text: Life http://www.mdpi.com/journal/life Latest open access articles published in Life at http://www.mdpi.com/journal/life http://www.mdpi.com/2075-1729/7/1/8 The tRNA identity elements for some amino acids are distinct between the bacterial and archaeal domains. Searching in recent genomic and metagenomic sequence data, we found some candidate phyla radiation (CPR) bacteria with archaeal tRNA identity for Tyr-tRNA and Trp-tRNA synthesis. These bacteria possess genes for tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase (TrpRS) predicted to be derived from DPANN superphylum archaea, while the cognate tRNATyr and tRNATrp genes reveal bacterial or archaeal origins. We identified a trace of domain fusion and swapping in the archaeal-type TyrRS gene of a bacterial lineage, suggesting that CPR bacteria may have used this mechanism to create diverse proteins. Archaeal-type TrpRS of bacteria and a few TrpRS species of DPANN archaea represent a new phylogenetic clade (named TrpRS-A). The TrpRS-A open reading frames (ORFs) are always associated with another ORF (named ORF1) encoding an unknown protein without global sequence identity to any known protein. However, our protein structure prediction identified a putative HIGH-motif and KMSKS-motif as well as many α-helices that are characteristic of class I aminoacyl-tRNA synthetase (aaRS) homologs. These results provide another example of the diversity of molecular components that implement the genetic code and provide a clue to the early evolution of life and the genetic code. Life 2017-02-21 7 1 Article 10.3390/life7010008 8 2075-1729 2017-02-21 doi: 10.3390/life7010008 Takahito Mukai Noah Reynolds Ana Crnković Dieter Söll http://www.mdpi.com/2075-1729/7/1/7 In this work, we determine the biological and mathematical properties that are sufficient and necessary to uniquely determine both the primeval RNY (purine-any base-pyrimidine) code and the standard genetic code (SGC). These properties are: the evolution of the SGC from the RNY code; the degeneracy of both codes, and the non-degeneracy of the assignments of aminoacyl-tRNA synthetases (aaRSs) to amino acids; the wobbling property; the consideration that glycine was the first amino acid; the topological and symmetrical properties of both codes. Life 2017-02-13 7 1 Article 10.3390/life7010007 7 2075-1729 2017-02-13 doi: 10.3390/life7010007 Gabriel Zamudio Marco José http://www.mdpi.com/2075-1729/7/1/6 Aminoacyl-tRNA synthetases (AARSs) have evolved "quality control" mechanisms which prevent tRNA aminoacylation with non-protein amino acids, such as homocysteine, homoserine, and ornithine, and thus their access to the Genetic Code. Of the ten AARSs that possess editing function, five edit homocysteine: Class I MetRS, ValRS, IleRS, LeuRS, and Class II LysRS. Studies of their editing function reveal that catalytic modules of these AARSs have a thiol-binding site that confers the ability to catalyze the aminoacylation of coenzyme A, pantetheine, and other thiols. Other AARSs also catalyze aminoacyl-thioester synthesis. Amino acid selectivity of AARSs in the aminoacyl thioesters formation reaction is relaxed, characteristic of primitive amino acid activation systems that may have originated in the Thioester World. With homocysteine and cysteine as thiol substrates, AARSs support peptide bond synthesis. Evolutionary origin of these activities is revealed by genomic comparisons, which show that AARSs are structurally related to proteins involved in coenzyme A/sulfur metabolism and non-coded peptide bond synthesis. These findings suggest that the extant AARSs descended from ancestral forms that were involved in non-coded Thioester-dependent peptide synthesis, functionally similar to the present-day non-ribosomal peptide synthetases. Life 2017-02-09 7 1 Review 10.3390/life7010006 6 2075-1729 2017-02-09 doi: 10.3390/life7010006 Hieronim Jakubowski http://www.mdpi.com/2075-1729/7/1/5 At some point in early evolution, life became cellular. Assuming that this step was required for the origin of life, there would necessarily be a pre-existing source of amphihilic compounds capable of assembling into membranous compartments. It is possible to make informed guesses about the properties of such compounds and the conditions most conducive to their self-assembly into boundary structures. The membranes were likely to incorporate mixtures of hydrocarbon derivatives between 10 and 20 carbons in length with carboxylate or hydroxyl head groups. Such compounds can be synthesized by chemical reactions and small amounts were almost certainly present in the prebiotic environment. Membrane assembly occurs most readily in low ionic strength solutions with minimal content of salt and divalent cations, which suggests that cellular life began in fresh water pools associated with volcanic islands rather than submarine hydrothermal vents. Life 2017-01-17 7 1 Review 10.3390/life7010005 5 2075-1729 2017-01-17 doi: 10.3390/life7010005 David Deamer http://www.mdpi.com/2075-1729/7/1/4 The editors of Life would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2016. Life 2017-01-10 7 1 Editorial 10.3390/life7010004 4 2075-1729 2017-01-10 doi: 10.3390/life7010004 Life Editorial Office http://www.mdpi.com/2075-1729/7/1/3 A basic problem in all postulated pathways of prebiotic chemistry is the low concentration which generally is expected for interesting reactants in fluid environments. Even though compounds, like nucleobases, sugars or peptides, principally may form spontaneously under environmental conditions, they will always be rapidly diluted in an aqueous environment. In addition, any such reaction leads to side products which often exceed the desired compound and generally hamper the first steps of a subsequent molecular evolution. Therefore, a mechanism of selection and accumulation of relevant prebiotic compounds seems to be crucial for molecular evolution. A very efficient environment for selection and accumulation can be found in the fluid continuum circulating in tectonic fault zones. Vesicles which form spontaneously at a depth of approximately 1 km present a selective trap for amphiphilic molecules, especially for peptides composed of hydrophilic and hydrophobic amino acids in a suitable sequence. The accumulation effect is shown in a numeric simulation on a simplified model. Further, possible mechanisms of a molecular evolution in vesicle membranes are discussed. Altogether, the proposed scenario can be seen as an ideal environment for constant, undisturbed molecular evolution in and on cell-like compartments. Life 2017-01-07 7 1 Article 10.3390/life7010003 3 2075-1729 2017-01-07 doi: 10.3390/life7010003 Christian Mayer Ulrich Schreiber María Dávila http://www.mdpi.com/2075-1729/7/1/2 An essential question in studies on the origins of life is how nucleic acids were first synthesized and then incorporated into compartments about 4 billion years ago. A recent discovery is that guided polymerization within organizing matrices could promote a non-enzymatic condensation reaction allowing the formation of RNA-like polymers, followed by encapsulation in lipid membranes. Here, we used neutron scattering and deuterium labelling to investigate 5′-adenosine monophosphate (AMP) molecules captured in a multilamellar phospholipid matrix. The aim of the research was to determine and compare how mononucleotides are captured and differently organized within matrices and multilamellar phospholipid structures and to explore the role of water in organizing the system to determine at which level the system becomes sufficiently anhydrous to lock the AMP molecules into an organized structure and initiate ester bond synthesis. Elastic incoherent neutron scattering experiments were thus employed to investigate the changes of the dynamic properties of AMP induced by embedding the molecules within the lipid matrix. The influence of AMP addition to the lipid membrane organization was determined through diffraction measurement, which also helped us to define the best working Q range for dynamical data analysis with respect to specific hydration. The use of different complementary instruments allowed coverage of a wide time-scale domain, from ns to ps, of atomic mean square fluctuations, providing evidence of a well-defined dependence of the AMP dynamics on the hydration level. Life 2017-01-04 7 1 Article 10.3390/life7010002 2 2075-1729 2017-01-04 doi: 10.3390/life7010002 Loreto Misuraca Francesca Natali Laura da Silva Judith Peters Bruno Demé Jacques Ollivier Tilo Seydel Valerie Laux-Lesourd Michael Haertlein Giuseppe Zaccai David Deamer Marie Maurel http://www.mdpi.com/2075-1729/7/1/1 Three novel strains of photosynthetic bacteria from the family Ectothiorhodospiraceae were isolated from soda lakes of the Great Basin Desert, USA by employing arsenite (As(III)) as the sole electron donor in the enrichment/isolation process. Strain PHS-1 was previously isolated from a hot spring in Mono Lake, while strain MLW-1 was obtained from Mono Lake sediment, and strain BSL-9 was isolated from Big Soda Lake. Strains PHS-1, MLW-1, and BSL-9 were all capable of As(III)-dependent growth via anoxygenic photosynthesis and contained homologs of arxA, but displayed different phenotypes. Comparisons were made with three related species: Ectothiorhodospira shaposhnikovii DSM 2111, Ectothiorhodospira shaposhnikovii DSM 243T, and Halorhodospira halophila DSM 244. All three type cultures oxidized arsenite to arsenate but did not grow with As(III) as the sole electron donor. DNA–DNA hybridization indicated that strain PHS-1 belongs to the same species as Ect. shaposhnikovii DSM 2111 (81.1% sequence similarity), distinct from Ect. shaposhnikovii DSM 243T (58.1% sequence similarity). These results suggest that the capacity for light-driven As(III) oxidation is a common phenomenon among purple photosynthetic bacteria in soda lakes. However, the use of As(III) as a sole electron donor to sustain growth via anoxygenic photosynthesis is confined to novel isolates that were screened for by this selective cultivation criterion. Life 2016-12-26 7 1 Article 10.3390/life7010001 1 2075-1729 2016-12-26 doi: 10.3390/life7010001 Shelley Hoeft McCann Alison Boren Jaime Hernandez-Maldonado Brendon Stoneburner Chad Saltikov John Stolz Ronald Oremland http://www.mdpi.com/2075-1729/6/4/43 The origin of biomolecular machinery likely centered around an ancient and central molecule capable of interacting with emergent macromolecular complexity. tRNA is the oldest and most central nucleic acid molecule of the cell. Its co-evolutionary interactions with aminoacyl-tRNA synthetase protein enzymes define the specificities of the genetic code and those with the ribosome their accurate biosynthetic interpretation. Phylogenetic approaches that focus on molecular structure allow reconstruction of evolutionary timelines that describe the history of RNA and protein structural domains. Here we review phylogenomic analyses that reconstruct the early history of the synthetase enzymes and the ribosome, their interactions with RNA, and the inception of amino acid charging and codon specificities in tRNA that are responsible for the genetic code. We also trace the age of domains and tRNA onto ancient tRNA homologies that were recently identified in rRNA. Our findings reveal a timeline of recruitment of tRNA building blocks for the formation of a functional ribosome, which holds both the biocatalytic functions of protein biosynthesis and the ability to store genetic memory in primordial RNA genomic templates. Life 2016-12-02 6 4 Opinion 10.3390/life6040043 43 2075-1729 2016-12-02 doi: 10.3390/life6040043 Derek Caetano-Anollés Gustavo Caetano-Anollés http://www.mdpi.com/2075-1729/6/4/42 Cyanobacteria hold significant potential as industrial biotechnology (IB) platforms for the production of a wide variety of bio-products ranging from biofuels such as hydrogen, alcohols and isoprenoids, to high-value bioactive and recombinant proteins. Underpinning this technology, are the recent advances in cyanobacterial "omics" research, the development of improved genetic engineering tools for key species, and the emerging field of cyanobacterial synthetic biology. These approaches enabled the development of elaborate metabolic engineering programs aimed at creating designer strains tailored for different IB applications. In this review, we provide an overview of the current status of the fields of cyanobacterial omics and genetic engineering with specific focus on the current molecular tools and technologies that have been developed in the past five years. The paper concludes by giving insights on future commercial applications of cyanobacteria and highlights the challenges that need to be addressed in order to make cyanobacterial industrial biotechnology more feasible in the near future. Life 2016-11-30 6 4 Review 10.3390/life6040042 42 2075-1729 2016-11-30 doi: 10.3390/life6040042 Lamya Al-Haj Yuen Lui Raeid Abed Mohamed Gomaa Saul Purton http://www.mdpi.com/2075-1729/6/4/41 Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella—structures required for motility—share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air–liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876—also critical for both motility and adhesion—will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects. Life 2016-11-26 6 4 Article 10.3390/life6040041 41 2075-1729 2016-11-26 doi: 10.3390/life6040041 Georgio Legerme Evan Yang Rianne Esquivel Saija Kiljunen Harri Savilahti Mechthild Pohlschroder http://www.mdpi.com/2075-1729/6/4/40 Cellular life is based on interacting polymer networks that serve as catalysts, genetic information and structural molecules. The complexity of the DNA, RNA and protein biochemistry suggests that it must have been preceded by simpler systems. The RNA world hypothesis proposes RNA as the prime candidate for such a primal system. Even though this proposition has gained currency, its investigations have highlighted several challenges with respect to bulk aqueous media: (1) the synthesis of RNA monomers is difficult; (2) efficient pathways for monomer polymerization into functional RNAs and their subsequent, sequence-specific replication remain elusive; and (3) the evolution of the RNA function towards cellular metabolism in isolation is questionable in view of the chemical mixtures expected on the early Earth. This review will address the question of the possible roles of heterogeneous media and catalysis as drivers for the emergence of RNA-based polymer networks. We will show that this approach to non-enzymatic polymerizations of RNA from monomers and RNA evolution cannot only solve some issues encountered during reactions in bulk aqueous solutions, but may also explain the co-emergence of the various polymers indispensable for life in complex mixtures and their organization into primitive networks. Life 2016-11-04 6 4 Review 10.3390/life6040040 40 2075-1729 2016-11-04 doi: 10.3390/life6040040 Pierre-Alain Monnard http://www.mdpi.com/2075-1729/6/3/39 Gene duplication followed by mutation is a classic mechanism of neofunctionalization, producing gene families with functional diversity. In some cases, a single point mutation is sufficient to change the substrate specificity and/or the chemistry performed by an enzyme, making it difficult to accurately separate enzymes with identical functions from homologs with different functions. Because sequence similarity is often used as a basis for assigning functional annotations to genes, non-isofunctional gene families pose a great challenge for genome annotation pipelines. Here we describe how integrating evolutionary and functional information such as genome context, phylogeny, metabolic reconstruction and signature motifs may be required to correctly annotate multifunctional families. These integrative analyses can also lead to the discovery of novel gene functions, as hints from specific subgroups can guide the functional characterization of other members of the family. We demonstrate how careful manual curation processes using comparative genomics can disambiguate subgroups within large multifunctional families and discover their functions. We present the COG0720 protein family as a case study. We also discuss strategies to automate this process to improve the accuracy of genome functional annotation pipelines. Life 2016-09-08 6 3 Article 10.3390/life6030039 39 2075-1729 2016-09-08 doi: 10.3390/life6030039 Rémi Zallot Katherine Harrison Bryan Kolaczkowski Valérie de Crécy-Lagard http://www.mdpi.com/2075-1729/6/3/38 Increasing evidence suggests that bdelloid rotifers regularly undergo horizontal gene transfer, apparently as a surrogate mechanism of genetic exchange in the absence of true sexual reproduction, in part because of their ability to withstand desiccation. We provide empirical support for this latter hypothesis using the bdelloid Philodina roseola, which we demonstrate to readily internalize environmental DNA in contrast to a representative monogonont rotifer (Brachionus rubens), which, like other monogononts, is facultative sexual and cannot withstand desiccation. In addition, environmental DNA that was more similar to the host DNA was retained more often and for a longer period of time. Indirect evidence (increased variance in the reproductive output of the untreated F1 generation) suggests that environmental DNA can be incorporated into the genome during desiccation and is thus heritable. Our observed fitness effects agree with sexual theory and also occurred when the animals were desiccated in groups (thereby acting as DNA donors), but not individually, indicating the mechanism could occur in nature. Thus, although DNA uptake and its genomic incorporation appears proximally related to anhydrobiosis in bdelloids, it might also facilitate accidental genetic exchange with closely related taxa, thereby maintaining higher levels of genetic diversity than is otherwise expected for this group of "ancient asexuals". Life 2016-09-06 6 3 Article 10.3390/life6030038 38 2075-1729 2016-09-06 doi: 10.3390/life6030038 Olaf Bininda-Emonds Claus Hinz Wilko Ahlrichs http://www.mdpi.com/2075-1729/6/3/37 Halophilic archaea experience high levels of ultraviolet (UV) light in their environments and demonstrate resistance to UV irradiation. DNA repair systems and carotenoids provide UV protection but do not account for the high resistance observed. Herein, we consider genomic signatures as an additional photoprotective strategy. The predominant forms of UV-induced DNA damage are cyclobutane pyrimidine dimers, most notoriously thymine dimers (T^Ts), which form at adjacent Ts. We tested whether the high G + C content seen in halophilic archaea serves a photoprotective function through limiting T nucleotides, and thus T^T lesions. However, this speculation overlooks the other bipyrimidine sequences, all of which capable of forming photolesions to varying degrees. Therefore, we designed a program to determine the frequencies of the four bipyrimidine pairs (5' to 3': TT, TC, CT, and CC) within genomes of halophilic archaea and four other randomized sample groups for comparison. The outputs for each sampled genome were weighted by the intrinsic photoreactivities of each dinucleotide pair. Statistical methods were employed to investigate intergroup differences. Our findings indicate that the UV-resistance seen in halophilic archaea can be attributed in part to a genomic strategy: high G + C content and the resulting bipyrimidine signature reduces the genomic photoreactivity. Life 2016-09-02 6 3 Article 10.3390/life6030037 37 2075-1729 2016-09-02 doi: 10.3390/life6030037 Daniel Jones Bonnie Baxter http://www.mdpi.com/2075-1729/6/3/36 Francis Crick (Figure 1) was born on 8 June 1916, in Northampton, England, and passed away on 28 July 2004, in La Jolla, California, USA.[...] Life 2016-08-25 6 3 Editorial 10.3390/life6030036 36 2075-1729 2016-08-25 doi: 10.3390/life6030036 Koji Tamura http://www.mdpi.com/2075-1729/6/3/35 In recent years, biodiesel, a substitute for fossil fuels, has led to the excessive production of crude glycerol. The resulting crude glycerol can possess a high concentration of salts and an alkaline pH. Moreover, current crude glycerol purification methods are expensive, rendering this former commodity a waste product. However, Halanaerobium hydrogeniformans, a haloalkaliphilic bacterium, possesses the metabolic capability to convert glycerol into 1,3-propanediol, a valuable commodity compound, without the need for salt dilution or adjusting pH when grown on this waste. Experiments were performed with different combinations of 24 medium components to determine their impact on the production of 1,3-propanediol by using a fractional factorial design. Tested medium components were selected based on data from the organism's genome. Analysis of HPLC data revealed enhanced production of 1,3-propanediol with additional glycerol, pH, vitamin B12, ammonium ions, sodium sulfide, cysteine, iron, and cobalt. However, other selected components; nitrate ions, phosphate ions, sulfate ions, sodium:potassium ratio, chloride, calcium, magnesium, silicon, manganese, zinc, borate, nickel, molybdenum, tungstate, copper and aluminum, did not enhance 1,3-propanediol production. The use of a fractional factorial design enabled the quick and efficient assessment of the impact of 24 different medium components on 1,3-propanediol production from glycerol from a haloalkaliphilic bacterium. Life 2016-08-20 6 3 Article 10.3390/life6030035 35 2075-1729 2016-08-20 doi: 10.3390/life6030035 Shivani Kalia Jordan Trager Oliver Sitton Melanie Mormile http://www.mdpi.com/2075-1729/6/3/34 Facts concerning the stability and functioning of key biomolecular components suggest that cellular life should no longer be viable above a few thousand atmospheres (200–300 MPa). However, organisms are seen to survive in the laboratory to much higher pressures, extending into the GPa or even tens of GPa ranges. This is causing main questions to be posed concerning the survival mechanisms of simple to complex organisms. Understanding the ultimate pressure survival of organisms is critical for food sterilization and agricultural products conservation technologies. On Earth the deep biosphere is limited in its extent by geothermal gradients but if life forms exist in cooler habitats elsewhere then survival to greater depths must be considered. The extent of pressure resistance and survival appears to vary greatly with the timescale of the exposure. For example, shock experiments on nanosecond timescales reveal greatly enhanced survival rates extending to higher pressure. Some organisms could survive bolide impacts thus allowing successful transport between planetary bodies. We summarize some of the main questions raised by recent results and their implications for the survival of life under extreme compression conditions and its possible extent in the laboratory and throughout the universe. Life 2016-08-17 6 3 Concept Paper 10.3390/life6030034 34 2075-1729 2016-08-17 doi: 10.3390/life6030034 Rachael Hazael Filip Meersman Fumihisa Ono Paul McMillan http://www.mdpi.com/2075-1729/6/3/33 We propose that the first step in the origin of cellular life on Earth was the self-assembly of fatty acids with the building blocks of RNA and protein, resulting in a stable aggregate. This scheme provides explanations for the selection and concentration of the prebiotic components of cells; the stabilization and growth of early membranes; the catalysis of biopolymer synthesis; and the co-localization of membranes, RNA and protein. In this article, we review the evidence and rationale for the formation of the proposed aggregate: (i) the well-established phenomenon of self-assembly of fatty acids to form vesicles; (ii) our published evidence that nucleobases and sugars bind to and stabilize such vesicles; and (iii) the reasons why amino acids likely do so as well. We then explain how the conformational constraints and altered chemical environment due to binding of the components to the membrane could facilitate the formation of nucleosides, oligonucleotides and peptides. We conclude by discussing how the resulting oligomers, even if short and random, could have increased vesicle stability and growth more than their building blocks did, and how competition among these vesicles could have led to longer polymers with complex functions. Life 2016-08-11 6 3 Review 10.3390/life6030033 33 2075-1729 2016-08-11 doi: 10.3390/life6030033 Roy Black Matthew Blosser http://www.mdpi.com/2075-1729/6/3/32 Gene duplication is an important mechanism of molecular evolution. It offers a fast track to modification, diversification, redundancy or rescue of gene function. However, duplication may also be neutral or (slightly) deleterious, and often ends in pseudo-geneisation. Here, we investigate the phylogenetic distribution of ultra large gene families on long and short evolutionary time scales. In particular, we focus on a family of NACHT-domain and leucine-rich-repeat-containing (NLR)-genes, which we previously found in large numbers to occupy one chromosome arm of the zebrafish genome. We were interested to see whether such a tight clustering is characteristic for ultra large gene families. Our data reconfirm that most gene family inflations are lineage-specific, but we can only identify very few gene clusters. Based on our observations we hypothesise that, beyond a certain size threshold, ultra large gene families continue to proliferate in a mechanism we term "run-away evolution". This process might ultimately lead to the failure of genomic integrity and drive species to extinction. Life 2016-08-08 6 3 Opinion 10.3390/life6030032 32 2075-1729 2016-08-08 doi: 10.3390/life6030032 Philipp Schiffer Jan Gravemeyer Martina Rauscher Thomas Wiehe http://www.mdpi.com/2075-1729/6/3/31 Haloferax volcanii AglM and Halobacterium salinarum VNG1048G are UDP-glucose dehydrogenases involved in N-glycosylation in each species. Despite sharing >60% sequence identity and the ability of VNG1048G to functionally replace AglM in vivo, these proteins behaved differently as salinity changed. Whereas AglM was active in 2–4 M NaCl, VNG1048G lost much of its activity when salinity dropped below 3 M NaCl. To understand the molecular basis of this phenomenon, each protein was examined by size exclusion chromatrography in 2 M NaCl. Whereas AglM appeared as a dodecamer, VNG1048G was essentially detected as a dodecamer and a dimer. The specific activity of the VNG1048G dodecamer was only a sixth of that of AglM, while the dimer was inactive. As such, not only was the oligomeric status of VNG1048G affected by lowered salinity, so was the behavior of the individual dodecamer subunits. Analyzing surface-exposed residues in homology models of the two UDP-glucose dehydrogenases revealed the more acidic and less basic VNG1048G surface, further explaining the greater salt-dependence of the Hbt. salinarum enzyme. Life 2016-08-03 6 3 Article 10.3390/life6030031 31 2075-1729 2016-08-03 doi: 10.3390/life6030031 Lina Kandiba Jerry Eichler http://www.mdpi.com/2075-1729/6/3/30 The existence of multiple copies of genes is a well-known phenomenon. A gene family is a set of sufficiently similar genes, formed by gene duplication. In earlier works conducted on a limited number of completely sequenced and annotated genomes it was found that size of gene family and size of genome are positively correlated. Additionally, it was found that several atypical microbes deviated from the observed general trend. In this study, we reexamined these associations on a larger dataset consisting of 1484 prokaryotic genomes and using several ranking approaches. We applied ranking methods in such a way that genomes with lower numbers of gene copies would have lower rank. Until now only simple ranking methods were used; we applied the Kemeny optimal aggregation approach as well. Regression and correlation analysis were utilized in order to accurately quantify and characterize the relationships between measures of paralog indices and genome size. In addition, boxplot analysis was employed as a method for outlier detection. We found that, in general, all paralog indexes positively correlate with an increase of genome size. As expected, different groups of atypical prokaryotic genomes were found for different types of paralog quantities. Mycoplasmataceae and Halobacteria appeared to be among the most interesting candidates for further research of evolution through gene duplication. Life 2016-08-03 6 3 Article 10.3390/life6030030 30 2075-1729 2016-08-03 doi: 10.3390/life6030030 Gon Carmi Alexander Bolshoy http://www.mdpi.com/2075-1729/6/3/29 RNA played a central role in the emergence of the first life-like system on primitive Earth since RNA molecules contain both genetic information and catalytic activity. However, there are several drawbacks regarding the RNA world hypothesis. Here, I briefly discuss the feasibility of the RNA world hypothesis to deduce the RNA functions that are essential for forming a life-like system. At the same time, I have conducted a conceptual analysis of the characteristics of biosystems as a useful approach to deduce a realistic life-like system in relation to the definition of life. For instance, an RNA-based life-like system should possess enough stability to resist environmental perturbations, by developing a cell-like compartment, for instance. Here, a conceptual viewpoint is summarized to provide a realistic life-like system that is compatible with the primitive Earth environment and the capabilities of RNA molecules. According to the empirical and conceptual analysis, I propose the hypothesis that the first life-like system could have initiated from only two genes. Life 2016-08-02 6 3 Article 10.3390/life6030029 29 2075-1729 2016-08-02 doi: 10.3390/life6030029 Kunio Kawamura http://www.mdpi.com/2075-1729/6/3/28 The endoergic nature of protein and nucleic acid assembly in aqueous media presents two questions that are fundamental to the understanding of life's origins: (i) how did the polymers arise in an aqueous prebiotic world; and (ii) once formed in some manner, how were they sufficiently persistent to engage in further chemistry. We propose here a quantitative resolution of these issues that evolved from recent accounts in which RNA-like polymers were produced in evaporation/rehydration cycles. The equilibrium Nm + Nn ↔ Nm+n + H2O is endoergic by about 3.3 kcal/mol for polynucleotide formation, and the system thus lies far to the left in the starting solutions. Kinetic simulations of the evaporation showed that simple Le Châtelier's principle shifts were insufficient, but the introduction of oligomer-stabilizing factors of 5–10 kcal/mol both moved the process to the right and respectively boosted and retarded the elongation and hydrolysis rates. Molecular crowding and excluded volume effects in present-day cells yield stabilizing factors of that order, and we argue here that the crowded conditions in the evaporites generate similar effects. Oligomer formation is thus energetically preferred in those settings, but the process is thwarted in each evaporation step as diffusion becomes rate limiting. Rehydration dissipates disordered oligomer clusters in the evaporites, however, and subsequent dry/wet cycling accordingly "ratchets up" the system to an ultimate population of kinetically trappedthermodynamically preferred biopolymers. Life 2016-07-26 6 3 Article 10.3390/life6030028 28 2075-1729 2016-07-26 doi: 10.3390/life6030028 David Ross David Deamer http://www.mdpi.com/2075-1729/6/3/27 The abundance of mammalian long intergenic non-coding RNA (lincRNA) genes is high, yet their functions remain largely unknown. One possible way to study this important question is to use large-scale comparisons of various characteristics of lincRNA with those of protein-coding genes for which a large body of functional information is available. A prominent feature of mammalian protein-coding genes is the high evolutionary conservation of the exon-intron structure. Comparative analysis of putative intron positions in lincRNA genes from various mammalian genomes suggests that some lincRNA introns have been conserved for over 100 million years, thus the primary and/or secondary structure of these molecules is likely to be functionally important. Life 2016-07-15 6 3 Article 10.3390/life6030027 27 2075-1729 2016-07-15 doi: 10.3390/life6030027 Diana Chernikova David Managadze Galina Glazko Wojciech Makalowski Igor Rogozin http://www.mdpi.com/2075-1729/6/3/26 The mystery of the origin of life can be divided into two parts. The first part is the origin of biomolecules: under what physicochemical conditions did biomolecules such as amino acids, nucleotides, and their polymers arise? The second part of the mystery is the origin of life-specific functions such as the replication of genetic information, the reproduction of cellular structures, metabolism, and evolution. These functions require the coordination of many different kinds of biological molecules. A direct strategy to approach the second part of the mystery is the constructive approach, in which life-specific functions are recreated in a test tube from specific biological molecules. Using this approach, we are able to employ design principles to reproduce life-specific functions, and the knowledge gained through the reproduction process provides clues as to their origins. In this mini-review, we introduce recent insights gained using this approach, and propose important future directions for advancing our understanding of the origins of life. Life 2016-07-13 6 3 Review 10.3390/life6030026 26 2075-1729 2016-07-13 doi: 10.3390/life6030026 Norikazu Ichihashi Tetsuya Yomo http://www.mdpi.com/2075-1729/6/3/25 Life on Earth provides a unique biological record from single-cell microbes to technologically intelligent life forms. Our evolution is marked by several major steps or innovations along a path of increasing complexity from microbes to space-faring humans. Here we identify various major key innovations, and use an analytical toolset consisting of a set of models to analyse how likely each key innovation is to occur. Our conclusion is that once the origin of life is accomplished, most of the key innovations can occur rather readily. The conclusion for other worlds is that if the origin of life can occur rather easily, we should live in a cosmic zoo, as the innovations necessary to lead to complex life will occur with high probability given sufficient time and habitat. On the other hand, if the origin of life is rare, then we might live in a rather empty universe. Life 2016-06-30 6 3 Essay 10.3390/life6030025 25 2075-1729 2016-06-30 doi: 10.3390/life6030025 William Bains Dirk Schulze-Makuch http://www.mdpi.com/2075-1729/6/2/24 A long-standing problem for the origins of life is that polymerization of many biopolymers, including nucleic acids and peptides, is thermodynamically unfavourable in aqueous solution. If bond making and breaking is reversible, monomers and very short oligomers predominate. Recent experiments have shown that wetting and drying cycles can overcome this problem and drive the formation of longer polymers. In the dry phase, bond formation is favourable, but diffusion is restricted, and bonds only form between monomers that are initially close together. In the wet phase, some of the bonds are hydrolyzed. However, repositioning of the molecules allows new bonds to form in the next dry phase, leading to an increase in mean polymer length. Here, we consider a simple theoretical model that explains the effect of cycling. There is an equilibrium length distribution with a high mean length that could be achieved if diffusion occurred freely in the dry phase. This equilibrium is inaccessible without diffusion. A single dry cycle without diffusion leads to mean lengths much shorter than this. Repeated cycling leads to a significant increase in polymerization relative to a single cycle. In the most favourable case, cycling leads to the same equilibrium length distribution as would be achieved if free diffusion were possible in the dry phase. These results support the RNA World scenario by explaining a potential route to synthesis of long RNAs; however, they also imply that cycling would be beneficial to the synthesis of other kinds of polymers, including peptides, where bond formation involves a condensation reaction. Life 2016-06-08 6 2 Article 10.3390/life6020024 24 2075-1729 2016-06-08 doi: 10.3390/life6020024 Paul Higgs http://www.mdpi.com/2075-1729/6/2/23 We have explored the use of optical oxygen electrodes to study oxygenic photosynthesis and heterotrophic activities in crystallizer brines of the salterns in Eilat, Israel. Monitoring oxygen uptake rates in the dark enables the identification of organic substrates that are preferentially used by the community. Addition of glycerol (the osmotic solute synthesized by Dunaliella) or dihydroxyacetone (produced from glycerol by Salinibacter) enhanced respiration rates. Pyruvate, produced from glycerol or from some sugars by certain halophilic Archaea also stimulated community respiration. Fumarate had a sparing effect on respiration, possibly as many halophilic Archaea can use fumarate as a terminal electron acceptor in respiration. Calculating the photosynthetic activity of Dunaliella by monitoring oxygen concentration changes during light/dark incubations is not straightforward as light also affects respiration of some halophilic Archaea and Bacteria due to action of light-driven proton pumps. When illuminated, community respiration of brine samples in which oxygenic photosynthesis was inhibited by DCMU decreased by ~40%. This effect was interpreted as the result of competition between two energy yielding systems: the bacteriorhodopsin proton pump and the respiratory chain of the prokaryotes. These findings have important implications for the interpretation of other published data on photosynthetic and respiratory activities in hypersaline environments. Life 2016-06-08 6 2 Review 10.3390/life6020023 23 2075-1729 2016-06-08 doi: 10.3390/life6020023 Aharon Oren http://www.mdpi.com/2075-1729/6/2/22 We used Ar plasma-sterilization at a temperature below 80 °C to examine its effects on the viability of microorganisms when intermixed with tested soil. Due to a relatively low temperature, this method is not thought to affect the properties of a soil, particularly its organic component, to a significant degree. The method has previously been shown to work well on spacecraft parts. The selected microorganism for this test was Deinococcus radiodurans R1, which is known for its remarkable resistance to radiation effects. Our results showed a reduction in microbial counts after applying a low temperature plasma, but not to a degree suitable for a sterilization of the soil. Even an increase of the treatment duration from 1.5 to 45 min did not achieve satisfying results, but only resulted in in a mean cell reduction rate of 75% compared to the untreated control samples. Life 2016-05-26 6 2 Article 10.3390/life6020022 22 2075-1729 2016-05-26 doi: 10.3390/life6020022 Janosch Schirmack Marcel Fiebrandt Katharina Stapelmann Dirk Schulze-Makuch http://www.mdpi.com/2075-1729/6/2/21 Charles Darwin's original intuition that life began in a "warm little pond" has for the last three decades been eclipsed by a focus on marine hydrothermal vents as a venue for abiogenesis. However, thermodynamic barriers to polymerization of key molecular building blocks and the difficulty of forming stable membranous compartments in seawater suggest that Darwin's original insight should be reconsidered. I will introduce the terrestrial origin of life hypothesis, which combines field observations and laboratory results to provide a novel and testable model in which life begins as protocells assembling in inland fresh water hydrothermal fields. Hydrothermal fields are associated with volcanic landmasses resembling Hawaii and Iceland today and could plausibly have existed on similar land masses rising out of Earth's first oceans. I will report on a field trip to the living and ancient stromatolite fossil localities of Western Australia, which provided key insights into how life may have emerged in Archaean, fluctuating fresh water hydrothermal pools, geological evidence for which has recently been discovered. Laboratory experimentation and fieldwork are providing mounting evidence that such sites have properties that are conducive to polymerization reactions and generation of membrane-bounded protocells. I will build on the previously developed coupled phases scenario, unifying the chemical and geological frameworks and proposing that a hydrogel of stable, communally supported protocells will emerge as a candidate Woese progenote, the distant common ancestor of microbial communities so abundant in the earliest fossil record. Life 2016-05-25 6 2 Essay 10.3390/life6020021 21 2075-1729 2016-05-25 doi: 10.3390/life6020021 Bruce Damer http://www.mdpi.com/2075-1729/6/2/20 Is it unrealistic to presuppose that all of the steps that could lead to the formation of life could occur in one setting?[...] Life 2016-05-16 6 2 Editorial 10.3390/life6020020 20 2075-1729 2016-05-16 doi: 10.3390/life6020020 Sohan Jheeta http://www.mdpi.com/2075-1729/6/2/19 Prof. Hans Georg Trüper, one of the most important scientists in the field of halophile research, passed away on 9 March 2016 at the age of 79. I here present a brief obituary with special emphasis on Prof. Trüper's contributions to our understanding of the halophilic prokaryotes and their adaptations to life in hypersaline environments. He has pioneered the study of the halophilic anoxygenic phototrophic sulfur bacteria of the Ectothiorhodospira—Halorhodospira group. Some of the species he and his group isolated from hypersaline and haloalkaline environments have become model organisms for the study of the mechanisms of haloadaptation: the functions of three major organic compounds – glycine betaine, ectoine, and trehalose – known to serve as "compatible solutes" in halophilic members of the Bacteria domain, were discovered during studies of these anoxygenic phototrophs. Prof. Trüper's studies of hypersaline alkaline environments in Egypt also led to the isolation of the first known extremely halophilic archaeon (Natronomonas pharaonis). The guest editors dedicate this special volume of Life to the memory of Prof. Hans Georg Trüper. Life 2016-05-12 6 2 Editorial 10.3390/life6020019 19 2075-1729 2016-05-12 doi: 10.3390/life6020019 Aharon Oren http://www.mdpi.com/2075-1729/6/2/18 Carbonaceous Chondrite (CC) meteorites are fragments of asteroids, solar planetesimals that never became large enough to separate matter by their density, like terrestrial planets. CC contains various amounts of organic carbon and carry a record of chemical evolution as it came to be in the Solar System, at the time the Earth was formed and before the origins of life. We review this record as it pertains to the chiral asymmetry determined for several organic compounds in CC, which reaches a broad molecular distribution and enantiomeric excesses of up to 50%–60%. Because homochirality is an indispensable attribute of extant polymers and these meteoritic enantiomeric excesses are still, to date, the only case of chiral asymmetry in organic molecules measured outside the biosphere, the possibility of an exogenous delivery of primed prebiotic compounds to early Earth from meteorites is often proposed. Whether this exogenous delivery held a chiral advantage in molecular evolution remains an open question, as many others regarding the origins of life are. Life 2016-04-13 6 2 Review 10.3390/life6020018 18 2075-1729 2016-04-13 doi: 10.3390/life6020018 Sandra Pizzarello http://www.mdpi.com/2075-1729/6/2/17 It is still uncertain how the first minimal cellular systems evolved to the complexity required for life to begin, but it is obvious that the role of amphiphilic compounds in the origin of life is one of huge relevance. Over the last four decades a number of studies have demonstrated how amphiphilic molecules can be synthesized under plausibly prebiotic conditions. The majority of these experiments also gave evidence for the ability of so formed amphiphiles to assemble in closed membranes of vesicles that, in principle, could have compartmented first biological processes on early Earth, including the emergence of self-replicating systems. For a competitive selection of the best performing molecular replicators to become operative, some kind of bounded units capable of harboring them are indispensable. Without the competition between dynamic populations of different compartments, life itself could not be distinguished from an otherwise disparate array or network of molecular interactions. In this review, we describe experiments that demonstrate how different prebiotically-available building blocks can become precursors of phospholipids that form vesicles. We discuss the experimental conditions that resemble plausibly those of the early Earth (or elsewhere) and consider the analytical methods that were used to characterize synthetic products. Two brief sections focus on phosphorylating agents, catalysts and coupling agents with particular attention given to their geochemical context. In Section 5, we describe how condensing agents such as cyanamide and urea can promote the abiotic synthesis of phospholipids. We conclude the review by reflecting on future studies of phospholipid compartments, particularly, on evolvable chemical systems that include giant vesicles composed of different lipidic amphiphiles. Life 2016-03-28 6 2 Review 10.3390/life6020017 17 2075-1729 2016-03-28 doi: 10.3390/life6020017 Michele Fiore Peter Strazewski http://www.mdpi.com/2075-1729/6/2/16 tRNAs are the fundamental components of the translation machinery as they deliver amino acids to the ribosomes during protein synthesis. Beyond their essential function in translation, tRNAs also function in regulating gene expression, modulating apoptosis and several other biological processes. There are multiple layers of regulatory mechanisms in each step of tRNA biogenesis. For example, tRNA 3′ trailer processing is altered upon nutrient stress; tRNA modification is reprogrammed under various stresses; nuclear accumulation of tRNAs occurs upon nutrient deprivation; tRNA halves accumulate upon oxidative stress. Here we address how environmental stresses can affect nearly every step of tRNA biology and we describe the possible regulatory mechanisms that influence the function or expression of tRNAs under stress conditions. Life 2016-03-23 6 2 Review 10.3390/life6020016 16 2075-1729 2016-03-23 doi: 10.3390/life6020016 Hsiao-Yun Huang Anita Hopper http://www.mdpi.com/2075-1729/6/2/15 Herein we present the tRNA core hypothesis, which emphasizes the central role of tRNAs molecules in the origin and evolution of fundamental biological processes. tRNAs gave origin to the first genes (mRNA) and the peptidyl transferase center (rRNA), proto-tRNAs were at the core of a proto-translation system, and the anticodon and operational codes then arose in tRNAs molecules. Metabolic pathways emerged from evolutionary pressures of the decoding systems. The transitions from the RNA world to the ribonucleoprotein world to modern biological systems were driven by three kinds of tRNAs transitions, to wit, tRNAs leading to both mRNA and rRNA. Life 2016-03-23 6 2 Article 10.3390/life6020015 15 2075-1729 2016-03-23 doi: 10.3390/life6020015 Savio de Farias Thais Rêgo Marco José http://www.mdpi.com/2075-1729/6/1/12 The origins of the components of genetic coding are examined in the present study. Genetic information arose from replicator induction by metabolite in accordance with the metabolic expansion law. Messenger RNA and transfer RNA stemmed from a template for binding the aminoacyl-RNA synthetase ribozymes employed to synthesize peptide prosthetic groups on RNAs in the Peptidated RNA World. Coevolution of the genetic code with amino acid biosynthesis generated tRNA paralogs that identify a last universal common ancestor (LUCA) of extant life close to Methanopyrus, which in turn points to archaeal tRNA introns as the most primitive introns and the anticodon usage of Methanopyrus as an ancient mode of wobble. The prediction of the coevolution theory of the genetic code that the code should be a mutable code has led to the isolation of optional and mandatory synthetic life forms with altered protein alphabets. Life 2016-03-16 6 1 Article 10.3390/life6010012 12 2075-1729 2016-03-16 doi: 10.3390/life6010012 J. Wong Siu-Kin Ng Wai-Kin Mat Taobo Hu Hong Xue http://www.mdpi.com/2075-1729/6/1/14 In 1957, Francis Crick et al. suggested an ingenious explanation for the process of frame maintenance. The idea was based on the notion of comma-free codes. Although Crick's hypothesis proved to be wrong, in 1996, Arquès and Michel discovered the existence of a weaker version of such codes in eukaryote and prokaryote genomes, namely the so-called circular codes. Since then, circular code theory has invariably evoked great interest and made significant progress. In this article, the codon distributions in maximal comma-free, maximal self-complementary C3 and maximal self-complementary circular codes are discussed, i.e., we investigate in how many of such codes a given codon participates. As the main (and surprising) result, it is shown that the codons can be separated into very few classes (three, or five, or six) with respect to their frequency. Moreover, the distribution classes can be hierarchically ordered as refinements from maximal comma-free codes via maximal self-complementary C3 codes to maximal self-complementary circular codes. Life 2016-03-15 6 1 Article 10.3390/life6010014 14 2075-1729 2016-03-15 doi: 10.3390/life6010014 Elena Fimmel Lutz Strüngmann http://www.mdpi.com/2075-1729/6/1/13 All nucleic acids in cells are subject to post-transcriptional chemical modifications. These are catalyzed by a myriad of enzymes with exquisite specificity and that utilize an often-exotic array of chemical substrates. In no molecule are modifications more prevalent than in transfer RNAs. In the present document, we will attempt to take a chemical rollercoaster ride from prebiotic times to the present, with nucleoside modifications as key players and tRNA as the centerpiece that drove the evolution of biological systems to where we are today. These ideas will be put forth while touching on several examples of tRNA modification enzymes and their modus operandi in cells. In passing, we submit that the choice of tRNA is not a whimsical one but rather highlights its critical function as an essential invention for the evolution of protein enzymes. Life 2016-03-14 6 1 Review 10.3390/life6010013 13 2075-1729 2016-03-14 doi: 10.3390/life6010013 Katherine McKenney Juan Alfonzo http://www.mdpi.com/2075-1729/6/1/11 We have recently developed a distance metric for efficiently estimating the number of substitutions per site between unaligned genome sequences. These substitution rates are called "anchor distances" and can be used for phylogeny reconstruction. Most phylogenies come with bootstrap support values, which are computed by resampling with replacement columns of homologous residues from the original alignment. Unfortunately, this method cannot be applied to anchor distances, as they are based on approximate pairwise local alignments rather than the full multiple sequence alignment necessary for the classical bootstrap. We explore two alternatives: pairwise bootstrap and quartet analysis, which we compare to classical bootstrap. With simulated sequences and 53 human primate mitochondrial genomes, pairwise bootstrap gives better results than quartet analysis. However, when applied to 29 E. coli genomes, quartet analysis comes closer to the classical bootstrap. Life 2016-03-07 6 1 Article 10.3390/life6010011 11 2075-1729 2016-03-07 doi: 10.3390/life6010011 Fabian Klötzl Bernhard Haubold http://www.mdpi.com/2075-1729/6/1/10 We have previously proposed that tRNAGly was the first tRNA and glycine was the first amino acid incorporated into the genetic code. The next two amino acids incorporated would have been the other two small hydrophilic amino acids serine and aspartic acid, which occurred through the duplication of the tRNAGly sequence, followed by mutation of its anticodon by single C to U transition mutations, possibly through spontaneous deamination. Interestingly, however, tRNASer has a different structure than most other tRNAs, possessing a long variable arm; because of this tRNASer is classified as a class II tRNA. Also, serine codons are found not only in the bottom right-hand corner of the genetic code table next to those for glycine and aspartic acid, but also in the top row of the table, next to those for two of the most hydrophobic amino acids, leucine and phenylalanine. In the following, I propose that the class II tRNA structure of tRNASer and the arrangement of serine codons in the genetic code provide clues to the early evolution of tRNA and the genetic code. In addition, I address Di Giulio's recent criticism of our proposal that tRNAGly was the first tRNA, and discuss how early peptides produced from a restricted amino acid alphabet of glycine, serine and aspartic acid might have possessed proteolytic activity, which is possibly important for the early recycling of amino acid monomers. Life 2016-02-24 6 1 Hypothesis 10.3390/life6010010 10 2075-1729 2016-02-24 doi: 10.3390/life6010010 Harold Bernhardt http://www.mdpi.com/2075-1729/6/1/9 Silent mutations are being intensively studied. We previously showed that the estrogen receptor alpha Ala87's synonymous polymorphism affects its functional properties. Whereas a link has been clearly established between the effect of silent mutations, tRNA abundance and protein folding in prokaryotes, this connection remains controversial in eukaryotic systems. Although a synonymous polymorphism can affect mRNA structure or the interaction with specific ligands, it seems that the relative frequencies of isoacceptor tRNAs could play a key role in the protein-folding process, possibly through modulation of translation kinetics. Conformational changes could be subtle but enough to cause alterations in solubility, proteolysis profiles, functional parameters or intracellular targeting. Interestingly, recent advances describe dramatic changes in the tRNA population associated with proliferation, differentiation or response to chemical, physical or biological stress. In addition, several reports reveal changes in tRNAs' posttranscriptional modifications in different physiological or pathological conditions. In consequence, since changes in the cell state imply quantitative and/or qualitative changes in the tRNA pool, they could increase the likelihood of protein conformational variants, related to a particular codon usage during translation, with consequences of diverse significance. These observations emphasize the importance of genetic code flexibility in the co-translational protein-folding process. Life 2016-02-17 6 1 Review 10.3390/life6010009 9 2075-1729 2016-02-17 doi: 10.3390/life6010009 Tamara Fernández-Calero Florencia Cabrera-Cabrera Ricardo Ehrlich Mónica Marín http://www.mdpi.com/2075-1729/6/1/8 Titan is the only world we know, other than Earth, that has a liquid on its surface. It also has a thick atmosphere composed of nitrogen and methane with a thick organic haze. There are lakes, rain, and clouds of methane and ethane. Here, we address the question of carbon-based life living in Titan liquids. Photochemically produced organics, particularly acetylene, in Titan's atmosphere could be a source of biological energy when reacted with atmospheric hydrogen. Light levels on the surface of Titan are more than adequate for photosynthesis, but the biochemical limitations due to the few elements available in the environment may lead only to simple ecosystems that only consume atmospheric nutrients. Life on Titan may make use of the trace metals and other inorganic elements produced by meteorites as they ablate in its atmosphere. It is conceivable that H2O molecules on Titan could be used in a biochemistry that is rooted in hydrogen bonds in a way that metals are used in enzymes by life on Earth. Previous theoretical work has shown possible membrane structures, azotosomes, in Titan liquids, azotosomes, composed of small organic nitrogen compounds, such as acrylonitrile. The search for a plausible information molecule for life in Titan liquids remains an open research topic—polyethers have been considered and shown to be insoluble at Titan temperatures. Possible search strategies for life on Titan include looking for unusual concentrations of certain molecules reflecting biological selection. Homochirality is a special and powerful example of such biology selection. Environmentally, a depletion of hydrogen in the lower atmosphere may be a sign of metabolism. A discovery of life in liquid methane and ethane would be our first compelling indication that the universe is full of diverse and wondrous life forms. Life 2016-02-03 6 1 Concept Paper 10.3390/life6010008 8 2075-1729 2016-02-03 doi: 10.3390/life6010008 Christopher McKay http://www.mdpi.com/2075-1729/6/1/7 A novel algorithm and original software were used to cluster all proteins encoded in plastids of 72 species of the rhodophytic branch. The results are publicly available at http://lab6.iitp.ru/ppc/redline72/ in a database that allows fast identification of clusters (protein families) both by a fragment of an amino acid sequence and by a phylogenetic profile of a protein. No such integral clustering with the corresponding functions can be found in the public domain. The putative regulons of the transcription factors Ycf28 and Ycf29 encoded in the plastids were identified using the clustering and the database. A regulation of translation initiation was proposed for the ycf24 gene in plastids of certain red algae and apicomplexans as well as a regulation of a putative gene in apicoplasts of Babesia spp. and Theileria parva. The conserved regulation of the ycf24 gene expression and specificity alternation of the transcription factor Ycf28 were shown in the plastids. A phylogenetic tree of plastids was generated for the rhodophytic branch. The hypothesis of the origin of apicoplasts from the common ancestor of all apicomplexans from plastids of red algae was confirmed. Life 2016-01-29 6 1 Article 10.3390/life6010007 7 2075-1729 2016-01-29 doi: 10.3390/life6010007 Oleg Zverkov Alexandr Seliverstov Vassily Lyubetsky http://www.mdpi.com/2075-1729/6/1/6 It is no doubt quite difficult to solve the riddle of the origin of life. So, firstly, I would like to point out the kinds of obstacles there are in solving this riddle and how we should tackle these difficult problems, reviewing the studies that have been conducted so far. After that, I will propose that the consecutive evolutionary steps in a timeline can be rationally deduced by using a common event as a juncture, which is obtained by two counter-directional approaches: one is the bottom-up approach through which many researchers have studied the origin of life, and the other is the top-down approach, through which I established the [GADV]-protein world hypothesis or GADV hypothesis on the origin of life starting from a study on the formation of entirely new genes in extant microorganisms. Last, I will describe the probable evolutionary process from the formation of Earth to the emergence of life, which was deduced by using a common event—the establishment of the first genetic code encoding [GADV]-amino acids—as a juncture for the results obtained from the two approaches. Life 2016-01-26 6 1 Review 10.3390/life6010006 6 2075-1729 2016-01-26 doi: 10.3390/life6010006 Kenji Ikehara http://www.mdpi.com/2075-1729/6/1/5 The editors of Life would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2015. Life 2016-01-21 6 1 Editorial 10.3390/life6010005 5 2075-1729 2016-01-21 doi: 10.3390/life6010005 Life Editorial Office http://www.mdpi.com/2075-1729/6/1/4 Transfer RNAs (tRNAs) are powerful small RNA entities that are used to translate nucleotide language of genes into the amino acid language of proteins. Their near-uniform length and tertiary structure as well as their high nucleotide similarity and post-transcriptional modifications have made it difficult to characterize individual species quantitatively. However, due to the central role of the tRNA pool in protein biosynthesis as well as newly emerging roles played by tRNAs, their quantitative assessment yields important information, particularly relevant for virus research. Viruses which depend on the host protein expression machinery have evolved various strategies to optimize tRNA usage—either by adapting to the host codon usage or encoding their own tRNAs. Additionally, several viruses bear tRNA-like elements (TLE) in the 5′- and 3′-UTR of their mRNAs. There are different hypotheses concerning the manner in which such structures boost viral protein expression. Furthermore, retroviruses use special tRNAs for packaging and initiating reverse transcription of their genetic material. Since there is a strong specificity of different viruses towards certain tRNAs, different strategies for recruitment are employed. Interestingly, modifications on tRNAs strongly impact their functionality in viruses. Here, we review those intersection points between virus and tRNA research and describe methods for assessing the tRNA pool in terms of concentration, aminoacylation and modification. Life 2016-01-19 6 1 Review 10.3390/life6010004 4 2075-1729 2016-01-19 doi: 10.3390/life6010004 Suki Albers Andreas Czech http://www.mdpi.com/2075-1729/6/1/3 Prominent in the L-shaped three-dimensional structure of tRNAs is the "elbow" where their two orthogonal helical stacks meet. It has a conserved structure arising from the interaction of the terminal loops of the D- and T-stem-loops, and presents to solution a flat face of a tertiary base pair between the D- and T-loops. In addition to the ribosome, which interacts with the elbow in all three of its tRNA binding sites, several cellular RNAs and many proteins are known to recognize the elbow. At least three classes of non-coding RNAs, namely 23S rRNA, ribonuclease P, and the T-box riboswitches, recognize the tRNA elbow employing an identical structural motif consisting of two interdigitated T-loops. In contrast, structural solutions to tRNA-elbow recognition by proteins are varied. Some enzymes responsible for post-transcriptional tRNA modification even disrupt the elbow structure in order to access their substrate nucleotides. The evolutionary origin of the elbow is mysterious, but, because it does not explicitly participate in the flow of genetic information, it has been proposed to be a late innovation. Regardless, it is biologically essential. Even some viruses that hijack the cellular machinery using tRNA decoys have convergently evolved near-perfect mimics of the tRNA elbow. Life 2016-01-12 6 1 Review 10.3390/life6010003 3 2075-1729 2016-01-12 doi: 10.3390/life6010003 Jinwei Zhang Adrian Ferré-D'Amaré http://www.mdpi.com/2075-1729/6/1/2 Aminoacyl-tRNA protein transferases catalyze the transfer of amino acids from aminoacyl-tRNAs to polypeptide substrates. Different forms of these enzymes are found in the different kingdoms of life and have been identified to be central to a wide variety of cellular processes. L/F-transferase is the sole member of this class of enzyme found in Escherichia coli and catalyzes the transfer of leucine to the N-termini of proteins which result in the targeted degradation of the modified protein. Recent investigations on the tRNA specificity of L/F-transferase have revealed the unique recognition nucleotides for a preferred Leu-tRNALeu isoacceptor substrate. In addition to discussing this tRNA selectivity by L/F-transferase, we present and discuss a hypothesis and its implications regarding the apparent competition for this aminoacyl-tRNA between L/F-transferase and the translational machinery. Our discussion reveals a hypothetical involvement of the bacterial stringent response that occurs upon amino acid limitation as a potential cellular event that may reduce this competition and provide the opportunity for L/F-transferase to readily increase its access to the pool of aminoacylated tRNA substrates. Life 2015-12-31 6 1 Review 10.3390/life6010002 2 2075-1729 2015-12-31 doi: 10.3390/life6010002 Angela Fung Roshani Payoe Richard Fahlman http://www.mdpi.com/2075-1729/6/1/1 How could modern life have evolved? The answer to that question still remains unclear. However, evidence is growing that, since the origin of life, RNA could have played an important role throughout evolution, right up to the development of complex organisms and even highly sophisticated features such as human cognition. RNA mediated RNA-aminoacylation can be seen as a first landmark on the path from the RNA world to modern DNA- and protein-based life. Likewise, the generation of the RNA modifications that can be found in various RNA species today may already have started in the RNA world, where such modifications most likely entailed functional advantages. This association of modification patterns with functional features was apparently maintained throughout the further course of evolution, and particularly tRNAs can now be seen as paradigms for the developing interdependence between structure, modification and function. It is in this spirit that this review highlights important stepping stones of the development of (t)RNAs and their modifications (including aminoacylation) from the ancient RNA world up until their present role in the development and maintenance of human cognition. The latter can be seen as a high point of evolution at its present stage, and the susceptibility of cognitive features to even small alterations in the proper structure and functioning of tRNAs underscores the evolutionary relevance of this RNA species. Life 2015-12-23 6 1 Review 10.3390/life6010001 1 2075-1729 2015-12-23 doi: 10.3390/life6010001 Darko Balke Andreas Kuss Sabine Müller http://www.mdpi.com/2075-1729/5/4/1703 Aminoacyl-tRNA synthetases (aaRSs) are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories. Life 2015-12-08 5 4 Review 10.3390/life5041703 1703 1725 2075-1729 2015-12-08 doi: 10.3390/life5041703 Pengfei Fang Min Guo http://www.mdpi.com/2075-1729/5/4/1700 n/a Life 2015-12-08 5 4 Editorial 10.3390/life5041700 1700 1702 2075-1729 2015-12-08 doi: 10.3390/life5041700 Pasquale Stano Fabio Mavelli http://www.mdpi.com/2075-1729/5/4/1687 Modern transfer RNAs (tRNAs) are composed of ~76 nucleotides and play an important role as "adaptor" molecules that mediate the translation of information from messenger RNAs (mRNAs). Many studies suggest that the contemporary full-length tRNA was formed by the ligation of half-sized hairpin-like RNAs. A minihelix (a coaxial stack of the acceptor stem on the T-stem of tRNA) can function both in aminoacylation by aminoacyl tRNA synthetases and in peptide bond formation on the ribosome, indicating that it may be a vestige of the ancestral tRNA. The universal CCA-3′ terminus of tRNA is also a typical characteristic of the molecule. "Why CCA?" is the fundamental unanswered question, but several findings give a comprehensive picture of its origin. Here, the origins and early evolution of tRNA are discussed in terms of various perspectives, including nucleotide ligation, chiral selectivity of amino acids, genetic code evolution, and the organization of the ribosomal peptidyl transferase center (PTC). The proto-tRNA molecules may have evolved not only as adaptors but also as contributors to the composition of the ribosome. Life 2015-12-03 5 4 Review 10.3390/life5041687 1687 1699 2075-1729 2015-12-03 doi: 10.3390/life5041687 Koji Tamura http://www.mdpi.com/2075-1729/5/4/1652 Among all known microbes capable of thriving under extreme and, therefore, potentially extraterrestrial environmental conditions, methanogens from the domain Archaea are intriguing organisms. This is due to their broad metabolic versatility, enormous diversity, and ability to grow under extreme environmental conditions. Several studies revealed that growth conditions of methanogens are compatible with environmental conditions on extraterrestrial bodies throughout the Solar System. Hence, life in the Solar System might not be limited to the classical habitable zone. In this contribution we assess the main ecophysiological characteristics of methanogens and compare these to the environmental conditions of putative habitats in the Solar System, in particular Mars and icy moons. Eventually, we give an outlook on the feasibility and the necessity of future astrobiological studies concerning methanogens. Life 2015-12-03 5 4 Review 10.3390/life5041652 1652 1686 2075-1729 2015-12-03 doi: 10.3390/life5041652 Ruth-Sophie Taubner Christa Schleper Maria Firneis Simon Rittmann http://www.mdpi.com/2075-1729/5/4/1638 This review will summarise the recent discoveries and current state of research on short noncoding RNAs derived from tRNAs—known as tRNA-derived fragments (tRFs). It will describe the features of the known subtypes of these RNAs; including sequence characteristics, protein interactors, expression characteristics, biogenesis, and similarity to canonical miRNA pathways. Also their role in regulating gene expression; including mediating translational suppression, will be discussed. We also highlight their potential use as biomarkers, functions in gene regulation and links to disease. Finally, this review will speculate as to the origin and rationale for the conservation of this novel class of noncoding RNAs amongst both prokaryotes and eukaryotes. Life 2015-11-27 5 4 Review 10.3390/life5041638 1638 1651 2075-1729 2015-11-27 doi: 10.3390/life5041638 Simon Keam Gyorgy Hutvagner http://www.mdpi.com/2075-1729/5/4/1629 Three aspects which make planet Earth special, and which must be taken in consideration with respect to the emergence of peptides, are the mineralogical composition, the Moon which is in the same size class, and the triple environment consisting of ocean, atmosphere, and continent. GlyGly is a remarkable peptide because it stimulates peptide bond formation in the Salt-Induced Peptide Formation reaction. The role glycine and aspartic acid play in the active site of RNA polymerase is remarkable too. GlyGly might have been the original product of coded peptide synthesis because of its importance in stimulating the production of oligopeptides with a high aspartic acid content, which protected small RNA molecules by binding Mg2+ ions. The feedback loop, which is closed by having RNA molecules producing GlyGly, is proposed as the essential element fundamental to life. Having this system running, longer sequences could evolve, gradually solving the problem of error catastrophe. The basic structure of the standard genetic code (8 fourfold degenerate codon boxes and 8 split codon boxes) is an example of the way information concerning the emergence of life is frozen in the biological constitution of organisms: the structure of the code contains historical information. Life 2015-11-16 5 4 Review 10.3390/life5041629 1629 1637 2075-1729 2015-11-16 doi: 10.3390/life5041629 Peter Gulik http://www.mdpi.com/2075-1729/5/4/1610 The essential feature of the genetic code is the strict one-to-one correspondence between codons and amino acids. The canonical code consists of three stop codons and 61 sense codons that encode 20% of the amino acid repertoire observed in nature. It was originally designated as immutable and universal due to its conservation in most organisms, but sequencing of genes from the human mitochondrial genomes revealed deviations in codon assignments. Since then, alternative codes have been reported in both nuclear and mitochondrial genomes and genetic code engineering has become an important research field. Here, we review the most recent concepts arising from the study of natural non-standard genetic codes with special emphasis on codon re-assignment strategies that are relevant to engineering genetic code in the laboratory. Recent tools for synthetic biology and current attempts to engineer new codes for incorporation of non-standard amino acids are also reviewed in this article. Life 2015-11-12 5 4 Review 10.3390/life5041610 1610 1628 2075-1729 2015-11-12 doi: 10.3390/life5041610 Ana Bezerra Ana Guimarães Manuel Santos http://www.mdpi.com/2075-1729/5/4/1587 Recent studies claiming to revive ancient microorganisms trapped in fluid inclusions in halite have warranted an investigation of long-term microbial persistence. While starvation-survival is widely reported for bacteria, it is less well known for halophilic archaea—microorganisms likely to be trapped in ancient salt crystals. To better understand microbial survival in fluid inclusions in ancient evaporites, laboratory experiments were designed to simulate growth of halophilic archaea under media-rich conditions, complete nutrient deprivation, and a controlled substrate condition (glycerol-rich) and record their responses. Haloarchaea used for this work included Hbt. salinarum and isolate DV582A-1 (genus Haloterrigena) sub-cultured from 34 kyear Death Valley salt. Hbt. salinarum and DV582A-1 reacted to nutrient limitation with morphological and population changes. Starved populations increased and most cells converted from rods to small cocci within 56 days of nutrient deprivation. The exact timing of starvation adaptations and the physical transformations differed between species, populations of the same species, and cells of the same population. This is the first study to report the timing of starvation strategies for Hbt. salinarum and DV582A-1. The morphological states in these experiments may allow differentiation between cells trapped with adequate nutrients (represented here by early stages in nutrient-rich media) from cells trapped without nutrients (represented here by experimental starvation) in ancient salt. The hypothesis that glycerol, leaked from Dunaliella, provides nutrients for the survival of haloarchaea trapped in fluid inclusions in ancient halite, is also tested. Hbt. salinarum and DV582A-1 were exposed to a mixture of lysed and intact Dunaliella for 56 days. The ability of these organisms to utilize glycerol from Dunaliella cells was assessed by documenting population growth, cell length, and cell morphology. Hbt. salinarum and DV582A-1 experienced size reductions and shape transitions from rods to cocci. In the short-term, these trends more closely resembled the response of these organisms to starvation conditions than to nutrient-rich media. Results from this experiment reproduced the physical state of cells (small cocci) in ancient halite where prokaryotes co-exist with single-celled algae. We conclude that glycerol is not the limiting factor in the survival of haloarchaea for thousands of years in fluid inclusions in halite. Life 2015-11-12 5 4 Article 10.3390/life5041587 1587 1609 2075-1729 2015-11-12 doi: 10.3390/life5041587 Yaicha Winters Tim Lowenstein Michael Timofeeff http://www.mdpi.com/2075-1729/5/4/1583 The RNA World is now some four billion years behind us, but only recently turned 50 as a human hypothesis. [...] Life 2015-10-22 5 4 Editorial 10.3390/life5041583 1583 1586 2075-1729 2015-10-22 doi: 10.3390/life5041583 Niles Lehman http://www.mdpi.com/2075-1729/5/4/1567 The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient antitermination. Life 2015-09-28 5 4 Article 10.3390/life5041567 1567 1582 2075-1729 2015-09-28 doi: 10.3390/life5041567 Liang-Chun Liu Frank Grundy Tina Henkin http://www.mdpi.com/2075-1729/5/3/1539 The functionality of the plasma membrane is essential for all organisms. Adaption to high growth temperatures imposes challenges and Bacteria, Eukarya, and Archaea have developed several mechanisms to cope with these. Hyperthermophilic archaea have earlier been shown to synthesize tetraether membrane lipids with an increased number of cyclopentane moieties at higher growth temperatures. Here we used shotgun lipidomics to study this effect as well as the influence of growth phase on the lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii for the first time. Both species were cultivated at three different temperatures, with samples withdrawn during lag, exponential, and stationary phases. Three abundant tetraether lipid classes and one diether lipid class were monitored. Beside the expected increase in the number of cyclopentane moieties with higher temperature in both archaea, we observed previously unreported changes in the average cyclization of the membrane lipids throughout growth. The average number of cyclopentane moieties showed a significant dip in exponential phase, an observation that might help to resolve the currently debated biosynthesis pathway of tetraether lipids. Life 2015-08-25 5 3 Article 10.3390/life5031539 1539 1566 2075-1729 2015-08-25 doi: 10.3390/life5031539 Sara Jensen Vinnie Neesgaard Sandra Skjoldbjerg Martin Brandl Christer Ejsing Alexander Treusch http://www.mdpi.com/2075-1729/5/3/1518 Recent evidence suggests that quantum mechanics is relevant in photosynthesis, magnetoreception, enzymatic catalytic reactions, olfactory reception, photoreception, genetics, electron-transfer in proteins, and evolution; to mention few. In our recent paper published in Life, we have derived the operator-sum representation of a biological channel based on codon basekets, and determined the quantum channel model suitable for study of the quantum biological channel capacity. However, this model is essentially memoryless and it is not able to properly model the propagation of mutation errors in time, the process of aging, and evolution of genetic information through generations. To solve for these problems, we propose novel quantum mechanical models to accurately describe the process of creation spontaneous, induced, and adaptive mutations and their propagation in time. Different biological channel models with memory, proposed in this paper, include: (i) Markovian classical model, (ii) Markovian-like quantum model, and (iii) hybrid quantum-classical model. We then apply these models in a study of aging and evolution of quantum biological channel capacity through generations. We also discuss key differences of these models with respect to a multilevel symmetric channel-based Markovian model and a Kimura model-based Markovian process. These models are quite general and applicable to many open problems in biology, not only biological channel capacity, which is the main focus of the paper. We will show that the famous quantum Master equation approach, commonly used to describe different biological processes, is just the first-order approximation of the proposed quantum Markov chain-like model, when the observation interval tends to zero. One of the important implications of this model is that the aging phenotype becomes determined by different underlying transition probabilities in both programmed and random (damage) Markov chain-like models of aging, which are mutually coupled. Life 2015-08-24 5 3 Article 10.3390/life5031518 1518 1538 2075-1729 2015-08-24 doi: 10.3390/life5031518 Ivan Djordjevic http://www.mdpi.com/2075-1729/5/3/1497 The notion of fitness landscapes, a map between genotype and fitness, was proposed more than 80 years ago. For most of this time data was only available for a few alleles, and thus we had only a restricted view of the whole fitness landscape. Recently, advances in genetics and molecular biology allow a more detailed view of them. Here we review experimental and theoretical studies of fitness landscapes of functional RNAs, especially aptamers and ribozymes. We find that RNA structures can be divided into critical structures, connecting structures, neutral structures and forbidden structures. Such characterisation, coupled with theoretical sequence-to-structure predictions, allows us to construct the whole fitness landscape. Fitness landscapes then can be used to study evolution, and in our case the development of the RNA world. Life 2015-08-21 5 3 Review 10.3390/life5031497 1497 1517 2075-1729 2015-08-21 doi: 10.3390/life5031497 Ádám Kun Eörs Szathmáry http://www.mdpi.com/2075-1729/5/3/1487 Halophilic archaebacteria (Haloarchaea) can survive extreme desiccation, starvation and radiation, sometimes apparently for millions of years. Several of the strategies that are involved appear specific for Haloarchaea (for example, the formation of halomucin, survival in fluid inclusions of halite), and some are known from other prokaryotes (dwarfing of cells, reduction of ATP). Several newly-discovered haloarchaeal strategies that were inferred to possibly promote long-term survival—halomucin, polyploidy, usage of DNA as a phosphate storage polymer, production of spherical dormant stages—remain to be characterized in detail. More information on potential strategies is desirable, since evidence for the presence of halite on Mars and on several moons in the solar system increased interest in halophiles with respect to the search for extraterrestrial life. This review deals in particular with novel findings and hypotheses on haloarchaeal long-term survival. Life 2015-07-28 5 3 Review 10.3390/life5031487 1487 1496 2075-1729 2015-07-28 doi: 10.3390/life5031487 Helga Stan-Lotter Sergiu Fendrihan http://www.mdpi.com/2075-1729/5/3/1472 Life on Earth displays an incredible diversity in form and function, which allows it to survive not only physical extremes, but also periods of time when it is exposed to non-habitable conditions. Extreme physiological adaptations to bridge non-habitable conditions include various dormant states, such as spores or tuns. Here, we advance the hypothesis that if the environmental conditions are different on some other planetary body, a deviating biochemistry would evolve with types of adaptations that would manifest themselves with different physical and chemical limits of life. In this paper, we discuss two specific examples: putative life on a Mars-type planet with a hydrogen peroxide-water solvent and putative life on a Titan-type planetary body with liquid hydrocarbons as a solvent. Both examples would have the result of extending the habitable envelope of life in the universe. Life 2015-07-17 5 3 Hypothesis 10.3390/life5031472 1472 1486 2075-1729 2015-07-17 doi: 10.3390/life5031472 Dirk Schulze-Makuch Alexander Schulze-Makuch Joop Houtkooper http://www.mdpi.com/2075-1729/5/2/1454 Acetate and acetyl-CoA play fundamental roles in all of biology, including anaerobic prokaryotes from the domains Bacteria and Archaea, which compose an estimated quarter of all living protoplasm in Earth's biosphere. Anaerobes from the domain Archaea contribute to the global carbon cycle by metabolizing acetate as a growth substrate or product. They are components of anaerobic microbial food chains converting complex organic matter to methane, and many fix CO2 into cell material via synthesis of acetyl-CoA. They are found in a diversity of ecological habitats ranging from the digestive tracts of insects to deep-sea hydrothermal vents, and synthesize a plethora of novel enzymes with biotechnological potential. Ecological investigations suggest that still more acetate-metabolizing species with novel properties await discovery. Life 2015-06-09 5 2 Review 10.3390/life5021454 1454 1471 2075-1729 2015-06-09 doi: 10.3390/life5021454 James Ferry http://www.mdpi.com/2075-1729/5/2/1445 How did life emerge on Earth? The aim of the Network of Researchers on Horizontal Gene Transfer and the Last Universal Cellular Ancestor (NoR HGT & LUCA) is to understand how the genetics of LUCAs were reorganised prior to the advent of the three domains of life. This paper reports the research of eminent scientists who have come together within the network and are making significant contributions to the wider knowledge base surrounding this, one of science's remaining mysteries. I also report on their relevance in relation to LUCAs and life's origins, as well as ask a question: what next? Life 2015-06-05 5 2 Conference Report 10.3390/life5021445 1445 1453 2075-1729 2015-06-05 doi: 10.3390/life5021445 Sohan Jheeta http://www.mdpi.com/2075-1729/5/2/1427 Genome annotation errors are a persistent problem that impede research in the biosciences. A manual curation effort is described that attempts to produce high-quality genome annotations for a set of haloarchaeal genomes (Halobacterium salinarum and Hbt. hubeiense, Haloferax volcanii and Hfx. mediterranei, Natronomonas pharaonis and Nmn. moolapensis, Haloquadratum walsbyi strains HBSQ001 and C23, Natrialba magadii, Haloarcula marismortui and Har. hispanica, and Halohasta litchfieldiae). Genomes are checked for missing genes, start codon misassignments, and disrupted genes. Assignments of a specific function are preferably based on experimentally characterized homologs (Gold Standard Proteins). To avoid overannotation, which is a major source of database errors, we restrict annotation to only general function assignments when support for a specific substrate assignment is insufficient. This strategy results in annotations that are resistant to the plethora of errors that compromise public databases. Annotation consistency is rigorously validated for ortholog pairs from the genomes surveyed. The annotation is regularly crosschecked against the UniProt database to further improve annotations and increase the level of standardization. Enhanced genome annotations are submitted to public databases (EMBL/GenBank, UniProt), to the benefit of the scientific community. The enhanced annotations are also publically available via HaloLex. Life 2015-06-02 5 2 Article 10.3390/life5021427 1427 1444 2075-1729 2015-06-02 doi: 10.3390/life5021427 Friedhelm Pfeiffer Dieter Oesterhelt http://www.mdpi.com/2075-1729/5/2/1405 The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria. Life 2015-05-19 5 2 Review 10.3390/life5021405 1405 1426 2075-1729 2015-05-19 doi: 10.3390/life5021405 R. Papke Paulina Corral Nikhil Ram-Mohan Rafael Haba Cristina Sánchez-Porro Andrea Makkay Antonio Ventosa http://www.mdpi.com/2075-1729/5/2/1396 In their natural habitats, microorganisms are often exposed to periods of starvation if their substrates for energy generation or other nutrients are limiting. Many microorganisms have developed strategies to adapt to fluctuating nutrients and long-term starvation. In the environment, ammonia oxidizers have to compete with many different organisms for ammonium and are often exposed to long periods of ammonium starvation. We investigated the effect of ammonium starvation on ammonia-oxidizing archaea (AOA) and bacteria (AOB) enriched from freshwater lake sediments. Both AOA and AOB were able to recover even after almost two months of starvation; however, the recovery time differed. AOA and AOB retained their 16S rRNA (ribosomes) throughout the complete starvation period. The AOA retained also a small portion of the mRNA of the ammonia monooxygenase subunit A (amoA) for the complete starvation period. However, after 10 days, no amoA mRNA was detected anymore in the AOB. These results indicate that AOA and AOB are able to survive longer periods of starvation, but might utilize different strategies. Life 2015-05-19 5 2 Article 10.3390/life5021396 1396 1404 2075-1729 2015-05-19 doi: 10.3390/life5021396 Elizabeth French Annette Bollmann http://www.mdpi.com/2075-1729/5/2/1381 Due to the limited cultivability of the vast majority of microorganisms, researchers have applied environmental genomics and other state-of-the-art technologies to gain insights into the biology of uncultivated Archaea and bacteria in their natural biotope. In this review, we summarize the scientific findings on a recently proposed order-level lineage of uncultivated Archaea called Altiarchaeales, which includes "Candidatus Altiarchaeum hamiconexum" as the most well-described representative. Ca. A. hamiconexum possesses a complex biology: thriving strictly anaerobically, this microorganism is capable of forming highly-pure biofilms, connecting the cells by extraordinary cell surface appendages (the "hami") and has other highly unusual traits, such as a double-membrane-based cell wall. Indicated by genomic information from different biotopes, the Altiarchaeales seem to proliferate in deep, anoxic groundwater of Earth's crust bearing a potentially very important function: carbon fixation. Although their net carbon fixation rate has not yet been determined, they appear as highly abundant organisms in their biotopes and may thus represent an important primary producer in the subsurface. In sum, the research over more than a decade on Ca. A. hamiconexum has revealed many interesting features of its lifestyle, its genomic information, metabolism and ultrastructure, making this archaeon one of the best-studied uncultivated Archaea in the literature. Life 2015-05-12 5 2 Review 10.3390/life5021381 1381 1395 2075-1729 2015-05-12 doi: 10.3390/life5021381 Alexander Probst Christine Moissl-Eichinger http://www.mdpi.com/2075-1729/5/2/1346 Missisquoi Bay (MB) is a temperate eutrophic freshwater lake that frequently experiences toxic Microcystis-dominated cyanobacterial blooms. Non-point sources are responsible for the high concentrations of phosphorus and nitrogen in the bay. This study combined data from environmental parameters, E. coli counts, high-throughput sequencing of 16S rRNA gene amplicons, quantitative PCR (16S rRNA and mcyD genes) and toxin analyses to identify the main bloom-promoting factors. In 2009, nutrient concentrations correlated with E. coli counts, abundance of total cyanobacterial cells, Microcystis 16S rRNA and mcyD genes and intracellular microcystin. Total and dissolved phosphorus also correlated significantly with rainfall. The major cyanobacterial taxa were members of the orders Chroococcales and Nostocales. The genus Microcystis was the main mcyD-carrier and main microcystin producer. Our results suggested that increasing nutrient concentrations and total nitrogen:total phosphorus (TN:TP) ratios approaching 11:1, coupled with an increase in temperature, promoted Microcystis-dominated toxic blooms. Although the importance of nutrient ratios and absolute concentrations on cyanobacterial and Microcystis dynamics have been documented in other laboratories, an optimum TN:TP ratio for Microcystis dominance has not been previously observed in situ. This observation provides further support that nutrient ratios are an important determinant of species composition in natural phytoplankton assemblages. Life 2015-05-12 5 2 Article 10.3390/life5021346 1346 1380 2075-1729 2015-05-12 doi: 10.3390/life5021346 Nathalie Fortin Valentina Munoz-Ramos David Bird Benoît Lévesque Lyle Whyte Charles Greer http://www.mdpi.com/2075-1729/5/2/1333 Archaea are well-recognized components of the human microbiome. However, they appear to be drastically underrepresented compared to the high diversity of bacterial taxa which can be found on various human anatomic sites, such as the gastrointestinal environment, the oral cavity and the skin. As our "microbial" view of the human body, including the methodological concepts used to describe them, has been traditionally biased on bacteria, the question arises whether our current knowledge reflects the actual ratio of archaea versus bacteria or whether we have failed so far to unravel the full diversity of human-associated archaea. This review article hypothesizes that distinct archaeal lineages within humans exist, which still await our detection. First, previously unrecognized taxa might be quite common but they have eluded conventional detection methods. Two recent prime examples are described that demonstrate that this might be the case for specific archaeal lineages. Second, some archaeal taxa might be overlooked because they are rare and/or in low abundance. Evidence for this exists for a broad range of phylogenetic lineages, however we currently do not know whether these sporadically appearing organisms are mere transients or important members of the so called "rare biosphere" with probably basic ecosystem functions. Lastly, evidence exists that different human populations harbor different archaeal taxa and/or the abundance and activity of shared archaeal taxa may differ and thus their impact on the overall microbiome. This research line is rather unexplored and warrants further investigation. While not recapitulating exhaustively all studies on archaeal diversity in humans, this review highlights pertinent recent findings that show that the choice of appropriate methodological approaches and the consideration of different human populations may lead to the detection of archaeal lineages previously not associated with humans. This in turn will help understand variations found in the overall microbiomes from different individuals and ultimately may lead to the emergence of novel concepts/mechanisms impacting human health. Life 2015-05-05 5 2 Review 10.3390/life5021333 1333 1345 2075-1729 2015-05-05 doi: 10.3390/life5021333 Hans-Peter Horz http://www.mdpi.com/2075-1729/5/2/1301 The standard genetic code (SGC) is central to molecular biology and its origin and evolution is a fundamental problem in evolutionary biology, the elucidation of which promises to reveal much about the origins of life. In addition, we propose that study of its origin can also reveal some fundamental and generalizable insights into mechanisms of molecular evolution, utilizing concepts from complexity theory. The first is that beneficial traits may arise by non-adaptive processes, via a process of "neutral emergence". The structure of the SGC is optimized for the property of error minimization, which reduces the deleterious impact of point mutations. Via simulation, it can be shown that genetic codes with error minimization superior to the SGC can emerge in a neutral fashion simply by a process of genetic code expansion via tRNA and aminoacyl-tRNA synthetase duplication, whereby similar amino acids are added to codons related to that of the parent amino acid. This process of neutral emergence has implications beyond that of the genetic code, as it suggests that not all beneficial traits have arisen by the direct action of natural selection; we term these "pseudaptations", and discuss a range of potential examples. Secondly, consideration of genetic code deviations (codon reassignments) reveals that these are mostly associated with a reduction in proteome size. This code malleability implies the existence of a proteomic constraint on the genetic code, proportional to the size of the proteome (P), and that its reduction in size leads to an "unfreezing" of the codon – amino acid mapping that defines the genetic code, consistent with Crick's Frozen Accident theory. The concept of a proteomic constraint may be extended to propose a general informational constraint on genetic fidelity, which may be used to explain variously, differences in mutation rates in genomes with differing proteome sizes, differences in DNA repair capacity and genome GC content between organisms, a selective pressure in the evolution of sexual reproduction, and differences in translational fidelity. Lastly, the utility of the concept of an informational constraint to other diverse fields of research is explored. Life 2015-04-24 5 2 Article 10.3390/life5021301 1301 1332 2075-1729 2015-04-24 doi: 10.3390/life5021301 Steven Massey http://www.mdpi.com/2075-1729/5/2/1282 Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion. Life 2015-04-23 5 2 Article 10.3390/life5021282 1282 1300 2075-1729 2015-04-23 doi: 10.3390/life5021282 Rafael Pernil Silvia Picossi Antonia Herrero Enrique Flores Vicente Mariscal http://www.mdpi.com/2075-1729/5/2/1264 Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism. Sm-based regulation is diverse and can range in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in these processes being mediated by an RNA-associated molecular assembly built on Sm proteins. Despite the availability of several 3D-structures of Sm-like archaeal proteins (SmAPs), their function has remained elusive. The aim of this study was to shed light on the function of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso). Using co-purification followed by RNASeq different classes of non-coding RNAs and mRNAs were identified that co-purified either with both paralogues or solely with Sso-SmAP1 or Sso-SmAP2. The large number of associated intron-containing tRNAs and tRNA/rRNA modifying RNAs may suggest a role of the two Sso-SmAPs in tRNA/rRNA processing. Moreover, the 3D structure of Sso-SmAP2 was elucidated. Like Sso-SmAP1, Sso-SmAP2 forms homoheptamers. The binding of both proteins to distinct RNA substrates is discussed in terms of surface conservation, structural differences in the RNA binding sites and differences in the electrostatic surface potential of the two Sso-SmAP proteins. Taken together, this study may hint to common and different functions of both Sso-SmAPs in Sso RNA metabolism. Life 2015-04-21 5 2 Article 10.3390/life5021264 1264 1281 2075-1729 2015-04-21 doi: 10.3390/life5021264 Birgit Märtens Gustavo Bezerra Mathias Kreuter Irina Grishkovskaya Andrea Manica Valentina Arkhipova Kristina Djinovic-Carugo Udo Bläsi http://www.mdpi.com/2075-1729/5/2/1239 Contemporary biological cells are highly sophisticated dynamic compartment systems which separate an internal volume from the external medium through a boundary, which controls, in complex ways, the exchange of matter and energy between the cell's interior and the environment. Since such compartmentalization is a fundamental principle of all forms of life, scenarios have been elaborated about the emergence of prebiological compartments on early Earth, in particular about their likely structural characteristics and dynamic features. Chemical systems that consist of potentially prebiological compartments and chemical reaction networks have been designed to model pre-cellular systems. These systems are often referred to as "protocells". Past and current protocell model systems are presented and compared. Since the prebiotic formation of cell-like compartments is directly linked to the prebiotic availability of compartment building blocks, a few aspects on the likely chemical inventory on the early Earth are also summarized. Life 2015-04-10 5 2 Review 10.3390/life5021239 1239 1263 2075-1729 2015-04-10 doi: 10.3390/life5021239 Pierre-Alain Monnard Peter Walde http://www.mdpi.com/2075-1729/5/2/1218 Exopolysaccharides (EPSs) are an important class of biopolymers with great ecological importance. In natural environments, they are a common feature of microbial biofilms, where they play key protective and structural roles. As the primary colonizers of constrained environments, such as desert soils and lithic and exposed substrates, cyanobacteria are the first contributors to the synthesis of the EPSs constituting the extracellular polymeric matrix that favors the formation of microbial associations with varying levels of complexity called biofilms. Cyanobacterial colonization represents the first step for the formation of biofilms with different levels of complexity. In all of the possible systems in which cyanobacteria are involved, the synthesis of EPSs contributes a structurally-stable and hydrated microenvironment, as well as chemical/physical protection against biotic and abiotic stress factors. Notwithstanding the important roles of cyanobacterial EPSs, many aspects related to their roles and the relative elicited biotic and abiotic factors have still to be clarified. The aim of this survey is to outline the state-of-the-art of the importance of the cyanobacterial EPS excretion, both for the producing cells and for the microbial associations in which cyanobacteria are a key component. Life 2015-04-01 5 2 Review 10.3390/life5021218 1218 1238 2075-1729 2015-04-01 doi: 10.3390/life5021218 Federico Rossi Roberto De Philippis http://www.mdpi.com/2075-1729/5/2/1204 We simulate pH-dependent growth of cyanobacteria with an ecosystem model for the central Baltic Sea. Four model components—a life cycle model of cyanobacteria, a biogeochemical model, a carbonate chemistry model and a water column model—are coupled via the framework for aquatic biogeochemical models. The coupled model is forced by the output of a regional climate model, based on the A1B emission scenario. With this coupled model, we perform simulations for the period 1968–2098. Our simulation experiments suggest that in the future, cyanobacteria growth is hardly affected by the projected pH decrease. However, in the simulation phase prior to 1980, cyanobacteria growth and N2-fixation are limited by the relatively high pH. The observed absence of cyanobacteria before the 1960s may thus be explained not only by lower eutrophication levels, but also by a higher alkalinity. Life 2015-03-30 5 2 Article 10.3390/life5021204 1204 1217 2075-1729 2015-03-30 doi: 10.3390/life5021204 Jana Hinners Richard Hofmeister Inga Hense http://www.mdpi.com/2075-1729/5/2/1172 Chlorophyll a (Chl) is a light-absorbing tetrapyrrole pigment that is essential for photosynthesis. The molecule is produced from glutamate via a complex biosynthetic pathway comprised of at least 15 enzymatic steps. The first half of the Chl pathway is shared with heme biosynthesis, and the latter half, called the Mg-branch, is specific to Mg-containing Chl a. Bilin pigments, such as phycocyanobilin, are additionally produced from heme, so these light-harvesting pigments also share many common biosynthetic steps with Chl biosynthesis. Some of these common steps in the biosynthetic pathways of heme, Chl and bilins require molecular oxygen for catalysis, such as oxygen-dependent coproporphyrinogen III oxidase. Cyanobacteria thrive in diverse environments in terms of oxygen levels. To cope with Chl deficiency caused by low-oxygen conditions, cyanobacteria have developed elaborate mechanisms to maintain Chl production, even under microoxic environments. The use of enzymes specialized for low-oxygen conditions, such as oxygen-independent coproporphyrinogen III oxidase, constitutes part of a mechanism adapted to low-oxygen conditions. Another mechanism adaptive to hypoxic conditions is mediated by the transcriptional regulator ChlR that senses low oxygen and subsequently activates the transcription of genes encoding enzymes that work under low-oxygen tension. In diazotrophic cyanobacteria, this multilayered regulation also contributes in Chl biosynthesis by supporting energy production for nitrogen fixation that also requires low-oxygen conditions. We will also discuss the evolutionary implications of cyanobacterial tetrapyrrole biosynthesis and regulation, because low oxygen-type enzymes also appear to be evolutionarily older than oxygen-dependent enzymes. Life 2015-03-30 5 2 Review 10.3390/life5021172 1172 1203 2075-1729 2015-03-30 doi: 10.3390/life5021172 Yuichi Fujita Ryoma Tsujimoto Rina Aoki http://www.mdpi.com/2075-1729/5/2/1141 The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed. Life 2015-03-27 5 2 Article 10.3390/life5021141 1141 1171 2075-1729 2015-03-27 doi: 10.3390/life5021141 Fei Cai Zhicheng Dou Susan Bernstein Ryan Leverenz Eric Williams Sabine Heinhorst Jessup Shively Gordon Cannon Cheryl Kerfeld http://www.mdpi.com/2075-1729/5/2/1127 Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as "topologically important." Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as "functionally important" genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles. Life 2015-03-27 5 2 Article 10.3390/life5021127 1127 1140 2075-1729 2015-03-27 doi: 10.3390/life5021127 Hyun-Seob Song Ryan McClure Hans Bernstein Christopher Overall Eric Hill Alexander Beliaev http://www.mdpi.com/2075-1729/5/2/1111 Cyanobacterial lipid droplets (LDs) are packed with hydrophobic energy-dense compounds and have great potential for biotechnological expression and the compartmentalization of high value compounds. Nostoc punctiforme normally accumulates LDs containing neutral lipids, and small amounts of heptadecane, during the stationary phase of growth. In this study, we further enhanced heptadecane production in N. punctiforme by introducing extrachromosomal copies of aar/adc genes, and report the discovery of a putative novel lipase encoded by Npun_F5141, which further enhanced alkane production. Extra copies of all three genes in high light conditions resulted in a 16-fold higher accumulation of heptadecane compared to the wild type strain in the exponential phase. LD accumulation during exponential phase also increased massively to accommodate the heptadecane production. A large number of small, less fluorescent LDs were observed at the cell periphery in exponential growth phase, whereas fewer number of highly fluorescent, much larger LDs were localized towards the center of the cell in the stationary phase. These advances demonstrate that cyanobacterial LDs are an ideal model platform to make industrially relevant compounds, such as alkanes, during exponential growth, and provide insight into LD formation in cyanobacteria. Life 2015-03-26 5 2 Article 10.3390/life5021111 1111 1126 2075-1729 2015-03-26 doi: 10.3390/life5021111 Anantha Peramuna Ray Morton Michael Summers http://www.mdpi.com/2075-1729/5/2/1101 Thermally induced shape fluctuations were used to study elastic properties of giant vesicles composed of archaeal lipids C25,25-archetidyl (glucosyl) inositol and C25,25-archetidylinositol isolated from lyophilised Aeropyrum pernix K1 cells. Giant vesicles were created by electroformation in pure water environment. Stroboscopic illumination using a xenon flash lamp was implemented to remove the blur effect due to the finite integration time of the camera and to obtain an instant picture of the fluctuating vesicle shape. The mean weighted value of the bending elasticity modulus kc of the archaeal membrane determined from the measurements meeting the entire set of qualification criteria was (1.89 ± 0.18) × 10−19 J, which is similar to the values obtained for a membrane composed of the eukaryotic phospholipids SOPC (1.88 ± 0.17) × 10−19 J and POPC (2.00 ± 0.21) ´ 10−19 J. We conclude that membranes composed of archaeal lipids isolated from Aeropyrum pernix K1 cells have similar elastic properties as membranes composed of eukaryotic lipids. This fact, together with the importance of the elastic properties for the normal circulation through blood system, provides further evidence in favor of expectations that archaeal lipids could be appropriate for the design of drug delivery systems. Life 2015-03-26 5 2 Article 10.3390/life5021101 1101 1110 2075-1729 2015-03-26 doi: 10.3390/life5021101 Julia Genova Nataša Ulrih Veronika Kralj-Iglič Aleš Iglič Isak Bivas http://www.mdpi.com/2075-1729/5/2/1054 The components of life must survive in a cell long enough to perform their function in that cell. Because the rate of attack by water increases with temperature, we can, in principle, predict a maximum temperature above which an active terrestrial metabolism cannot function by analysis of the decomposition rates of the components of life, and comparison of those rates with the metabolites' minimum metabolic half-lives. The present study is a first step in this direction, providing an analytical framework and method, and analyzing the stability of 63 small molecule metabolites based on literature data. Assuming that attack by water follows a first order rate equation, we extracted decomposition rate constants from literature data and estimated their statistical reliability. The resulting rate equations were then used to give a measure of confidence in the half-life of the metabolite concerned at different temperatures. There is little reliable data on metabolite decomposition or hydrolysis rates in the literature, the data is mostly confined to a small number of classes of chemicals, and the data available are sometimes mutually contradictory because of varying reaction conditions. However, a preliminary analysis suggests that terrestrial biochemistry is limited to environments below ~150–180 °C. We comment briefly on why pressure is likely to have a small effect on this. Life 2015-03-26 5 2 Article 10.3390/life5021054 1054 1100 2075-1729 2015-03-26 doi: 10.3390/life5021054 William Bains Yao Xiao Changyong Yu http://www.mdpi.com/2075-1729/5/2/1019 The increasing ease of producing nucleic acids and proteins to specification offers potential for design and fabrication of artificial synthetic "organisms" with a myriad of possible capabilities. The prospects for these synthetic organisms are significant, with potential applications in diverse fields including synthesis of pharmaceuticals, sources of renewable fuel and environmental cleanup. Until now, artificial cell technology has been largely restricted to the modification and metabolic engineering of living unicellular organisms. This review discusses emerging possibilities for developing synthetic protocell "machines" assembled entirely from individual biological components. We describe a host of recent technological advances that could potentially be harnessed in design and construction of synthetic protocells, some of which have already been utilized toward these ends. More elaborate designs include options for building self-assembling machines by incorporating cellular transport and assembly machinery. We also discuss production in miniature, using microfluidic production lines. While there are still many unknowns in the design, engineering and optimization of protocells, current technologies are now tantalizingly close to the capabilities required to build the first prototype protocells with potential real-world applications. Life 2015-03-25 5 2 Review 10.3390/life5021019 1019 1053 2075-1729 2015-03-25 doi: 10.3390/life5021019 David Miller Jacqueline Gulbis http://www.mdpi.com/2075-1729/5/1/997 Photobiological production of H2 by cyanobacteria is considered to be an ideal source of renewable energy because the inputs, water and sunlight, are abundant. The products of photobiological systems are H2 and O2; the H2 can be used as the energy source of fuel cells, etc., which generate electricity at high efficiencies and minimal pollution, as the waste product is H2O. Overall, production of commercially viable algal fuels in any form, including biomass and biodiesel, is challenging, and the very few systems that are operational have yet to be evaluated. In this paper we will: briefly review some of the necessary conditions for economical production, summarize the reports of photobiological H2 production by cyanobacteria, present our schemes for future production, and discuss the necessity for further progress in the research needed to achieve commercially viable large-scale H2 production. Life 2015-03-18 5 1 Review 10.3390/life5010997 997 1018 2075-1729 2015-03-18 doi: 10.3390/life5010997 Hidehiro Sakurai Hajime Masukawa Masaharu Kitashima Kazuhito Inoue http://www.mdpi.com/2075-1729/5/1/969 How did primitive living cells originate? The formation of early cells, which were probably solute-filled vesicles capable of performing a rudimentary metabolism (and possibly self-reproduction), is still one of the big unsolved questions in origin of life. We have recently used lipid vesicles (liposomes) as primitive cell models, aiming at the study of the physical mechanisms for macromolecules encapsulation. We have reported that proteins and ribosomes can be encapsulated very efficiently, against statistical expectations, inside a small number of liposomes. Moreover the transcription-translation mixture, which realistically mimics a sort of minimal metabolic network, can be functionally reconstituted in liposomes owing to a self-concentration mechanism. Here we firstly summarize the recent advancements in this research line, highlighting how these results open a new vista on the phenomena that could have been important for the formation of functional primitive cells. Then, we present new evidences on the non-random entrapment of macromolecules (proteins, dextrans) in phospholipid vesicle, and in particular we show how enzymatic reactions can be accelerated because of the enhancement of their concentration inside liposomes. Life 2015-03-18 5 1 Article 10.3390/life5010969 969 996 2075-1729 2015-03-18 doi: 10.3390/life5010969 Erica D'Aguanno Emiliano Altamura Fabio Mavelli Alfred Fahr Pasquale Stano Pier Luisi http://www.mdpi.com/2075-1729/5/1/949 A tripartite comparison of Archaea phylogeny and taxonomy at and above the rank order is reported: (1) the whole-genome-based and alignment-free CVTree using 179 genomes; (2) the 16S rRNA analysis exemplified by the All-Species Living Tree with 366 archaeal sequences; and (3) the Second Edition of Bergey's Manual of Systematic Bacteriology complemented by some current literature. A high degree of agreement is reached at these ranks. From the newly proposed archaeal phyla, Korarchaeota, Thaumarchaeota, Nanoarchaeota and Aigarchaeota, to the recent suggestion to divide the class Halobacteria into three orders, all gain substantial support from CVTree. In addition, the CVTree helped to determine the taxonomic position of some newly sequenced genomes without proper lineage information. A few discrepancies between the CVTree and the 16S rRNA approaches call for further investigation. Life 2015-03-17 5 1 Article 10.3390/life5010949 949 968 2075-1729 2015-03-17 doi: 10.3390/life5010949 Guanghong Zuo Zhao Xu Bailin Hao http://www.mdpi.com/2075-1729/5/1/921 The sequence GCGATCGC (Highly Iterated Palindrome, HIP1) is commonly found in high frequency in cyanobacterial genomes. An important clue to its function may be the presence of two orphan DNA methyltransferases that recognize internal sequences GATC and CGATCG. An examination of genomes from 97 cyanobacteria, both free-living and obligate symbionts, showed that there are exceptional cases in which HIP1 is at a low frequency or nearly absent. In some of these cases, it appears to have been replaced by a different GC-rich palindromic sequence, alternate HIPs. When HIP1 is at a high frequency, GATC- and CGATCG-specific methyltransferases are generally present in the genome. When an alternate HIP is at high frequency, a methyltransferase specific for that sequence is present. The pattern of 1-nt deviations from HIP1 sequences is biased towards the first and last nucleotides, i.e., those distinguish CGATCG from HIP1. Taken together, the results point to a role of DNA methylation in the creation or functioning of HIP sites. A model is presented that postulates the existence of a GmeC-dependent mismatch repair system whose activity creates and maintains HIP sequences. Life 2015-03-17 5 1 Article 10.3390/life5010921 921 948 2075-1729 2015-03-17 doi: 10.3390/life5010921 Jeff Elhai http://www.mdpi.com/2075-1729/5/1/905 MicroRNAs are important regulatory small RNAs in many eukaryotes. Due to their small size and simple structure, they are readily innovated de novo. Throughout the evolution of animals, the emergence of novel microRNA families traces key morphological innovations. Here, we use a computational approach based on homology search and parsimony-based presence/absence analysis to draw a comprehensive picture of microRNA evolution in 159 animal species. We confirm previous observations regarding bursts of innovations accompanying the three rounds of genome duplications in vertebrate evolution and in the early evolution of placental mammals. With a much better resolution for the invertebrate lineage compared to large-scale studies, we observe additional bursts of innovation, e.g., in Rhabditoidea. More importantly, we see clear evidence that loss of microRNA families is not an uncommon phenomenon. The Enoplea may serve as a second dramatic example beyond the tunicates. The large-scale analysis presented here also highlights several generic technical issues in the analysis of very large gene families that will require further research. Life 2015-03-13 5 1 Article 10.3390/life5010905 905 920 2075-1729 2015-03-13 doi: 10.3390/life5010905 Jana Hertel Peter Stadler http://www.mdpi.com/2075-1729/5/1/888 Nitrogen starvation acclimation in non-diazotrophic cyanobacteria is characterized by a process termed chlorosis, where the light harvesting pigments are degraded and the cells gradually tune down photosynthetic and metabolic activities. The chlorosis response is governed by a complex and poorly understood regulatory network, which converges at the expression of the nblA gene, the triggering factor for phycobiliprotein degradation. This study established a method that allows uncoupling metabolic and redox-signals involved in nitrogen-starvation acclimation. Inhibition of glutamine synthetase (GS) by a precise dosage of l-methionine-sulfoximine (MSX) mimics the metabolic situation of nitrogen starvation. Addition of nitrate to such MSX-inhibited cells eliminates the associated redox-stress by enabling electron flow towards nitrate/nitrite reduction and thereby, prevents the induction of nblA expression and the associated chlorosis response. This study demonstrates that nitrogen starvation is perceived not only through metabolic signals, but requires a redox signal indicating over-reduction of PSI-reduced electron acceptors. It further establishes a cryptic role of nitrate/nitrite reductases as electron sinks to balance conditions of over-reduction. Life 2015-03-13 5 1 Article 10.3390/life5010888 888 904 2075-1729 2015-03-13 doi: 10.3390/life5010888 Alexander Klotz Edgar Reinhold Sofía Doello Karl Forchhammer http://www.mdpi.com/2075-1729/5/1/872 Hydrothermal fields on the prebiotic Earth are candidate environments for biogenesis. We propose a model in which molecular systems driven by cycles of hydration and dehydration in such sites undergo chemical evolution in dehydrated films on mineral surfaces followed by encapsulation and combinatorial selection in a hydrated bulk phase. The dehydrated phase can consist of concentrated eutectic mixtures or multilamellar liquid crystalline matrices. Both conditions organize and concentrate potential monomers and thereby promote polymerization reactions that are driven by reduced water activity in the dehydrated phase. In the case of multilamellar lipid matrices, polymers that have been synthesized are captured in lipid vesicles upon rehydration to produce a variety of molecular systems. Each vesicle represents a protocell, an "experiment" in a natural version of combinatorial chemistry. Two kinds of selective processes can then occur. The first is a physical process in which relatively stable molecular systems will be preferentially selected. The second is a chemical process in which rare combinations of encapsulated polymers form systems capable of capturing energy and nutrients to undergo growth by catalyzed polymerization. Given continued cycling over extended time spans, such combinatorial processes will give rise to molecular systems having the fundamental properties of life. Life 2015-03-13 5 1 Article 10.3390/life5010872 872 887 2075-1729 2015-03-13 doi: 10.3390/life5010872 Bruce Damer David Deamer http://www.mdpi.com/2075-1729/5/1/861 Acetate kinase (ACK), which catalyzes the reversible phosphorylation of acetate by ATP, is a member of the acetate and sugar kinase/heat shock cognate/actin (ASKHA) superfamily. ASKHA family members share a common core fold that includes an ATPase domain with five structural motifs. The PHOSPHATE1 motif has previously been shown to be important for catalysis. We have investigated the role of two of these motifs in the Methanosarcina thermophila ACK (MtACK) and have shown that residues projecting into the ACK active site from the PHOSPHATE2 and ADENOSINE loops and a third highly conserved loop designated here as LOOP3 play key roles in nucleotide triphosphate (NTP) selection and utilization. Alteration of Asn211 of PHOSPHATE2, Gly239 of LOOP3, and Gly331 of ADENOSINE greatly reduced catalysis. In particular, Gly331, which is highly conserved throughout the ASKHA superfamily, has the greatest effect on substrate selection. Alteration at this site strongly skewed MtACK toward utilization of purines over pyrimidines, unlike the wild type enzyme that shows broad NTP utilization. Further investigation into differences between the ATPase domain in MtACK and other acetate kinases that show different substrate preferences will provide us with a better understanding of the diversity of phosphoryl donor selection in this enzyme family. Life 2015-03-12 5 1 Communication 10.3390/life5010861 861 871 2075-1729 2015-03-12 doi: 10.3390/life5010861 Cheryl Ingram-Smith Jeffrey Wharton Christian Reinholz Tara Doucet Rachel Hesler Kerry Smith http://www.mdpi.com/2075-1729/5/1/841 In this contribution, we address the question of iron bioavailability to cyanobacteria by measuring Fe uptake rates and probing for a reductive uptake pathway in diverse cyanobacterial species. We examined three Fe-substrates: dissolved inorganic iron (Fe') and the Fe-siderophores Ferrioxamine B (FOB) and FeAerobactin (FeAB). In order to compare across substrates and strains, we extracted uptake rate constants (kin = uptake rate/[Fe-substrate]). Fe' was the most bioavailable Fe form to cyanobacteria, with kin values higher than those of other substrates. When accounting for surface area (SA), all strains acquired Fe' at similar rates, as their kin/SA were similar. We also observed homogeneity in the uptake of FOB among strains, but with 10,000 times lower kin/SA values than Fe'. Uniformity in kin/SA suggests similarity in the mechanism of uptake and indeed, all strains were found to employ a reductive step in the uptake of Fe' and FOB. In contrast, different uptake pathways were found for FeAB along with variations in kin/SA. Our data supports the existence of a common reductive Fe uptake pathway amongst cyanobacteria, functioning alone or in addition to siderophore-mediated uptake. Cyanobacteria combining both uptake strategies benefit from increased flexibility in accessing different Fe-substrates. Life 2015-03-11 5 1 Article 10.3390/life5010841 841 860 2075-1729 2015-03-11 doi: 10.3390/life5010841 Hagar Lis Chana Kranzler Nir Keren Yeala Shaked http://www.mdpi.com/2075-1729/5/1/818 With the continuously accelerating genome sequencing from diverse groups of archaea and bacteria, accurate identification of gene orthology and availability of readily expandable clusters of orthologous genes are essential for the functional annotation of new genomes. We report an update of the collection of archaeal Clusters of Orthologous Genes (arCOGs) to cover, on average, 91% of the protein-coding genes in 168 archaeal genomes. The new arCOGs were constructed using refined algorithms for orthology identification combined with extensive manual curation, including incorporation of the results of several completed and ongoing research projects in archaeal genomics. A new level of classification is introduced, superclusters that untie two or more arCOGs and more completely reflect gene family evolution than individual, disconnected arCOGs. Assessment of the current archaeal genome annotation in public databases indicates that consistent use of arCOGs can significantly improve the annotation quality. In addition to their utility for genome annotation, arCOGs also are a platform for phylogenomic analysis. We explore this aspect of arCOGs by performing a phylogenomic study of the Thermococci that are traditionally viewed as the basal branch of the Euryarchaeota. The results of phylogenomic analysis that involved both comparison of multiple phylogenetic trees and a search for putative derived shared characters by using phyletic patterns extracted from the arCOGs reveal a likely evolutionary relationship between the Thermococci, Methanococci, and Methanobacteria. The arCOGs are expected to be instrumental for a comprehensive phylogenomic study of the archaea. Life 2015-03-10 5 1 Article 10.3390/life5010818 818 840 2075-1729 2015-03-10 doi: 10.3390/life5010818 Kira Makarova Yuri Wolf Eugene Koonin http://www.mdpi.com/2075-1729/5/1/783 The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed. Life 2015-03-10 5 1 Review 10.3390/life5010783 783 817 2075-1729 2015-03-10 doi: 10.3390/life5010783 Roger Garrett Shiraz Shah Susanne Erdmann Guannan Liu Marzieh Mousaei Carlos León-Sobrino Wenfang Peng Soley Gudbergsdottir Ling Deng Gisle Vestergaard Xu Peng Qunxin She http://www.mdpi.com/2075-1729/5/1/770 The aim of this study was to explore the possibility of using an archaeal microorganism as a host system for expressing mammalian olfactory receptors (ORs). We have selected the archaeon Haloferax volcanii as a cell host system and one of the most extensively investigated OR, namely I7-OR, whose preferred ligands are short-chain aldehydes, such as octanal, heptanal, nonanal. A novel plasmid has been constructed to express the rat I7-OR, fused with a hexahistidine-tag for protein immunodetection. The presence of the recombinant receptor at a membrane level was demonstrated by immunoblot of the membranes isolated from the transgenic archaeal strain. In addition, the lipid composition of archaeonanosomes containing ORs has been characterized in detail by High-Performance Thin-Layer Chromatography (HPTLC) in combination with Matrix-Assisted Laser Desorption Ionization—Time-Of-Flight/Mass Spectrometry (MALDI-TOF/MS) analysis. Life 2015-03-09 5 1 Communication 10.3390/life5010770 770 782 2075-1729 2015-03-09 doi: 10.3390/life5010770 Simona Lobasso Rita Vitale Patrizia Lopalco Angela Corcelli http://www.mdpi.com/2075-1729/5/1/744 Marine cyanobacterial mats were cultured on coastal sediments (Nivå Bay, Øresund, Denmark) for over three years in a closed system. Carbonate particles formed in two different modes in the mat: (i) through precipitation of submicrometer-sized grains of Mg calcite within the mucilage near the base of living cyanobacterial layers, and (ii) through precipitation of a variety of mixed Mg calcite/aragonite morphs in layers of degraded cyanobacteria dominated by purple sulfur bacteria. The d13C values were about 2‰ heavier in carbonates from the living cyanobacterial zones as compared to those generated in the purple bacterial zones. Saturation indices calculated with respect to calcite, aragonite, and dolomite inside the mats showed extremely high values across the mat profile. Such high values were caused by high pH and high carbonate alkalinity generated within the mats in conjunction with increased concentrations of calcium and magnesium that were presumably stored in sheaths and extracellular polymer substances (EPS) of the living cyanobacteria and liberated during their post-mortem degradation. The generated CaCO3 morphs were highly similar to morphs reported from heterotrophic bacterial cultures, and from bacterially decomposed cyanobacterial biomass emplaced in Ca-rich media. They are also similar to CaCO3 morphs precipitated from purely inorganic solutions. No metabolically (enzymatically) controlled formation of particular CaCO3 morphs by heterotrophic bacteria was observed in the studied mats. The apparent alternation of in vivo and post-mortem generated calcareous layers in the studied cyanobacterial mats may explain the alternation of fine-grained (micritic) and coarse-grained (sparitic) laminae observed in modern and fossil calcareous cyanobacterial microbialites as the result of a probably similar multilayered mat organization. Life 2015-03-09 5 1 Article 10.3390/life5010744 744 769 2075-1729 2015-03-09 doi: 10.3390/life5010744 Józef Kaźmierczak Tom Fenchel Michael Kühl Stephan Kempe Barbara Kremer Bożena Łącka Krzysztof Małkowski