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Introduction
Publications
Publications (81)
Type III CRISPR immune systems bind viral or plasmid RNA transcripts and activate Csm3/Cmr4 and Cas10 nucleases to uniquely cleave both invader RNA and DNA, respectively. Additionally, type III effector complexes generate cyclic oligoadenylate (cOA) signaling molecules to activate trans-acting, auxiliary Csm6/Csx1 ribonucleases, previously proposed...
The mechanisms underpinning the replication of genomic DNA have recently been challenged in Archaea . Indeed, the lack of origin of replication has no deleterious effect on growth, suggesting that replication initiation relies on homologous recombination. Recombination-dependent replication (RDR) appears to be based on the recombinase RadA, which i...
CRISPR–Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5′ end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host...
Conjugative plasmids are self-transmissible mobile genetic elements that transfer DNA between host cells via type IV secretion systems (T4SS). While T4SS-mediated conjugation has been well-studied in bacteria, information is sparse in Archaea and known representatives exist only in the Sulfolobales order of Crenarchaeota. Here we present the first...
All life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. A handful of obligate intracellular species are known to lack ribonucleotide reduction and are instead dependent on their host for deoxyribonucleotide synthesis. As ribonucleotide reduction has on occasion been lost in obligate intracellular parasites and endo...
Thermophiles are microorganisms that thrive at high temperature. Studying them can provide valuable information on how life has adapted to extreme conditions. However, high temperature conditions are difficult to achieve on conventional optical microscopes. Some home-made solutions have been proposed, all based on local resistive electric heating,...
DNA gyrase is a type II topoisomerase with the unique capacity to introduce negative supercoiling in DNA. In bacteria, DNA gyrase has an essential role in the homeostatic regulation of supercoiling. While ubiquitous in Bacteria, DNA gyrase was previously reported to have a patchy distribution in Archaea but its emergent function and evolutionary hi...
In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Arch...
The mechanisms underpinning replication of genomic DNA in Archaea have recently been challenged. Species belonging to two different taxonomic orders grow well in the absence of an origin of replication, challenging the role of the replication origin in these organisms. Here, we pursue the investigation of the particular way some archaea manage thei...
Culturing cells confined in microscale geometries has been reported in many studies this last decade, in particular following the development of microfluidic-based applications and lab-on-a-chip devices. Such studies usually examine growth of Escherichia coli. In this article, we show that E. coli may be a poor model and that spatial confinement ca...
Culturing cells confined in microscale geometries has been reported in many studies this last decade, in particular following the development of microfluidic-based applications and lab-on-a-chip devices. Such studies usually examine growth of Escherichia coli. In this article, we show that E. coli may be a poor model and that spatial confinement ca...
Motivation:
Comparative plasmid genome analyses require complex tools, the manipulation of large numbers of sequences and constitute a daunting task for the wet bench experimentalist. Dedicated plasmid databases are sparse, only comprise bacterial plasmids and provide exclusively access to sequence similarity searches.
Results:
We have developed...
Although plasmids play an important role in biological evolution, the number of plasmid families well characterized in terms of geographical distribution and evolution remains limited, especially in Archaea. Here, we describe the first systematic study of an archaeal plasmid family, the pT26‐2 plasmid family. The in‐depth analysis of the distributi...
Reverse gyrase (RG) is the only protein found ubiquitously in hyperthermophilic organisms, but absent from mesophiles. As such, its simple presence or absence allows us to deduce information about the optimal growth temperature of long-extinct organisms, even as far as the last universal common ancestor of extant life (LUCA). The growth environment...
Reverse gyrase (RG) is the only protein found ubiquitously in hyperthermophilic organisms, but absent from mesophiles. As such, its simple presence or absence allows us to deduce information about the optimal growth temperature of long-extinct organisms, even as far as the last universal common ancestor of extant life (LUCA). The growth environment...
Diverse DNA repair mechanisms are essential to all living organisms. Some of the most widespread repair systems allow recovery of genome integrity in the face of UV radiation. Here, we show that the hyperthermophilic archaeon Thermococcus nautili possesses a remarkable ability to recovery from extreme chromosomal damage. Immediately following UV ir...
Cells from all three domains of life, Archaea, Bacteria and Eukarya, produce extracellular vesicles (EVs) which are sometimes associated to filamentous structures known as nanopods or nanotubes. The mechanisms of EV biogenesis in the three domains remain poorly understood, although studies in Bacteria and Eukarya indicate that the regulation of lip...
Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genet...
A newly described plasmid, which encodes proteins facilitating its packaging and cell-to-cell transfer via membrane vesicles, challenges the way we think about the delineation of viruses, plasmids and extracellular vesicles.
One of the major mechanisms driving the evolution of all organisms is genomic rearrangement. In hyperthermophilic Archaea of the order Thermococcales, large chromosomal inversions occur so frequently that even closely related genomes are difficult to align. Clearly not resulting from the native homologous recombination machinery, the causative agen...
Metagenomic reads mapping (T. nautili 66G).
(DOCX)
Classical site-specific recombination model.
A. The intermolecular site-specific integration between cognate attP and attB sites generates a co-integrate with recombined attL and attR sites in direct orientation. The reverse reaction of excision regenerates the original components. B. In the intramolecular site-specific inversion reaction, the att...
Intptn3 overexpression and purification.
A. Protein expression was induced with 1mM IPTG in 1L of LB medium; cells harvested by centrifugation, and lysed by sonication. The soluble fraction of the sonicate was heated at 65°C for 10 minutes, and denatured proteins removed by centrifugation and by passing through a 0.45 μm filter. Strep-tagged protei...
Mutated IntY428A assay.
Increasing amounts of wild type IntpTN3 and mutated IntpTN3Y428A enzymes were incubated with plasmid pMC477 as substrate to analyze the inversion properties. The experimental conditions are those of the standard integrase assay (see Material and methods) except that increasing amounts of enzyme were used: 0.5, 1, 1.5, 2.5 an...
Detailed characterization of IntpTN3-promoted in vitro inversion event by DNA sequencing.
Specific sequences surrounding tRNA gene BD01_1976 are blocked in red while specific sequences surrounding tRNA gene BD01_1557 are blocked in green. Relevant anticodon sequences are boxed in yellow color. Two nucleotide mismatches between these tRNA genes are...
Thermococcus nautili 66G nucleotide sequence.
Predominant T. nautili chromosome sequence obtained after sub-culturing for 66 generations.
(FASTA)
Thermococcus 5–4 36G nucleotide sequence.
Predominant T. 5–4 chromosome sequence obtained after sub-culturing for 36 generations.
(FASTA)
Detailed mapping of the IntpTN3-promoted in vivo inversions between four pairs of T. nautili paralogs.
The sequences corresponding to the four genomic crossovers observed in T. nautili 60G and 66G were identified each time in pairs of paralogous genes shown aligned here. The sequences blocked in grey throughout the figure refer to perfectly conserv...
Integrase-promoted double-strand cut at ori ColE1.
Circular plasmid pCB548 (4675bp) treated with IntpTN3 and digested with XhoI-NdeI endonucleases generates bands of 2966 and 1709bp due to integrase-promoted low sequence specificity recombination (white arrowheads). The original larger 3896bp XhoI-NdeI fragment undergoes an additional double-strand...
Plasmids used in this work.
(DOCX)
Oligonucleotides used in this work.
(DOCX)
AttB nested deletions.
The Integrase dimerization test was used to determine the minimal site required for IntpTN3 tRNALeu × tRNALeu recombination on nested deletions carried by plasmid templates. A. DNA sequence of the nested deletions. DNA segments corresponding to theses sequences were annealed and cloned directionally in pUC18. B. The resulting...
Subcultures genome comparisons.
Dotplot alignment of the prominent genomes obtained after T. nautili 60G and 66G subculturing (left) and T. 5–4 36G and 66G (right).
(PDF)
Integrase structure comparisons.
The catalytic domain of IntpTN3 (B) was modeled using Phyre2 [Reference 5 in S1 Text] and compared using PyMol (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.) with the tridimensional structure of the integrase of Sulfolobus solfataricus virus SSV1 (PDB 3VCF) (A) determined by Zhan et al [Referen...
Thermococcus 5–4 66G nucleotide sequence.
Predominant T. 5–4 chromosome sequence obtained after sub-culturing for 66 generations.
(FASTA)
Supporting information references.
(DOCX)
pTN3 integration.
A. The comparison between the replicative and the chromosomal integrated forms of plasmid pTN3 enabled us to reconstitute the integration event. A stretch of 41bp is shared by both attP and attB sites. The nucleotides corresponding to the leucine anticodon are underlined. Upon integration, the integrase gene is disrupted and a ful...
Tyrosine recombinases sequence comparison.
A. Alignment of IntpTN3 with tyrosine recombinases from the three domains of life. The protein sequence of IntpTN3 (WP_022547007.1) is aligned using Praline [Reference 4 in S1 Text] with the reconstituted integrase from T. kodakarensis TKV4 and other previously characterized tyrosine recombinases from the...
LacZ gene segments used for low sequence specificity reactions mimicking homologous recombination.
DNA sequence of the lacZ gene segments cloned in plasmids pCB538 (lac100), pCB572 (lac175) and pCB574 (lac250) (Fig 8B).
(PDF)
Thermococcus nautili 60G nucleotide sequence.
Predominant T. nautili chromosome sequence obtained after sub-culturing for 60 generations.
(FASTA)
Planctomycetes are distinguished from other Bacteria by compartmentalization of cells via internal membranes, interpretation of which has been subject to recent debate regarding potential relations to Gram-negative cell structure. In our interpretation of the available data, the planctomycete Gemmata obscuriglobus contains a nuclear body compartmen...
Pores embedded in internal membranes of Gemmata obscuriglobus.
(A)Transmission electron micrograph of a tomographic slice of high-pressure-frozen cryosubstituted thick-sectioned cell showing a pore (boxed region) embedded in internal membranes situated within the cytoplasm and bounding the nuclear body region containing the cell’s nucleoid. Bar, 1...
Gemmata CJuql4 internal membrane pores as seen in the freeze-fractured cells.
(A) Transmission electron micrograph of a platinum/carbon (Pt/C)-shadowed replica of a whole cell of Gemmata CJuql4 (ACM5157) which has been prepared via the freeze-fracture technique. The fractured whole cell contains a large spherical organelle taking up most of the cel...
Transmission electron microscopy of the membranes enriched in fraction 2 and SDS PAGE of the proteins obtained from this fraction.
(A) (1, 2 and 3)—transmission electron micrographs of negatively stained membranes of fraction 2 (see S6 Fig) containing membranes which do not display pore complexes. Bar A1, 1 μm, Bar A2, 500 nm, Bar A3, 200 nm. (B) S...
The antibodies 6670 binds specifically to the beta-propeller-containing protein from fraction 3.
(A) Amino acid sequence of the protein annotated as Na-Ca exchanger/integrin-beta4 (NCBI Reference Sequence: ZP_02736670, later renumbered as the synonymous WP_010049031.1) was used for generation of an antibody (antibody 6670). The protein was identifi...
3D reconstruction of an internal pore-containing membrane of Gemmata obscuriglobus.
The electron tomography membrane reconstruction shown here is derived from a representative of the fraction 3 membranes in the thick section tilt-series.
(AVI)
3D reconstruction of a pore embedded in the internal pore-containing membrane of Gemmata obscuriglobus.
The reconstruction is derived from a pore in the membrane shown in S1 and S2 Videos.
(AVI)
Sucrose gradient fractionation of Gemmata obscuriglobus membranes.
(A) Schematic diagram showing bands resulting from density gradient fractionation of membrane fractions released from cells of G. obscuriglobus lysed via sonication. On the left is the initial distribution of sucrose concentrations in the gradient before ultracentrifugation and the...
Transmission electron microscopy of the membranes enriched in fraction 6 and SDS PAGE of the proteins obtained from this fraction.
(A) Transmission electron microscopy of negatively stained membranes of fraction 6 (see S6 Fig) containing membranes which do not display pore complexes. Bar A1, 10 μm, Bar A2, 500 nm, Bar A3, 200 nm. (B) SDS-PAGE of me...
Cryo-EM of fraction 3 isolated membrane sheets.
Cryo-EM image of frozen-hydrated sucrose-purified membranes from fraction 3 isolated from lysed cell preparation by density gradient centrifugation. Membrane sheets are indicated by arrows and large pore structures are marked by arrowheads. The two pores in the boxed region are seen in Fig 4C.
(TIF)
Crateriform structures on the surfaces of G.obscuriglobus cell walls.
(A) TEM of cell walls of G.obscuriglobus isolated via boiling of bacteria in 10% SDS for 1hr. Bar, 2 μm. (B) One of the cell walls of G.obscuriglobus with clearly recognizable crateriform structures (arrowheads). The electron dense core regions are variable in shape. Bar, 200 nm....
Multiple alignment of the proteins with significant structure predictions in cluster 2.
The 10 pili proteins in cluster 2 (bottom left in S13 Fig and S5 Table) that gave significant structure predictions using Phyre2 were aligned using MAFFT, option L-ins-i, and the alignment was evaluated using the T-Coffee CORE server (see text for details). The...
Structures of the two α-solenoids.
Two proteins showed a potential α-solenoid structure with stacked α-helices. Left: ZP_02735673 (constituent of fraction 2 and fraction 3) and right: ZP_02736511, unique to pore-containing membrane fraction 3.
(TIF)
Immunogold particles distribution.
(A)Distribution of gold particles within Gemmata obscuriglobus cells. The bars represent a number of particles associated with the intracytoplasmic membranes (blue bars) vs with no visible association with the membranes (red bars). A total of 50 cells were used for the counting; 549 particles were found as associa...
Proteins identified in membrane fractions by MALDI-TOF.
(DOC)
Results from structural analysis for the C-terminal region of cluster 1 (β-propeller) protein constituents.
(DOC)
Bioinformatics analyses.
(DOCX)
Dimensions of the Gemmata obscuriglobus internal pores.
The dimensions were calculated from transmission electron micrographs of the membrane fragments released from lysed cells via sonication and negatively stained with ammonium molybdate. The pores usually appear as circular structures with dense pore centers surrounded by a thin lighter inner ri...
Pore-containing membrane of Gemmata obscuriglobus disintegrates and pores aggregate after detergent treatment.
(A) Transmission electron micrograph of negatively stained gradient-fractionated pore-containing membranes purified from sonicated G. obscuriglobus acting as control for detergent treatments shown in (B) and (C). A “canoe” structure with p...
Clustering of membrane-related proteins.
All 128 proteins associated with the membrane fractions were clustered using VisBLAST (E = 0.001, i = 2). Proteins are identified by Genbank accession numbers. Lines indicate detectable sequence similarity between proteins. Colour key indicates membrane fractions in which proteins were detected. The 91 singl...
Clustering of all proteins identified through proteomics.
All 512 identified in our proteomics analysis were clustered using VisBLAST (E = 0.001, i = 2). This revealed several large clusters, though only one of these contained proteins specific to fraction 3 (cluster 1). Proteins are identified by Genbank accession numbers. Lines indicate detectabl...
TEM of the membranes enriched in fraction 3 and SDS PAGE of the proteins obtained from this fraction.
(A) TEM of negatively stained membranes of fraction 3 (see S6 Fig) containing membranes which display pore complexes. 1 and 2 show appearance of aggregates of membranes at relatively low magnification while 3 shows the characteristic ‘canoe’ shape...
Multiple alignment of the proteins with significant beta-propeller structures.
The multiple alignment of the 8 proteins from cluster 1 (top left cluster in S13 Fig and S4 Table) that gave significant structure predictions using Phyre2 is shown. The alignment was made using MAFFT, option L-ins-i, and was evaluated using the T-Coffee CORE server (see...
Summary of the membrane proteome analysis.
(DOC)
Results from structural analysis of cluster 2 (pili) protein constituents.
(DOC)
Results from keyword analysis of Phyre output.
(DOC)
Comparison of Gemmata pores with the nuclear pores of eukaryotes.
(A) and (B) Reconstruction of architecture of a single pore from two different angles. In panel (A), a side view of the pore displays the basket structure with a series of struts (arrows) connecting with the main pore rings. In panel (B), a top view shows the ring-like element (arrow...
Structures of the pili-proteins from membrane fraction 3.
Using PyMOL, the predicted structures for the 11 proteins from membrane fraction 3 that were clustered together and shared a pili-like structure were visualized. None of these are unique to fraction 3 (S5 Table). The structures are for (from left to right and top to bottom): ZP_02731198, ZP_...
Bioinformatic analyses of proteins from membrane fractions used for cluster analysis.
(XLSX)
Planctomycetes are distinguished from other Bacteria by compartmentalization of cells via internal membranes, interpretation of which has been subject to recent debate regarding potential relations to Gram-negative cell structure. In our interpretation of the available data, the planctomycete Gemmata obscuriglobus contains a nuclear body compartmen...
All life generates deoxyribonucleotides, the building blocks of DNA, via ribonucleotide reductases (RNRs). The complexity of this reaction suggests it did not evolve until well after the advent of templated protein synthesis, which in turn suggests DNA evolved later than both RNA and templated protein synthesis. However, deoxyribonucleotides may ha...
Escherichia coli dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a natively homotetrameric enzyme was converted to a monomeric species through the introduction of destabilising interactions at two different subunit interfaces allowing exploration of the roles of the quaternary structure in affecting catalytic competency. The double mutant DHDP...
Questions
Question (1)
I am designing an experiment which requires quantifying the relative abundance of two sequences in a chromosome. Ideally, I would like to use the same experimental design across a family of prokaryotes. An alignment of the sequences shows no regions with perfect sequence identity that would work for both primers and TaqMan probes. However, I can find regions which have a single base difference that would work for primers and probes.
Has anyone had experience with ddPCR using degenerate primers and/or probes? Is it best to use a normal degenerate primer and/or probe mix, or something like inosine at the degenerate position?
Any advice would be greatly appreciated!
