Abstract
The initial relationships between organisms leading to endosymbiosis and the first eukaryote are currently a topic of hot debate. Here, I present a theory that offers a gradual scenario in ...which the origins of phagocytosis and mitochondria are intertwined in such a way that the evolution of one would not be possible without the other. In this scenario, the premitochondrial bacterial symbiont became initially associated with a protophagocytic host on the basis of cooperation to kill prey with symbiont‐produced toxins and reactive oxygen species (ROS). Subsequently, the cooperation was focused on the digestion stage, through the acidification of the protophagocytic cavities via exportation of protons produced by the aerobic respiration of the symbiont. The host gained an improved phagocytic capacity and the symbiont received organic compounds from prey. As the host gradually lost its membrane energetics to develop lysosomal digestion, respiration was centralized in the premitochondrial symbiont for energy production for the consortium.
Replication of DNA-encoded information and its conversion into functional proteins are universal properties of life. In an effort toward the construction of a synthetic minimal cell, we implement ...here the DNA replication machinery of the Φ29 virus in a cell-free gene expression system. Amplification of a linear DNA template by self-encoded, de novo synthesized Φ29 proteins is demonstrated. Complete information transfer is confirmed as the copied DNA can serve as a functional template for gene expression, which can be seen as an autocatalytic DNA replication cycle. These results show how the central dogma of molecular biology can be reconstituted and form a cycle in vitro. Finally, coupled DNA replication and gene expression is compartmentalized inside phospholipid vesicles providing the chassis for evolving functions in a prospective synthetic cell relying on the extant biology.
In vivo mutagenesis systems accelerate directed protein evolution but often show restricted capabilities and deleterious off-site mutations on cells. To overcome these limitations, here we report an ...in vivo platform to diversify specific DNA segments based on protein fusions between various base deaminases (BD) and the T7 RNA polymerase (T7RNAP) that recognizes a cognate promoter oriented towards the target sequence. Transcriptional elongation of these fusions generates transitions C to T or A to G on both DNA strands and in long DNA segments. To delimit the boundaries of the diversified DNA, the catalytically dead Cas9 (dCas9) is tethered with custom-designed crRNAs as a "roadblock" for BD-T7RNAP elongation. Using this T7-targeted dCas9-limited in vivo mutagenesis (T7-DIVA) system, rapid molecular evolution of the antibiotic resistance gene TEM-1 is achieved. While the efficiency is demonstrated in E. coli, the system can be adapted to a variety of bacterial and eukaryotic hosts.
The archaea-bacteria lipid divide is one of the big evolutionary enigmas concerning these two domains of life. In short, bacterial membranes are made of fatty-acid esters whereas archaeal ones ...contain isoprenoid ethers, though at present we do not have a good understanding on why they evolved differently. The lateral proton transfer mode of energy transduction in membranes posits that protons utilize the solvation layer of the membrane interface as the main route between proton pumps and ATPases, avoiding dissipation of energy to the bulk phase. In this article I present the hypothesis on a proton-transport route through the ester groups of bacterial phospholipids as an explanation for the evolutionary divergence seen between bacteria and archaea. REVIEWERS: This article was reviewed by Uri Gophna (Editorial Board member) and Víctor Sojo.
The initial relationships between organisms leading to endosymbiosis and the first eukaryote are currently a topic of hot debate. Here, I present a theory that offers a gradual scenario in which the ...origins of phagocytosis and mitochondria are intertwined in such a way that the evolution of one would not be possible without the other. In this scenario, the premitochondrial bacterial symbiont became initially associated with a protophagocytic host on the basis of cooperation to kill prey with symbiont‐produced toxins and reactive oxygen species (ROS). Subsequently, the cooperation was focused on the digestion stage, through the acidification of the protophagocytic cavities via exportation of protons produced by the aerobic respiration of the symbiont. The host gained an improved phagocytic capacity and the symbiont received organic compounds from prey. As the host gradually lost its membrane energetics to develop lysosomal digestion, respiration was centralized in the premitochondrial symbiont for energy production for the consortium.
ADMit hypothesis for the origin of eukaryotes. A bacterial symbiont became associated with a protophagocytic host that captured prey with membrane processes. The symbiont cooperated to kill and digest the prey with reactive oxygen species and the acidification of the microenvironment. Eventually, host's specialized membrane systems assumed the digestion, and the symbiont centralized membrane energetics to become the mitochondria.
DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium
strain HB27 encodes a ...Ppol protein among the genes present in mobile element ICETh2, absent in other
strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every
mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the
mutants in combination with
mutants. The results show that knockout
mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas
mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the
and/or
mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains. All these data support that Ppol appears to keep in balance the activity of AddAB as a part of the DNA housekeeping maintenance in
HB27, thus, playing a key role in its genome stability.
Many different DNA delivery vehicles have been developed and tested, all with their advantages and disadvantages. The bacteriophage phi29 terminal protein (TP) is covalently linked to the 5' ends of ...the phage genome during the DNA replication process. Our approach is to utilize this TP as a platform to incorporate different protein or peptide modules that can target the DNA to the interior of the cell, to the nucleus, or even to subcellular compartments. In order to be able to insert different peptide modules on the TP sequence to endow it with desired functions and/or eliminate unwanted regions of the protein, we have carried out a transposition screening to detect insertion-permissive points on the sequence of the TP. We report the functional characterization of 12 insertion mutants of the TP, and the identification of one site at position 38 that allows the insertion of peptides up to 17 amino acids in length while maintaining the ability of the TP to support DNA amplification in vitro. A protein with one insertion at that position containing a cysteine residue, a linker, and a thrombin recognition site was purified and its amplification activity was optimized.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Cell to cell DNA transfer between Thermus thermophilus, or transjugation, requires the natural competence apparatus (NCA) of the recipient cell and a DNA donation machinery in the donor. In ...T. thermophilus HB27, two mobile genetic elements with functional similarities to Integrative and Conjugative Elements (ICEs) coexist, ICETh1 encoding the DNA transfer apparatus and ICETh2, encoding a putative replication module. Here, we demonstrate that excision and integration of both elements depend on a single tyrosine recombinase encoded by ICETh2, and that excision is not required but improves the transfer of these elements to a recipient cell. These findings along with previous results suggest that ICETh1 and ICETh2 depend on each other for spreading among T. thermophilus by transjugation.
A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages ...prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of Φ29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of Φ29 TP led us to identify a bona fide NLS within residues 1–37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of Φ29 TP attached to the 5′ DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes.
Modular plasmid architectures have shown to be a very useful resource to standardize, build, share, and compare biological parts and functional vectors, and are being applied in an increasing number ...of microorganisms. Here, we present a modular plasmid toolkit for Thermus thermophilus, a species considered as a workhorse for biotechnology and a model for high-temperature biology. Apart from integrating improved versions of already existing parts, we have characterized specific promoters and developed a thermosensor-based palette that restricts the expression to Thermus and, at the same time, controls protein expression in this organism in a temperature-dependent manner.
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