γ-globulin (HGG). One T-cell subpopulation helps the response of B cells to determinants (e.g., haptens) bound to the same antigen to which the T cells are primed (specific help); the other helper T-cell subpopulation responds to the same priming antigen by secreting a nonspecific molecule which helps B-cell responses to erythrocyte antigens co-cultured with the priming antigen (nonspecific help). These subpopulations also differ in their frequency and dose response to antigen, both in vivo and in vitro. They are similarly susceptible to the induction of unresponsiveness to HGG. In order to determine whether these T-cell subpopulations share or differ in their ranges of antigen recognition, we have compared the reaction of these two HGG-primed helper T-cell subpopulations to a number of γ-globulins (γG's) from other species. Plaque-forming cells generated in response to HGG shared little or no cross-reactivity with any of the heterologous (γG's) tested. In contrast, HGG-primed nonspecific helper T cells responded with significant cross-reactivity when challenged in vitro with dog γG, but HGG-primed specific helper T cells did not respond with any such cross-reactivity. No other heterologous γG tested stimulated any significant cross-reactivity from either HGG-primed T-cell subpopulation. Thus, these two T-cell subpopulations differ in their antigenic recognition. Possible explanations of these data include: (i) a difference in receptor specificity; (ii) a difference in the receptor affinity; (iii) a difference in Ia determinants of the two subpopulations.     

Evidence for the conformation about the C(5′)-O(5′) bond of AMP complexed to AMP kinase: Substrate properties of a vinyl phosphonate analog of AMP

A vinyl phosphonate analog of adenosine 5′-phosphate (AMP) was synthesized in which the CH₂OP system of AMP is replaced by CHCHP. The V_max values of this analog relative to AMP were 0.7% with rabbit muscle AMP aminohydrolase, 13.4% with rabbit muscle AMP kinase, and 6.6% with pig muscle AMP kinase. The vinyl analog of ADP produced by the kinases was a substrate of rabbit muscle pyruvate kinase. These results, together with substrate specificity properties at the AMP sites of the enzymes indicate that the C(4′)-C(5′)-O(5′)-P system of AMP is of trans character during conversion of AMP to ADP by pig or rabbit AMP kinase.     

Identification of a naturally occurring methyl-ester of phosphate, methyl-phosphorylcholine (methyl-2-(N,N,N trimethylamino) ethyl phosphate), in the eggs of the sea urchin S. purpuratus

A novel metabolite of choline, phosphorylcholine methyl ester, has been identified in the eggs of S. purpuratus wherein it is present at approximately 1 mM concentration. To the best of our knowledge, this is the first instance of a phosphoryl-methyl-ester to be observed in nature. The compound appears to be species specific, since it has not been observed in other species such as L. pictus and P. depressus. In S. purpuratus its distribution is confined to the ovary, eggs and embryos, and is absent from young animals following metamorphosis.     

Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 microM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 +/- 0.003 nmol min(-1) mg of protein(-1) only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.     

A fast and robust quantitative time-lapse assay for cell migration

We describe a simple and widely applicable method to measure cell migration in time-lapse sequences of fluorescently labeled cells in culture. Briefly, binarized cell images obtained after thresholding were cumulatively projected, and the covered areas were measured. This procedure determines the time course of the track area successively covered by the cell population. Under conditions where cell growth is negligible, a robust index of cell motility is derived from normalized plots for the displacement of cells over time. We applied this method to quantitatively examine the migration of B35 neuroblastoma cells transiently expressing GFP and to C6 glioma cells after staining with Hoechst 33258. This sensitive assay detected the influence of agents which inhibit actin polymerization (cytochalasin B) or interfere with the maintenance of cell polarity (methyl-beta-cyclodextrin) on cell migration. Thus, this assay is a versatile tool to measure quickly the migration of different cell types using different labeling strategies.