Perspective: Researching the transition from non-living to the first microorganisms: Methods and experiments are major challenges

Methods to research the origin of microbial life are limited. However, microorganisms were the first organisms on the Earth capable of cell growth and division, and interactions with their environment, other microbial cells, and eventually with diverse eukaryotic organisms. The origin of microbial life and the supporting scientific evidence are both an enigma and a scientific priority. Numerous hypotheses have been proposed, scenarios imagined, speculations presented in papers, insights shared, and assumptions made without supporting experimentation, which have led to limited progress in understanding the origin of microbial life. The use of the human imagination to envision the origin of life events, without supporting experimentation, observation and independently replicated experiments required for science, is a significant constraint. The challenge remains how to better understand the origin of microbial life using observations and experimental methods as opposed to speculation, assumptions, scenarios, envisioning events and un-testable hypotheses. This is not an easy challenge as experimental design and plausible hypothesis testing are difficult. Since past approaches have been inconclusive in providing evidence for the origin of microbial life mechanisms and the manner in which genetic instructions was encoded into DNA/RNA, it is reasonable and logical to propose that progress will be made when testable, plausible hypotheses and methods are used in the origin of microbial life research, and the experimental observations are, or are not reproduced in independent laboratories. These perspectives will be discussed in this article as well as the possibility that a pre-biotic film preceded a microbial biofilm as a possible micro-location for the origin of microbial cells capable of growth and division.     

Suitable microscopic entropy for the origin of microbial life: Microbiological methods are challenges

A hypothesis is proposed that the first living microbial cell(s) on Earth assembled about 3.6-4 billion years ago when an environmental microscopic entropy (balance between order and disorder; suitable amount of randomness) was within a range suitable for the origin of microbial cell(s) in a hydrogel environment. An earlier origin of microbial life was not possible as the elements, molecules and entropy conditions necessary for life were not available at the microscopic level. Methodology limitations to study postulated past origin of microbial life events and to mimic these events in the laboratory, are still obstacles to understanding the origin of life.     

Setting the stage: the history, chemistry, and geobiology behind RNA

No community-accepted scientific methods are available today to guide studies on what role RNA played in the origin and early evolution of life on Earth. Further, a definition-theory for life is needed to develop hypotheses relating to the "RNA First" model for the origin of life. Four approaches are currently at various stages of development of such a definition-theory to guide these studies. These are (a) paleogenetics, in which inferences about the structure of past life are drawn from the structure of present life; (b) prebiotic chemistry, in which hypotheses with experimental support are sought that get RNA from organic and inorganic species possibly present on early Earth; (c) exploration, hoping to encounter life independent of terran life, which might contain RNA; and (d) synthetic biology, in which laboratories attempt to reproduce biological behavior with unnatural chemical systems.     

Quantitative High-Resolution Imaging of Live Microbial Cells at High Hydrostatic Pressure

The majority of the Earth’s microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth’s crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth’s surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches.     

How life began

Study of the origin of life has become a legitimate scientific inquiry, with an international, multidisciplinary membership and a cogent body of data. Experiments involving plausible early Earth conditions and biogeochemical analyses of carbonaceous meteorites imply a variety of available starting molecules. Biogeological evidence indicates microbial beginnings about 3800 million years (3.8 aeons) ago. By then the known universe had been in existence for perhaps 15 aeons and galaxies abundant for ten. Conditions suitable for the origin of life may require a long prior cosmic evolution. The natural origin of life on the early Earth is now widely agreed upon but not the pathways. The beginnings of catalysis, replication and a functional cell remain moot. Much discussion has centered on the templating role that crystals such as clays and zeolites might have played in prebiotic evolution. Recent discovery of the catalytic and replicative functions of RNA recommend it as the key molecule in the transition from chemical to biological evolution.     

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.     

Metabolic reconstruction and modeling of nitrogen fixation in Rhizobium etli

Rhizobiaceas are bacteria that fix nitrogen during symbiosis with plants. This symbiotic relationship is crucial for the nitrogen cycle, and understanding symbiotic mechanisms is a scientific challenge with direct applications in agronomy and plant development. Rhizobium etli is a bacteria which provides legumes with ammonia (among other chemical compounds), thereby stimulating plant growth. A genome-scale approach, integrating the biochemical information available for R. etli, constitutes an important step toward understanding the symbiotic relationship and its possible improvement. In this work we present a genome-scale metabolic reconstruction (iOR363) for R. etli CFN42, which includes 387 metabolic and transport reactions across 26 metabolic pathways. This model was used to analyze the physiological capabilities of R. etli during stages of nitrogen fixation. To study the physiological capacities in silico, an objective function was formulated to simulate symbiotic nitrogen fixation. Flux balance analysis (FBA) was performed, and the predicted active metabolic pathways agreed qualitatively with experimental observations. In addition, predictions for the effects of gene deletions during nitrogen fixation in Rhizobia in silico also agreed with reported experimental data. Overall, we present some evidence supporting that FBA of the reconstructed metabolic network for R. etli provides results that are in agreement with physiological observations. Thus, as for other organisms, the reconstructed genome-scale metabolic network provides an important framework which allows us to compare model predictions with experimental measurements and eventually generate hypotheses on ways to improve nitrogen fixation.     

On the origin of microbial ORFans: quantifying the strength of the evidence for viral lateral transfer

Background:  

The origin of microbial ORFans, ORFs having no detectable homology to other ORFs in the databases, is one of the unexplained puzzles of the post-genomic era. Several hypothesis on the origin of ORFans have been suggested in the last few years, most of which based on selected, relatively small, subsets of ORFans. One of the hypotheses for the origin of ORFans is that they have been acquired thru lateral transfer from viruses. Here we carry out a comprehensive, genome-wide study on the origins of ORFans to quantify the strength of current evidence supporting this hypothesis.     

High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.     

Origin of life: hypothesized roles of high-energy electrical discharges, infrared radiation, thermosynthesis and pre-photosynthesis

The hypothesis is proposed that during the organization of pre-biotic bacterial cell(s), high-energy electrical discharges, infrared radiation (IR), thermosynthesis and possibly pre-photosynthesis were central to the origin of life. High-energy electrical discharges generated some simple organic molecules available for the origin of life. Infrared radiation, both incoming to the Earth and generated on the cooling Earth with day/night and warming/cooling cycles, was a component of heat engine thermosynthesis before enzymes and the genetic code were present. Eventually, a primitive forerunner of photosynthesis and the capability to capture visible light emerged. In addition, the dual particle-wave nature of light is discussed from the perspective that life requires light acting both as a wave and particle.     

Single-cell genomics reveals the lifestyle of Poribacteria,a candidate phylum symbiotically associated with marine sponges

In this study, we present a single-cell genomics approach for the functional characterization of the candidate phylum Poribacteria, members of which are nearly exclusively found in marine sponges. The microbial consortia of the Mediterranean sponge Aplysina aerophoba were singularized by fluorescence-activated cell sorting, and individual microbial cells were subjected to phi29 polymerase-mediated ‘whole-genome amplification''. Pyrosequencing of a single amplified genome (SAG) derived from a member of the Poribacteria resulted in nearly 1.6 Mb of genomic information distributed among 554 contigs analyzed in this study. Approximately two-third of the poribacterial genome was sequenced. Our findings shed light on the functional properties and lifestyle of a possibly ancient bacterial symbiont of marine sponges. The Poribacteria are mixotrophic bacteria with autotrophic CO₂-fixation capacities through the Wood–Ljungdahl pathway. The cell wall is of Gram-negative origin. The Poribacteria produce at least two polyketide synthases (PKSs), one of which is the sponge-specific Sup-type PKS. Several putative symbiosis factors such as adhesins (bacterial Ig-like domains, lamininin G domain proteins), adhesin-related proteins (ankyrin, fibronectin type III) and tetratrico peptide repeat domain-encoding proteins were identified, which might be involved in mediating sponge–microbe interactions. The discovery of genes coding for 24-isopropyl steroids implies that certain fossil biomarkers used to date the origins of metazoan life on earth may possibly be of poribacterial origin. Single-cell genomic approaches, such as those shown herein, contribute to a better understanding of beneficial microbial consortia, of which most members are, because of the lack of cultivation, inaccessible by conventional techniques.     

Geochemical and microbiological evidence for a hydrogen-based,hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) beneath an active deep-sea hydrothermal field

Subsurface microbial communities supported by geologically and abiologically derived hydrogen and carbon dioxide from the Earths interior are of great interest, not only with regard to the nature of primitive life on Earth, but as potential analogs for extraterrestrial life. Here, for the first time, we present geochemical and microbiological evidence pointing to the existence of hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) dominated by hyperthermophilic methanogens beneath an active deep-sea hydrothermal field in the Central Indian Ridge. Geochemical and isotopic analyses of gaseous components in the hydrothermal fluids revealed heterogeneity of both concentration and carbon isotopic compositions of methane between the main hydrothermal vent (0.08 mM and –13.8 PDB, respectively) and the adjacent divergent vent site (0.2 mM and –18.5 PDB, respectively), representing potential subsurface microbial methanogenesis, at least in the divergent vent emitting more ¹³C-depleted methane. Extremely high abundance of magmatic energy sources such as hydrogen (2.5 mM) in the fluids also encourages a hydrogen-based, lithoautotrophic microbial activity. Both cultivation and cultivation-independent molecular analyses suggested the predominance of Methanococcales members in the superheated hydrothermal emissions and chimney interiors along with the other major microbial components of Thermococcales members. These results imply that a HyperSLiME, consisting of methanogens and fermenters, occurs in this tectonically active subsurface zone, strongly supporting the existence of hydrogen-driven subsurface microbial communities.     

An Open Question on the Origin of Life: The First Forms of Metabolism

The general framework of the origin of life on Earth is outlined, emphasizing that the so‐called prebiotic ‘RNA world’ is as yet on shaky scientific ground, and that one should any way ask the question of the structure of the first protocellular compartments capable of the initial forms of metabolism. This question is the basis of the research project on the minimal cells, containing the minimal and sufficient complexity capable of leading to life. Such research is briefly summarized, highlighting experiments with liposome‐based semisynthetic cells which are capable of ribosomal protein synthesis with a very minimal number of enzymes. The most recent finding in this area of research is the unexpected observation that the formation and closure of liposomes in situ acts as an attractor for the solute molecules in solution, bringing about a very high local concentration in some of the liposomes. It is argued that this spontaneous overcrowding, which permits reactions which are not possible in the original dilute solution, might be the origin of cellular metabolism for the origin of life on Earth.     

First ecosystems on Earth

Theories attempting to explain the origin of life on Earth should be based on the assumption that habitability precedes habitation. The hypotheses of the first organism should be based on the evaluation of its possible life-supporting ecosystem. The ecosystem should necessarily include primary autotrophic producers, and hydrogenotrophy appears to be an adequate physiological type for primitive ecosystems. Consideration of life on Earth should differentiate between the origin of organisms in situ and the transportation of organisms from outside with cosmic bodies in the framework of life as a widespread phenomenon of the Universe. In the case of transport of life with cosmic bodies, there are no limitations on the transfer of a community rather than an individual cell. In the case of the transport of the community with a large piece of “dirty ice,” the problem lies in the correspondence between the community and its ecosystem on the parent body and the conditions on the primeval Earth rather than functional divergence from a primary ancestor. Subsequent events are within the framework of paleontologically observed evolution and can be described as biogeochemical succession without any additional speculations.     

Pluralism or unity in biology: could microbes hold the secret to life?

Pluralism is popular among philosophers of biology. This essay argues that negative judgments about universal biology, while understandable, are very premature. Familiar life on Earth represents a single example of life and, most importantly, there are empirical as well as theoretical reasons for suspecting that it may be unrepresentative. Scientifically compelling generalizations about the unity of life (or lack thereof) must await the discovery of forms of life descended from an alternative origin, the most promising candidate being the discovery of extraterrestrial life. Nonetheless, in the absence of additional examples of life, we are best off exploring the microbial world for promising explanatory concepts, principles, and mechanisms rather than prematurely giving up on universal biology. Unicellular microbes (especially prokaryotes) are by far the oldest, metabolically most diverse, and environmentally tolerant form of life on our planet. Yet somewhat ironically, much of our theorizing about life still implicitly privileges complex multicellular eukaryotes, which are now understood to be highly specialized, fragile latecomers to Earth. The problem with pursuing a pluralist approach to understanding life is that it is likely to blind us to the significance of just those entities and causal processes most likely to shed light on the underlying nature of life.     

Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial,Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities

Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community''s bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.     

Marine invertebrate immunodefense responses: Molecular and cellular approaches in tunicates

The reason for analyzing tunicate recognition systems is two-fold. First, they can be established as primitive models for understanding fundamental immunological mechanisms by analyzing either their individual cells in vivo or in vitro. Discovered mechanisms could provide alternatives to traditional mammalian (mouse, rat) and emerging models (fish, amphibians) in answering basic questions concerning immunity and disease. Moreover, their advantages lie in: (a) the simplicity of primitive systems (minimal hemopoietic sites), allowing a more effective dissection of variables; (b) their limited expense when compared to experimentation using vertebrates; and (c) their being socially non-controversial. Second, techniques of cell and molecular biology are equally applicable to tunicates as invertebrate models. Despite the considerable advances of recent years, many fundamental or conceptual aspects of immunological reactivity remain unresolved. A clear understanding of the system's evolutionary past will help to elucidate mechanisms which are complex and difficult to decipher in mammals, as we postulate, the immunorecognition systems of vertebrates evolved from simpler systems. Then tunicate immunodefense responses are excellent examples for analyses.     

Assessing the Relationship Between Pfiesteria and Estuarine Fishkills

A recently identified dinoflagellate, Pfiesteria piscicida, has been implicated as a cause of fishkills in mid-Atlantic estuaries. To date, field evidence supporting this argument has consisted of samples, analyzed for the presence of the toxic Pfiesteria forms, gathered during a fishkill. I present a probabilistic approach to examine the use of this kind of a posteriori information as an indication of cause and effect relationships. The analysis shows that the conditional probability of the presence of Pfiesteria after a fishkill has begun provides little support for Pfiesteria as a cause of fishkills, without also knowing the probability of Pfiesteria's presence under all conditions. Documenting the relative presence of toxic life stages during fishkills and under non-fishkill conditions will provide supporting evidence to assess Pfiesteria's role in fishkills. However, proving that Pfiesteria causes estuarine fishkills using only ‘after the fact‘ information is essentially impossible.     

Extraterrestrial Flux of Potentially Prebiotic C, N, and P to the Early Earth

With growing evidence for a heavy bombardment period ending 4–3.8 billion years ago, meteorites and comets may have been an important source of prebiotic carbon, nitrogen, and phosphorus on the early Earth. Life may have originated shortly after the late-heavy bombardment, when concentrations of organic compounds and reactive phosphorus were enough to “kick life into gear”. This work quantifies the sources of potentially prebiotic, extraterrestrial C, N, and P and correlates these fluxes with a comparison to total Ir fluxes, and estimates the effect of atmosphere on the survival of material. We find (1) that carbonaceous chondrites were not a good source of organic compounds, but interplanetary dust particles provided a constant, steady flux of organic compounds to the surface of the Earth, (2) extraterrestrial metallic material was much more abundant on the early Earth, and delivered reactive P in the form of phosphide minerals to the Earth’s surface, and (3) large impacts provided substantial local enrichments of potentially prebiotic reagents. These results help elucidate the potential role of extraterrestrial matter in the origin of life.