The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission International ImmunopharmacologyCitation Excerpt :Molecular and cellular mechanisms involved in these processes were succinctly analyzed The simian-human immunodeficiency virus (SHIV)-AD8 can induce some pathological features quite similar to HIV-1-infected human individuals and a marked inhibition of SHIV-AD8 by 3BNC117 was revealed by several studies It has been shown that 3BNC117 neutralizes hundreds of HIV/SHIV strains until now and TZM-bl cell assays are usually used for measuring the activity of 3BNC117 in vitro [24,25,29–31,33,34,36,38,40,46,47] All content on this site: Copyright © 2025 Elsevier B.V. 2025 /CNW/ - An INRS team discovers a new family of enzymes capable of inducing targeted cuts in single-stranded DNA the advent of technology known as CRISPR was a major breakthrough in the scientific world Developed from a derivative of the immune system of bacteria CRISPR enables double strands of nucleotides in deoxyribonucleic acid (DNA) to be cut This makes it possible to specifically modify a targeted gene in plant Ultimately, CRISPR became a preferred method in the search for treatments for acquired or hereditary diseases Recently, Professor Frédéric Veyrier at the Institut national de la recherche scientifique (INRS) and his team developed a new genetic tool based on a family of specific enzymes called Ssn that allows targeted cuts to be induced exclusively in single-stranded DNA The results of their work were recently published in the journal Nature Communications This major breakthrough sheds light on a crucial genetic mechanism that could revolutionize a multitude of biotechnology applications Single-stranded DNA is less common than double-stranded DNA It is often found in some viruses and plays a key role in certain biological processes Single-stranded DNA is also used in many technologies (sequencing no endonuclease – enzyme that cuts DNA – has been described as exclusively targeting a single-stranded DNA sequence which has constituted a barrier to the development of technologies based on this type of DNA Professor Veyrier's team has identified a family of enzymes capable of cutting a specific sequence in single-stranded DNA: the family of Ssn endonucleases the research team at INRS's Armand-Frappier Santé Biotechnologie Research Centre first characterized a new family of endonucleases part of the GIY-YIG superfamily called Ssn researchers focused on one of these enzymes in the bacterium Neisseria meningitidis The enzyme targeted in the study is crucial to the exchange and alteration of genetic material we found that it recognizes a specific sequence that is found in many instances in its genome and plays a key role in the natural transformation of the bacterium This interaction directly influences the dynamics of the bacterial genome," explains Professor Veyrier a specialist in genomic bacteriology and evolution INRS's research scientists identified thousands of other similar enzymes "We demonstrated that they are able to recognize and specifically cut their own single-stranded DNA sequence Thousands of enzymes therefore have this property with their own specificity," adds Alex Rivera-Millot a postdoctoral fellow on Professor Veyrier's team and co-first author of the study which represent a new tool for DNA recognition and exchange They pave the way to many novel applications in biology and medicine understanding this mechanism could help better control the bacteria in question and the associated infections the discovery of enzymes specific to single-stranded DNA makes it possible to develop more precise and efficient genetic manipulation tools This could namely improve methods of gene editing These enzymes could also be used to detect and manipulate DNA in various medical and industrial applications such as pathogen detection or genetic manipulation for medical and therapeutic purposes All of these avenues hold significant promise for addressing many health issues there is a patent pending for the results of this work Chenal, M.*, Rivera-Millot, A.*, Harrison, L.B. et al. Discovery of the widespread site-specific single-stranded nuclease family Ssn. Nat Commun 16, 2388 (2025). https://doi.org/10.1038/s41467-025-57514-1 This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) the Canadian Institutes of Health Research (CIHR) and the Fonds de recherche du Québec – Santé (FRQS) The INRS community includes over 1,500 students View original content to download multimedia: http://www.newswire.ca/en/releases/archive/April2025/14/c8889.html Institut national de la recherche scientifique Newsfeed Subscription RSS Feed Newsfeed Subscription  RSS Feed  Metrics details Rod-shaped bacteria typically elongate and divide by transverse fission several bacterial species can form rod-shaped cells that divide longitudinally we study the evolution of cell shape and division mode within the family Neisseriaceae which includes Gram-negative coccoid and rod-shaped species which can be found in the oral cavity of mammals are multicellular and divide longitudinally We use comparative genomics and ultrastructural microscopy to infer that longitudinal division within Neisseriaceae evolved from a rod-shaped ancestor In multicellular longitudinally-dividing species neighbouring cells within multicellular filaments are attached by their lateral peptidoglycan peptidoglycan insertion does not appear concentric but as a medial sheet guillotining each cell we identify genes and alleles associated with multicellularity and longitudinal division including the acquisition of amidase-encoding gene amiC2 and amino acid changes in proteins including MreB and FtsA Introduction of amiC2 and allelic substitution of mreB in a rod-shaped species that divides by transverse fission results in shorter cells with longer septa Our work sheds light on the evolution of multicellularity and longitudinal division in bacteria and suggests that members of the Neisseriaceae family may be good models to study these processes due to their morphological plasticity and genetic tractability Schematic representations and electron microscope images of a N Rightmost panels display extracted sacculi (peptidoglycan) of respective Neisseriaceae P proximal (host-attached) region of the cell The results are representative of at least three independent analyses we ask whether a similar path led to the evolution of Alysiella Simonsiella and Conchiformibius – henceforth collectively referred to as multicellular longitudinally dividing (MuLDi) Neisseriaceae Phylogenomic analysis coupled with both ultrastructural analysis and peptidoglycan (PG) mass spectrometry indicates that MuLDi Neisseriaceae evolved from a rod-shaped ancestor incubation with a palette of fluorescent D-amino acids (FDAAs) shows that nascent septa cross the cells medially as to guillotine them – from the proximal to the distal pole in A comparative genomics-informed recapitulation of MuLDi-specific allelic changes in the rod-shaped N suggesting that the transition from transverse to longitudinal division required the deletion of mraZ the acquisition of amiC2 and MreB amino acid permutations The capacity of oral cavity Neisseriaceae to have evolved – more than once – into coccoid or MuLDi cells from a rod-shaped ancestor together with their amenability to cultivation and genetic manipulation makes them ideal models to understand the evolution of bacterial cell division and that of animal-bacterium symbioses This let us envision two evolutionary scenarios: (1) M1 and M2 evolved independently from a rod-shaped bacterium with phenotypic convergence or (2) the common ancestor of M1 (Simonsiella/Alysiella) and M2 (Conchiformibius) evolved the MuLDi phenotype once from a rod-shaped bacterium reverted to unicellularity and transverse division corresponding epifluorescence images (middle panels) and enlarged selected regions (right panels; the white frames indicate the selected regions) of S Scale bars are 5 µm (middle panels) and 1 µm (right panels) BADA and TADA was plotted onto the long axis Scale bars are 5 µm (left and middle panel) and 1 µm (right panel) Source data are provided as a Source Data file Top left panel displays the filament from which the three septa shown in the bottom panels belong to Middle panel shows the same filament rotated by 30° of which an enlarged region of interest (white frame) is shown in the top right panel; arrowheads point to nascent septa Bottom panels: three septa at consecutive septation stages (septa 1–3 in top left panel) were rotated by 90° and ordered from the youngest to the oldest (left Scale bars are 5 µm (left upper corner) and 1 µm steedae septation mode (top view in left panel filiformis polarity in the absence of fimbriae localization in all the subsequent microscopic analyses filiformis septation is unidirectional (i.e. it proceeds from the distal to the proximal pole) and that the PG is not inserted concentrically from the periphery to the center of the cell but as a sheet that guillotined each cell from its distal to its proximal pole Summarizing, we propose that the two curved oral symbionts S. muelleri and C. steedae start septation at each pole independently, but synchronously, and septation ends when the two pole-originated PG sheets meet and merge at midcell (Fig. 4f) muelleri locus_tag (such as RS00570 for BWP33_RS00570) elongata locus_tag (such as RS02740 for NELON_RS02740) The putative encoded protein associated with each gene are also specified The green and black squares indicate genes that are present or absent of the 7 proteins displaying amino acid permutations rod-shaped or for MuLDi detected with amino acid permutations between rod-shaped and MuLDi Neisseriaceae c Volcano-plot: p value is plotted against fold change calculated using DeSeq2 Red and green points correspond to transcripts that are less or more abundant in MuLDi as compared to rod-shaped Neisseriaceae Source data and statistics are provided as a Source Data file ftsI and murE from the dcw cluster are highlighted In red are transcripts that are less abundant in MuLDi Neisseriaceae and in green are transcripts that are more abundant in MuLDi as compared to rod-shaped Neisseriaceae suggesting convergent evolution of these proteins or horizontal gene transfer between MuLDi species we also found two permutations in the efflux pump membrane transporter MtrD and one permutation in the DNA-directed RNA polymerase subunit RpoZ the single stranded DNA-binding protein Ssb the two-component regulator MisR and the long-chain-fatty-acid-CoA ligase FadD comparative genomics of rod-shaped versus MuLDi Neisseriaceae identified 18 genetic loci whose presence absence or mutation strictly correlate with the rod-to-MuLDi transition the dcw cluster regulator-encoding gene mraZ and the actin homolog-encoding gene mreB 12 genes appeared upregulated in MuLDi species the 19 downregulated genes in MuLDi species included murE and ftsI the absence of mraZ correlates with a downregulation of the dcw cluster (including ftsI) in MuLDi Neisseriaceae a Volcano plot of RNAseq analysis of an N p value is plotted against fold change and were calculated using DeSeq2 Red points represent genes upregulated in MraZ-overexpressing N mraZ under the control of the strong and constitutive porB promoter) c Transcript abundance of dcw cluster genes measured by qRT-PCR in N elongata expressing or not expressing MraZ Data represent mean (n = 3 biologically independent samples ± SD) and are representative of three independent experiments Statistical test used was Unpaired t test with Welch’s correction by comparing ΔmraZ to the parental wild-type and the ΔmraZ; porBp-mraZ to the parental ΔmraZ (ns not significant) elongata expressing or not expressing MraZ (n = 120 biologically independent cells) Data are presented with the median and are representative of at least two independent experiments with Bonferroni’s multiple comparisons test (***p < 0.001) Source data and statistics are provided as a Source Data file (for a we showed that mraZ is regulating transcription of the first five genes of the N elongata dcw cluster and that expression of these genes impacts N elongata (rpsL*) wild-type (left panels) or harboring multiple deletions (Δdgt with or without the mreBNe/merBSm allelic exchange with or without the addition of cdsA-amiC2 elongata (rpsL*) wild-type (left) or harboring the mreBNe/merBSm allelic exchange and cdsA-amiC2 (right) and c median length of the septum (n = 170 biologically independent cells) and of the cell axis perpendicular to the septum (n = 340 biologically independent cells) in N wild-type (left) or harboring the mreBNe/merBSm allelic exchange and cdsA-amiC2 (right) Statistical test used was Unpaired Two-Tailed T test (***p < 0.001) d Schematic representation of a septating wild-type (rpsL*) N elongata harboring the mreBNe/merBSm allelic exchange and cdsA-amiC2 (right) the ratio between the two cell axes changed from 0.61 ± 0.25 (n = 186) even if our attempt to genetically manipulate the rod-shaped N elongata into a MuLDi bacterium did not result into a complete transverse-to-longitudinal division switch (ratio between the two cell axes >1) the observed increase in septum length suggests that the genetic events identified by comparative genomics have participated in the rod-to-MuLDi Neisseriaceae transition full-scale comparative genomics revealed MuLDi-specific differences that set them apart from rod-shaped members of the Neisseriaceae (e.g. mraZ loss and amino acid changes in the cytoskeletal proteins MreB and FtsA) Supporting the role of specific genetic changes in the rod-to-MuLDi transition introduction of amiC2 and allelic substitution of mreB in the rod-shaped Neisseriaceae N elongata resulted in cells with longer septa we presented two novel modes of septal growth and we identified genetic events that likely contributed to the evolution of bacterial multicellularity polarization in a group of mammalian symbionts future studies are needed to clarify whether different cell types exist within each filament these studies suggest that MraZ controls cell division rate by regulating the dcw cluster and we speculate that its absence may have altered the balance between the divisome and the elongasome machineries (i.e the elongasome might contribute to PG synthesis at the septum) we can hypothesize that CdsA affects the composition of the membrane and or (2) an ancestral rod rotated its septation axis by 90 degrees the cell width of an ancestral rod increased (and its length decreased) perhaps following a misbalance between elongation and division genetic tools are needed to gain insights on MuLDi Neisseriaceae evolution by visualizing the localization pattern of FtsZ and MreB or by attempting reversion into unicellular into transversally dividing bacteria such as N most protein function studies have been conducted in either pathogenic or bacterial species that are easy to culture and manipulate in the laboratory such as E efforts to study other morphologies including commensal species are necessary to understand bacterial cell evolution but also to increase the pool of protein targets (e.g. antibiotic targets) for industrial and biopharmaceutical applications they are the only known multicellular longitudinally dividing bacteria that may thrive in humans We hence propose the use of Neisseriaceae as models to study how longitudinal division and multicellularity evolved as well as the molecular and cell biological mechanisms underlying the establishment of bacterium-animal symbioses Strains were streaked from −70 °C freezer stocks onto BSTSY agar plates and grown overnight at 37 °C with 5% CO2 Single colonies were transferred to liquid culture and grown to exponential phase (OD600 0.2) Cells were spotted onto pads made of 0.8% SeaKem LE Agarose (Lonza 50000) in BSTSY and topped with a glass coverslip Cells were transferred to an Okolab stage top chamber to control temperature (37 °C) and gas (CO2 5% and O2 18%) Images were recorded with inverted Nikon Ti-2 microscopes using a Plan Apo 100 × 1.40 NA oil Ph3 DM objective using Hamamatsu Orca FLASH 4 camera Images were processed with NIS Elements 5.02.01 software (Nikon) Representative images were processed using the Fiji 2.1.0/1.53c software package Strains were streaked from −70 °C freezer stocks onto PY (A steedae) agar plates grown overnight at 37 °C with 5% CO2 Single colonies were transferred to liquid culture and grown to exponential phase (OD600 0.2–0.5) at 37 °C shaking at 180 rpm agitation 250 μL of diluted exponential phase cultures (OD 0.025) were loaded into the cell loading well of a prepared (shipping solution removed and washed three times with sterile appropriate media) B04A-03 microfluidic plate (Merck-Millipore) Time-lapse imaging was performed using CellASIC® ONIX Microfuidic System The ONIX manifold was sealed to the B04A-03 plate CellASIC® ONIX2 System was used as the microfluidics control software a flow program was set up to prime flow channel and culture chamber by flowing medium from inlet wells 1–5 at 34.5 kPa for 2 min cells were loaded onto the plate at 13.8 kPa for 15 s Priming run was performed for 5 min with pressure set to 34.5 kPa The medium flow was set at 12 kPa throughout the experiment for 12 h with sterile appropriate media Images were recorded with an inverted Nikon Ti-E microscope using a Plan Apo 60XA oil Ph3 DM objective using Hamamatsu Orca FLASH 4 camera half a loopful of 6–8 h old bacterial cultures were fixed by direct resuspension in 500 μl of 2.5% glutaraldehyde in 0.1 M cacodylate buffer and incubated for at least 1 h at 4 °C Cells were then pelleted through centrifugation at 3075 × g for 3 min and washed 3 times in 500 μl 0.2 M cacodylate wash buffer solution (pH 7.2) 30–50 μl of wash solution containing bacterial cells was pipetted onto Formvar Carbon 200 mesh copper grids (Sigma-Aldrich) and negative staining done using 1% phosphotungstic acid (PTA) for 2 s before imaging at the INRS-CAFSB platform using a Hitachi H-7100 electron microscope with AMT Image Capture Engine (version 600.147) fresh bacterial cells were cultured for 6 h in liquid media containing poly-L-Lysine (Sigma) coated glass slides Cells were fixed using 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 h at 4 °C then rinsed 3 times in 0.2 M cacodylate wash buffer solution (pH 7.2) Post fixation was subsequently done using 1% osmium tetroxide (in 0.2 M cacodylate) before gradual dehydration through increasing ethanol concentrations (25% Carbon dioxide critical point drying (CPD) and gold sputtering were done on Leica EM CPD300 and Leica EM ACE600 instruments respectively The imaging was done at the electron Imaging Facility (Faculty of dental medicine Canada) using a Hitachi Regulas 8220 electron microscope with the SEM operation software Regulus 8200 series Samples were treated with Proteinase K (20 μg/mL The reaction was heat-inactivated and sacculi were further washed by ultracentrifugation samples were digested overnight with muramidase (100 μg/mL) at 37 °C Muramidase digestion was stopped by boiling and coagulated proteins were removed by centrifugation (3075 × g the pH of the supernatants was adjusted to pH 8.5–9.0 with sodium borate buffer and sodium borohydride was added to a final concentration of 10 mg/mL After incubating for 30 min at room temperature pH was adjusted to 3.5 with orthophosphoric acid The soluble muropeptides were analyzed by high-performance liquid chromatography (HPLC; Waters Corporation USA) on a Kinetex C18 column (150 × 4.6 mm; 2.6 µm particle size USA) and detected at 204 nm with UV detector (2489 UV/Visible Muropeptides were separated with organic buffers at 45 °C using a linear gradient from buffer A (formic acid 0.1% (v/v) in water) to buffer B (formic acid 0.1% (v/v) in 40% acetonitrile) in an 18-min-long run with a 1 ml/min flow Quantification of muropeptides was based on their relative abundances (relative area of the corresponding peak) normalized to their molar ratio The molar percentage was calculated for each muropeptide This relative molarity was also used to calculate the molar percentage of crosslinked muropeptides USA) was used for acquiring and managing the chromatographic information Muropeptide identity was confirmed by MS analysis using a UPLC–MS (UPLC system interfaced with a Xevo G2/XS Q-TOF mass spectrometer from Waters Corporation Data acquisition and processing was performed using UNIFI software platform (Waters Corporation After the first interval cells were washed twice with fresh medium (37 °C) and centrifuged between washes (7000 × g for 2 min at RT) the cell pellets were resuspended in pre-warmed medium containing label two cells were washed twice and resuspended in medium containing the third label Cells were then immediately treated with 70% ice-cold ethanol and incubated on ice for 1 h Ethanol-fixed cells were collected via centrifugation (7000 × g for 2 min at RT) washed twice with 4 °C 1 × Phosphate Buffered Saline (PBS To track symbiont cell wall growth followed by immunolabeling A filiformis cells were grown over night on PY plates Single colonies were incubated in 10 mM ethynyl-D-alanyl-D-alanine (EDA-DA a D-amino acid carrying a clickable ethynyl group) for 30 min washed twice (7000 × g for 2 min at RT) and treated with 70% ethanol like described before cells were rehydrated and washed in PBS containing 0.1% Tween 20 (PBT) Blocking was carried out for 30 min in PBS containing 0.1% Tween 20 (PBT) and 2% (wt/vol) bovine serum albumin (blocking solution) at room temperature An Alexa488 fluorophore was covalently bound to EDA-DA via copper catalyzed click-chemistry by following the user manual protocol for the Click-iT reaction cocktail (Click-iT EdU Imaging Kit The cells were incubated with the Click-iT reaction cocktail for 30 min at RT in the dark Unbound dye was removed by a 10-min wash in PBT and one wash in PBS For immunostaining of clicked bacterial cells cells were washed for 10 min in PBT and subsequently incubated with blocking solution for 30 min at room temperature in the dark immunostaining was performed as described below Proteins from bacteria cells were separated by reduced sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on NuPAGE 4%–12% Bis-Tris pre-cast MOPS gel (Invitrogen) and each blotted onto Hybond ECL nitrocellulose membranes (Amersham Biosciences) Membranes were blocked for 45 min in PBS containing 5% (wt/vol) nonfat milk (PBSM) at room temperature and incubated overnight at 4 °C with a 1:1,000 dilution of sheep polyclonal anti-E coli K88 fimbrial protein AB/FaeG antibody (ab35292 After five 6 min-long washes in PBSM and one final wash in PBS containing 0.1% Tween20 the blot was incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-sheep secondary antibody (1:10,000; Amersham Biosciences) in PBSM Protein-antibody complexes were visualized using ECL Plus detection reagents (Amersham Biosciences) Exponential phase cells were fixed overnight in 3% formaldehyde at 4 °C Cells were collected via centrifugation (7000 × g for 2 min at RT) washed twice with PBS and resuspended in PBS containing 0.1% Tween 20 (PBT) Blocking was carried out for 1 h in PBT containing 2% (wt/vol) bovine serum albumin (blocking solution) at room temperature cells were incubated with a 1:500 dilution of sheep polyclonal anti-E Abcam) overnight at 4 °C in blocking solution Upon incubation with primary antibody (or without in the case of the negative control) samples were washed three times in PBT and incubated with an Alexa555 conjugated anti-sheep antibody (Thermo Fisher Scientific) at 1:500 dilution in blocking solution for 1 h at room temperature Unbound secondary antibody was removed by two washing steps one in PBT and one in PBS Cell pellets were resuspended in PBS containing 5 µg/mL Hoechst for 20 min and subsequently washed and resuspended with PBS 1 µL of the bacterial solution was mixed with 0.5 µL of Vectashield mounting medium (Vector Labs) and mounted on an agarose slide Exponential phase cells were fixed overnight in 2% formaldehyde at 4 °C washed twice with PBS and resuspended in PBS containing 10 µg/mL Nile Red (Stock is prepared with DMSO; ThermoFisher N1142) and 5 µg/mL Hoechst for 15 min in the dark at room temperature Cells were washed and resuspended in PBS and subsequently 1 µL of the bacterial solution was mixed with 0.5 µL of Vectashield mounting medium (Vector Labs) and mounted on an agarose slide FDAA-labeled bacteria were visualized with a Leica TCS SP8 X confocal laser scanning microscope Images were taken with a 63X Plan-Apochromat glycerin objective with a NA of 1.30 and a refraction index of 1.46 (glass slide The Leica software LASX (3.7.2.22383) including the Lightning deconvolution software package (Leica) was used for image acquisition and post-processing if necessary For Fig. 7b and Supplementary Fig. 11b elongata wild type (rpsL*) and mutant (rpsL*; cdsA-amiCSM; mreBSM) were imaged at the INRS-CAFSB platform with a Zeiss LSM 780 AxioObserver confocal microscope equipped with a Zeiss Plan-Apochromat 100x/1.4 Oil M27 The Zeiss software Zen 2011 was used for image acquisition Figures were compiled using Adobe Photoshop 2021 and Adobe Illustrator 2021 (Adobe Systems The assembly with the least number of contigs and the greatest completeness was chosen and csv file associating genome name with morphology were used in PastML with default parameters to assess ancestral morphology The prediction method was maximum-likelihood-MPPA (marginal posterior probabilities approximation) “Absence” was defined as lower values than 50% and “presence” as higher values than 55% The proteins encoded by each genome under study here were further compared against these two references by TBLASTN and the groups to be compared were defined amino acid changes in proteins shared by both groups (identity threshold 60%) were investigated focusing on those amino acids conserved in the members of both groups but different between the two groups (I vs D option) Total RNA was extracted from 6 h cultures grown on GCB agar plates The cells were harvested in RNA protect reagent (Qiagen) RNeasy Mini Kit (Qiagen) with RNase Free DNAse set (Qiagen) was used for RNA extraction according to the manufacturer’s instructions Differential gene expression was calculated using ΔΔCT method using the mean CT value of each target obtained with the StepOneTM Software v2.3 normalization was done relative to gyrA gene Standard T-test using (GraphPad Prism v9.0; GraphPad Software CA) was used to ascertain statistical significance of gene expression between the strains where P < 0.05 was considered significant while the Mcherry cassette was obtained by PCR amplification of pMcherry10 (Addgene) using primer pairs pdcwsmMcherry F and McherryNsilR the 5’MraZ and Km cassette were fused using primer pairs 5KoMraZF and KmpSimR while Mcherry and 3’MraZ were fused using primer pairs pdcwMcherryF and 3’KoMraZ R pdcwSm and Mcherry-3’MraZ fragments were fused using primer pairs 5KoMraZ F and 3KoMraZ R and the resulting DNA was used for transformation in N coli DH5α cells to obtain the porBMraZ::p5nrq3::Cm plasmid The plasmid was subsequently linearized before transformation into the Neisseria elongata ΔmraZ strain Mutants were obtained by transforming either N elongata wild-type (single knockout) or an N elongata streptomycin-resistant strain (indicated rpsL*) (multiple knockout) with the linearized plasmid of the targeted gene that resulted in blue Markerless deletion was achieved by introducing DNA of the 5’−3’ homologous regions of the target gene thereby excising the RPLK cassette resulting in white kanamycin sensitive and streptomycin resistant clones Subsequent genes of interest were edited by repeating this procedure and verifications of the correct excision was done by PCR The plasmid was linearized with ScaI before transformation in N mreBsm positive and mreBne negative clones were confirmed by PCR we transformed the plasmid pUCNe::RPLK into N elongata RPLK (RPLK inserted at position 888015) we have replaced the RPLK cassette with cdsA-amiC2 genes by transforming the pUCcdsamiC2::ampR plasmid linearized using ScaI into N cdsamic2 positive transformants were confirmed by PCR Further information on research design is available in the Nature Research Reporting Summary linked to this article The codes used in this study have been reported previously and are available as described in the corresponding M&M section. 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biofilms: ten years of a paradigm up for review SepG coordinates sporulation-specific cell division and nucleoid organization in Streptomyces coelicolor Cyanobacterial septal junctions: properties and regulation At the heart of bacterial cytokinesis: the Z ring Chapter 5 molecular biology of bacterial septation Conservation of gene order amongst cell wall and cell division genes in Eubacteria and ribosomal genes in Eubacteria and Eukaryotic organelles Regulation of transcription of cell division genes in the Eschericia coli dcw cluster Genomic channeling in bacterial cell division Functional characterization of the cell division gene cluster of the wall-less bacterium Mycoplasma genitalium Functional insights of mraZ on the pathogenicity of Staphylococcus aureus Exclusion of assembled MreB by anionic phospholipids at cell poles confers cell polarity for bidirectional growth Analysis of gram-negative bacteria peptidoglycan by ultra-performance liquid chromatography NIH Image to ImageJ: 25 years of image analysis Fiji: an open-source platform for biological-image analysis finished microbial genome assemblies from long-read SMRT sequencing data Canu: scalable and accurate long-read assembly via adaptive κ-mer weighting and repeat separation Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement Fast and accurate de novo genome assembly from long uncorrected reads QUAST: quality assessment tool for genome assemblies BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic Prokka: rapid prokaryotic genome annotation Roary: Rapid large-scale prokaryote pan genome analysis ModelFinder: fast model selection for accurate phylogenetic estimates IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies UFBoot2: improving the ultrafast bootstrap approximation MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform The rise and fall of the Mycobacterium tuberculosis genome Composition-based statistics and translated nucleotide searches: Improving the TBLASTN module of BLAST Minimap2: pairwise alignment for nucleotide sequences FeatureCounts: an efficient general purpose program for assigning sequence reads to genomic features Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 The STRING database in 2021: customizable protein-protein networks and functional characterization of user-uploaded gene/measurement sets EMBOSS: the European molecular biology open software suite Isolation and characterization of Kingella negevensis sp a novel kingella species detected in a healthy paediatric population Antezack, A., Boxberger, M., Rolland, C., Monnet-Corti, V. & Scola, B. LA. Isolation and characterization of Kingella bonacorsii sp. nov., a novel Kingella species detected in a stable periodontitis subject. https://doi.org/10.3390/pathogens (2021) Transfer of some saccharolytic Moraxella species to Kingella Henriksen and Bovre 1976 with descriptions of Kingella indologenes sp emendation of Eikenella exigua Stormo et al 2019 and emendation of the genus Eikenella to include species which are strict anaerobes and emended description of the genus Crenobacter isolated from the intestinal tract of Konosirus punctatus Gapped BLAST and PSI-BLAST: A new generation of protein database search programs Download references The authors are extremely grateful to: Markus Christian Schmid and the Department of Microbial and Ecosystem Science of the University of Vienna for providing the Leica SP8 confocal laser scanning microscope and superb technical support; Belma Bejtovic and Mary Ward for performing preliminary experiments; Nathan Weyand for providing us with Neisseria sp DentCa1/247; Tanneke den Blaauwen (University of Amsterdam) for inspiring and constructive discussions; Antony Vincent (University Laval) for his help in genome assemblies; Dennis Claessen for valuable comments on the manuscript This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant (RGPIN-2016-04940) (F.V.) the Fonds De Recherche du Quebec Nature et technologies (FRQNT) Établissement de la relève professorale (205027) (F.V.) and the Austrian Science Fund (FWF) project P28593-B22 (S.B. also received DOC-fellowship 25240 from the Austrian Academy of Science and a PhD completion grant from the University of Vienna Fellowship from the Fondation Armand-Frappier was partially supported by a postdoctoral fellowship from the Swiss National Science Foundation (project #P2GEP3_191489) studentship Calmette & Yersin from the Institut Pasteur International Network Research in the Cava lab was supported by The Swedish Research Council (VR) The Knut and Alice Wallenberg Foundation (KAW) The Laboratory of Molecular Infection Medicine Sweden (MIMS) was funded by a postdoctoral fellowship from the Swedish Society for Medical Research (SSMF) is also supported by a Canada 150 Research Chair in Bacterial Cell Biology received a Junior 1 and Junior 2 research scholar salary award from the Fonds de Recherche du Québec - Santé These authors contributed equally: Sammy Nyongesa These authors contributed equally: Silvia Bulgheresi INRS-Centre Armand-Frappier Santé Biotechnologie Department of Functional and Evolutionary Ecology Vienna Doctoral School of Ecology and Evolution Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden (MIMS) Center for Microbiology and Environmental Systems Science E.B.; F.P; M.N.; M.D; C.N.; T.V.; N.K.; A.R.M did some experiments and formal analysis and critically revised the manuscript contributed ImageJ analysis tools (ObjectJ did formal analysis and revised manuscript Equally contributing authors were listed in alphabetical order The authors declare no competing interests Nature Communications thanks Pierre Garcia, Brian Hedlund and the other, anonymous, reviewers for their contribution to the peer review of this work. Peer reviewer reports are available Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-022-32260-w Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page On 12 December 2019 the Centenary of the presence (1919-2019) of the Daughters of Mary Help of Christians was celebrated in Veyrier in the presence of the Provincial of the Lombard Province of the Holy Family (ILO) Maria Teresa Cocco and of the Provincial Council the Centenary of the presence (1919-2019) of the Daughters of Mary Help of Christians in Veyrier The Eucharistic Celebration in the Church of Saint Paul in Geneva animated by the students of the La Salésienne Nursery School and with the participation of many parents and supporters of the work concluded the year of celebrations expressing her gratitude to the civil authorities Her words then accompanied the gesture made by a child who placed a vase containing the soil of Geneva at the foot of the altar:  “Thank you for the ‘Yes’ of our first missionaries who arrived here in 1919 leaving their Countries to take care of the children of Italian immigrants in Geneva and Sister Ida transmitted the Salesian charism by living the family spirit in everyday life In a hundred years of educational presence in Geneva thousands of hearts and minds have opened up to better serve God and their brothers and sisters in the contemporary world made herself present with a message sent to the Animator encouraging them to continue their mission in a renewed journey of holiness:  “I address the wish that in full synergy with those who take care of the human and Christian growth of young people especially those in situations of poverty or hardship a page of the living Gospel reflected in your community May yours be a presence that generates life and radiates hope so that the children and young people to whom you offer educational and any other person who shares your mission can look to the future with confidence and that the young people themselves may become responsible builders for a new humanity (…) I entrust your present and your future to Mary Help of Christians so that the community may continue to be fertile ground for the educational mission and for the proclamation of the Gospel of Jesus in this time of interesting and unprecedented possibilities You are given a precious heritage of 100 years of history I am sure that your heart is open to welcome it and honor it with your life witness” The centenary year opened on 31 January 2019 on the Solemnity of Don Bosco with the Eucharistic Celebration with the entire Educating Community there was a concert open to all on the theme of gratitude by the well-known Belgian singer-composer Théo Mertens in which even the pupils of the last year sang and made small videos two buses of families from the school went on pilgrimage to Colle Don Bosco in Morialdo All the initiatives were carried out thanks to the collaboration of the laity and the Committee of La Salésienne The story of the main historical stages of the House of Veyrier Luisa Gattiglia and Sister Ida Patrucco (Italian) to take care of the children of Italian immigrants who were numerous at the time The sisters from Turin were sent by the Superior General the mission was part of the French Sacred Heart Province The chronicle of that year reports that the Sisters had to wait at the border of 12 hours due to the passports and arrived in Geneva cold and hungry Miss Goria prepared the activities they were to carry out: looking after the Italian children and emigrants the oratory – a typically Salesian work in which the whole community was engaged due to the greater numbers of children became too small It was necessary to move to Rue de la Servette in Geneva At the train station an office is created specifically to deliver mail to Countries at war The sisters also have the task of getting the mail to the post office and having it arrive to the Superiors in Turin so they could give news to distant family members the house where they are living is put up for sale They had three months to look for another house and they find accommodation at No but fortunately a neighbor rents two rooms that serve as classrooms In this period they receive the visit of the Superior Fr Pietro Berruti who sees their difficulties and encourages them to continue to carry out the educational mission among the Geneva youth the Sisters arrive in Veyrier with some residents but they can accommodate a larger number of children The number of students increases rapidly: the majority are from immigrant families single-parent families or divorce petitioners who need to place their children safely in a boarding school The decision is made to build a prefabricated school and leave the house for the residents In 1966 the new school opened its doors in the presence of civil and religious authorities and took the name ‘La Salésienne’ The FMA meet many difficult situations of hardship and material and moral poverty During the summer they welcome young people who wish to learn French suited to the needs of the pedagogy of the time A large party organized by a promotion committee and the gift of an Italian benefactor’s villa cover a large part of the cost makes a large contribution to the construction of a big In 2017 there is a Nursery School with a new dining room and modern kitchen The Hans Wilsdorf Foundation still bears a large part of the expenditure Today the students who are preparing to be “good people of faith and honest citizens” are about 400 of all social strata and of different origins and religions and website in this browser for the next time I comment Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" This site uses Akismet to reduce spam. 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About us | Advertise with us | Contact us Posted: 25 November 2020 | | No comments yet An inexpensive molecule showed efficacy against antibiotic-resistant Neisseria gonorrhoeae and Neisseria meningitidis in an animal model A group of researchers has demonstrated the effectiveness of an inexpensive molecule to fight antibiotic-resistant strains of the bacteria responsible for gonorrhoea and meningococcal meningitis The study was conducted at the Institut national de la recherche scientifique (INRS) some strains of Neisseria gonorrhoeae have developed resistance to all effective antibiotics Resistant strains of Neisseria meningitidis have also emerged The researchers highlight that unlike other bacteria Neisseria that cause meningitis and gonorrhoea evolve very rapidly because of certain intrinsic properties they have a great capacity to acquire genes from other bacteria They also have a suboptimal DNA repair system that leads to mutations; antibiotic resistance can therefore easily emerge The fact that these diseases affect many people around the world also gives them many opportunities to evolve explaining why it is urgent to develop new ways to fight these bacteria simple molecule that demonstrated efficacy in bacterial cultures and in a model of infection “We noticed that the molecule only affects pathogenic Neisseria It does not affect other types of Neisseria that are found in the upper respiratory system and can be beneficial,” said Professor Frédéric Veyrier the research team tested whether there was any possible resistance to the molecule: “We were able to isolate strains of bacteria that were less sensitive to the treatment but this resistance was a double-edged sword because these mutants completely lost their virulence,” said Veyrier The team does not know exactly why the molecule reacts specifically with the two types of antibiotic-resistant Neisseria but they suspect a connection with the membrane of these pathogens The researchers say the next step will be to modify the structure of the molecule to make it more efficient the team wishes to identify an industrial partner to evaluate the possibility of developing a potential treatment The findings were published in Antimicrobial Agents and Chemotherapy Related topics, , , , Related conditions, Related organisations Related people By No comments yet , , , , , All subscriptions include online membership giving you access to the journal and exclusive content By By , Comment * document.getElementById("comment").setAttribute( "id" "af11bc028168b46decc38248aa00cb0e" );document.getElementById("a3f6228d48").setAttribute( "id" Write for us | Advertise with us Drug Target Review is published by: Russell Publishing Ltd.Court LodgeHogtrough HillBrasted © Russell Publishing Limited, 2010-2025. 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functionalities of the website CookieTypeDurationDescriptioncf_ob_infopersistent1 minuteThis cookie is set by Cloudflare content delivery network and in conjunction with the cookie 'cf_use_ob' Right-wing intimidation of activists in France has taken a new turn with a public death threat against Anasse Kazib — a trade union activist and a comrade of Left Voice’s sister organization a trade union leader in France and activist of the New Anticapitalist Party and Left Voice’s sister publication Révolution Permanente was the target on August 30 of a public death threat by an official of the National Rally (RN) the extreme right-wing party that was known as the National Front until June 2018 the regional head of the RN in Val-de-Marne It follows a series of intimidating acts undertaken by the extreme right against Anasse Kazib and other activists of immigrant origin Above a photo showing several people who have been hanged “In the #GrandRapatriement” [“Grand Repatriation”] we will begin with the letters A and K Little Anasse Kazib is to present himself at gate 12; his destination is a surprise.” You are witnessing the image of what they are preparing: fascism and now publicly threatens us with death.” Kazib was referencing several other recent intimidations unleashed by right-wing forces in France in recent days the French right-wing magazine Valeurs actuelles over the weekend published a fictional account of the return of Danièle Obono a deputy in the French National Assembly of Gabonese origin Obono — a member of the Left party La France Insoumise — is depicted in illustrations with chains around her neck She described the article as “racist shit.” In response, one of the magazine’s editors defended the story saying “it sought to show the ‘destroyers of history’ that an inter-African slave trade existed in tandem with the European one.” Veyrier’s threat against Anasse drove a huge wave of denunciation on social media Veyrier justified what he wrote by citing the alleged “Islamism” of Kazib Others have been subjected to similar intimidation Valeurs actuelles attacked the well-known French-Algerian journalist and activist Taha Bouhafs in a tweet calling him “scum” before it was deleted and replaced with “indigenous activist.” Bouhafs tweeted that he was being deluged with racist insults and death threats underneath photos of his vacation with Assa Traoré taken from Instagram by a “pseudo-journalist” from the magazine She is a highly visible activist against police racism and repression and the sister of Adama Traoré a young Malian-French man who was killed in police custody in 2016.  In Révolution Permanente Anasse emphasized that it “disturbs the fascistic right” when “young immigrants or young people from immigrant backgrounds … speak out in public denounce the injustices of a Republic that knew how to use our elders as cannon fodder in its wars or like coal in its factories or on the railroads but that today treats us as second-class citizens.” These threats against Anasse and others comes just as a racist national debate is unfolding over President Emmanuel Macron’s security policies The mainstream media is complicit: newspapers have been keeping these threats off their front pages where they would have been if they targeted representatives of the ruling class and the broadcast media have buried their reports Our comrades at Révolution Permanente have appealed to all trade unions and Left political organizations to denounce these relentless racist attacks they have demanded that the media and those in the bourgeois political class who claim to be antiracist to do the same — firmly condemn these racist death threats from the extreme Right and longtime socialist activist who lives in the Boston area Leftists who claim the mantle of internationalism must speak out against the reactionary nationalism that the Russian and Ukrainian regimes are using to crack down on dissent Kazib's trial comes amid global repression of the Palestine movement it is essential that we join in this solidarity to denounce state repression against pro-Palestine activists both in France and around the world Prominent figures of the French Left held a press conference on April 24 to denounce the attempt by the French government to jail Anasse Kazib for social media posts in solidarity with Palestine Increasing attacks by the Far Right in French universities has become dangerous for the student movement Our comrades from Le Poing Levé (Fist Up) are calling for the creation of action committees to combat these attacks and turn universities into battlegrounds against the Far Right The temporary disqualification of a current Detroit councilmember in the city’s upcoming elections is an undemocratic move that speaks to the flaws of the electoral process under capitalism workers across the world turned out to organize and demonstrate against the growing global threat posed by Trump and the Far Right The United States has already killed hundreds of civilians in its imperialist bombing of Yemen the Trump administration is indicating that it may back a ground invasion threatening to throw the country into a civil war and left organizations rallied across the United States showing the widespread anger at Trump’s reactionary agenda To defeat the Far Right workers and students need to organize from below — not rely on the Democratic Party that sabotages labor and capitulates to the right Ian Wylie selects her favourite stories in this weekly newsletter European business schools are a rich source of graduates for the global luxury goods sector No fewer than 19 of the 22 business schools with luxury programmes are in Europe according to the US-based AACSB accreditation body with more than half of these courses in France and a fifth in Italy But luxury brands — and those teaching about the industry — are under growing pressure from millennial and generation-Z consumers and students to promote sustainable and ethical practices, says Julia Pueschel, director of the MSc in luxury marketing at Neoma Business School in northern France This is a cohort conscious of climate change and keen to align with brands trying to make a difference and in a sector in which some businesses wear paper-thin eco-credentials on their sleeves engaged consumers want more than covetable products with snappy sustainability straplines Some 72 per cent of generation-Z consumers take into account companies’ commitment to sustainable development when deciding their purchases according to a survey this year by Boston Consulting Group and Altagamma “Many of our students on luxury programmes frequently highlight sustainability concerns when discussing luxury brands,” says Pueschel “In presentations given by representatives from luxury companies students consistently ask about sustainability initiatives and policies and when these students undertake research projects sustainability consistently emerges as a central theme.” academic director of the Master in Fashion Management at Iéseg in France business schools must equip students not only with sustainability awareness but also detailed understanding of ethics and social responsibility “They need knowledge of ethical business practices certifications and standards,” Slavich says “They need an understanding of sustainable materials and processes including eco-friendly packaging and product design as well as familiarity with key sustainability concepts such as the circular economy sustainable sourcing and carbon footprint reduction.” This story is from the ranking report publishing on December 4 Students must grasp concepts such as lifecycle analysis and new ownership business models from the circular economy, including renting, second-hand, recycling and upcycling, says Isabelle Chaboud, programme director of the MSc in fashion, design and luxury management at Grenoble Ecole de Management The programme includes a one-week study trip with the theme: Sustainability and Innovation in the Fashion and Luxury Sectors The history of ‘centenary maisons’ . . . could make it difficult to question existing frameworks “Generation Z vehemently condemns ‘fast fashion’ related to the incredible amount of waste and pollution, and it supports the idea of giving products a longer life,” Merk adds. “The concept of pre-loved brands, sold through increasingly professional second-hand channels, is perfectly in line with the values of this generation, which will represent the biggest group of luxury shoppers and talent by 2030. “Before Covid, just a few premium brands like Stella McCartney or Patagonia placed sustainability at the heart of their business strategies,” Merk notes. “Now many luxury brands want to become sustainable champions in record time, hiring chief sustainability officers, organising sustainability events, speaking in public about their sustainability efforts and fixing ambitious goals to reduce their carbon footprint.” Marie Veyrier-Montagneres graduated with a Masters in Management from Audencia in 2020 and is now an innovation and sustainability project manager for Christian Dior Couture in Paris. Dior parent LVMH’s Life 360 programme includes commitments to eliminate plastic from packaging by 2026, to reduce its overall water consumption by 30 per cent by 2030, and to use 100 per cent renewable energy in its factories by the same date. “My level of awareness around sustainability was relatively high when I went to Audencia but, while I understood actions I could take in my personal life, I lacked an understanding of how this translated into my work,” she recalls. “Sustainability was part of all the business cases I worked on as a student and the environmental and social stakes were evaluated and challenged with the same level of requirement and importance as for what we might call pure business.” During her induction week at Dior, two days out of the five were focused on sustainability, diversity, inclusion and “transmission of savoir-faire”. Veyrier-Montagneres says it has become common to hear clients ask about raw material sourcing conditions, traceability and production location. “The histories and legacies of ‘centenary maisons’ like Dior are so strong that, at first sight, students could feel it’s difficult to question existing frameworks,” concedes Veyrier-Montagneres. “Business schools can help students by providing concrete tools and examples of how early career professionals can make positive impacts at their level.” Students also need to understand the complexity of sustainability, says Alessandro Brun, director of two masters in luxury management at Polimi Graduate School of Management in Milan. “Faux-fur will produce less animal cruelty and by eating quinoa, we will kill fewer animals,” Brun says. “But faux-furs are made from microplastic, and washing them will result in a million tiny pieces of plastic in the oceans. And the sharp rise of quinoa prices forced large groups of Latin American populations to start feeding families with cheaper, but less healthy fast food. “Sustainability is a delicate balance of environmental, ethical and social aspects — students need to consider these as a whole to make the right decision,” he adds. Volume 11 - 2020 | https://doi.org/10.3389/fmicb.2020.558166 This article is part of the Research TopicRole of Sigma Factors of RNA Polymerase in Bacterial PhysiologyView all 7 articles the anti-Sigma factor K of Mycobacterium tuberculosis inhibits SigK and that mutations in RskA promote high expression of the SigK regulon The latter observation led us to hypothesize that RskA mutations lead to loss of the anti-Sigma factor function we used natural and artificial mutations in RskA to determine the basis of the SigK-RskA partnership the N-terminal cytoplasmic portion of RskA was sufficient on its own to inhibit SigK This activation depended on the same N-terminal region and was enhanced by the membrane-extracellular portion of RskA we engineered similar truncations in a Gram-negative bacterium Together these results support an alternative mechanism of anti-Sigma factor function that we could term modulator (activator-inhibitor) in both Actinobacteria and Gram-negative bacteria suggesting that Sigma factor activation by anti-Sigma factors could be under-recognized In this study, we used natural and artificial mutations in rskA (Supplementary Figure S1B) to improve our knowledge of the molecular mechanism of SigK regulation revealing an unexpected activating role of RskA Our findings indicate that RskA is a dual function activator-inhibitor whereby extracytosolic RskA mutations cause increased SigK activity we were able to reproduce these results using a SigK-RskA pair from the Gram-negative bacterium Yersinia enterocolitica suggesting that this dual function may be widespread in the SigK family We made a first observation that challenged the simple model of RskA as a pure inhibitor of SigK. Previously, we observed that wild-type RskA from M. tuberculosis partially suppressed the expression of the SigK regulon in M. bovis BCG Russia, but only when rskA was expressed under control of the highly active hsp60 promoter (Said-Salim et al., 2006) we were unable to suppress SigK activity in M it was expected that the absence of a functional RskA would liberate SigK to direct transcription of SigK-regulated genes so the introduction of wild-type RskA would restore this defect The difficulties encountered in these complementation studies stimulated an alternative hypothesis or X233S) may be dominant over RskAtuberculosis Our results show that the introduction of the dyadtuberculosis into M tuberculosis did not affect the expression of MPT70 and MPT83 the introduction of both dyadbovis and dyadorygis increased production of these proteins to a similar level than that observed in M these findings support the hypothesis that the mutated versions are dominant on the wild-type RskA Dominant effect of mutated RskA on wild-type RskA (A) Schematic representation of the plasmid backbone used in this study marinum harboring pMV::DyadRv-Lx70 (black) or pMV::Dyadbovis-Lx70 (white) which expressed Mutant forms of rskA result in over-expression of the SigK regulated genes even in the presence of the wild-type rskA Each bar represents the mean measurement for three independent clones (***p < 0.001) This representative data has been replicated at least twice (C) Comparison of the activity of dyadbovis and dyadorygis with daydtuberculosis assessed by microarray tuberculosis H37Rv daydtuberculosis (with pMV-Hyg::Dyad) compared with Dyadbovis representing average of two arrays) have been plotted against Z-scores obtained when M tuberculosis H37Rv daydtuberculosis (with pMV-Hyg::Dyad) has been compared with Dyadorygis Genes whose expression is up-regulated in both cases are for the majority part of the sigK or mpt70 locus (light blue) MPT83 and HSP65 (Loading control) measured by Western blot for M tuberculosis H37Rv in native state (lane 1) tuberculosis after introduction of different sigK-rskA dyads (lanes 2-4) and M indicates pMV::Dyadbovis and Dyadory indicates pMV::DyadOryx This representative data has been replicated twice therefore we have not quantified sigK expression by RT-qPCR tuberculosis is an activator and an inhibitor of SigK smegmatis harboring the indicated constructs Each bar represents the mean measurement for three independent clones (B–D) Effects of RskA truncation or mutation on SigK activity measured using luciferase under control of the promoter of mpt70 (B) Experiment conducted in the absence of a wild-type RskA (M (C) Experiment conducted in the absence of a wild-type RskA (M smegmatis) with over-expression of SigK and RskA and (D) Experiment conducted in the presence of a wild-type RskA (M Error bars represent standard deviations for measurement of three independent clones The background noise (line) was set at 1.8 smegmatis expressing the luciferase under the promoter of mpt70 but without sigK and rskA (data not shown) variable degrees of SigK modulation were observed This representative data has been replicated twice (***p < 0.001 compared with dyadtuberculosis) we could observe that the removal of a specific fragment of the C-terminal part of RskAtuberculosis (194 to 232 data point 8) led to the same phenotype observed with RskAbovis or RskAorygis (data points 2 and 3) whereas further truncation (122 to 232 This suggests that the extra cytoplasmic tail (194 to 232) is controlling the dual activity of RskA and removing it has the same effect as mutation (G107D RskA had the same effect on SigK: no activity with the full-length RskA or with 1 to 85 version (inhibition); some activity with SigK alone (low on); full activation with RskA from 1 to 179 (high on) These data suggest that the mechanism of SigK activation by RskA is conserved across these genera enterocolitica is an activator and an inhibitor of SigK (A) Schematic representation of the plasmid backbones used in this study Bold black arrow represents predicted SigK binding site enterocolitica and its effect on SigK activity measured by luciferase expression under control of mpt83 promoter in E Error bars represent standard deviations of three independent experiments Non-induction and induction with L-arabinose are shown in both cases (**p < 0.01 ***p < 0.001 compared with SigK and full-length RskA) SigK activity occurs without degradation of SigK protein was detected by immunoblot using monoclonal FLAG-tag antibodies in M or expressing SigK alone; Dyadtuberculosis Dyadbovis and Dyadorygis and the SigK-cysteine-less versions expression of SigK with a His-tag linked to the N-terminus was detected by immunoblot using monoclonal His-tag antibodies in E coli expressing SigK alone; SigK-RskA; SigK-RskA179 or SigK-RskA85 Purified SigK was used as a positive control and E coli harboring the empty plasmid as a negative control RpoB was used as a loading control in both cases using an anti-RpoB antibody that react with both species SigK activity requires two conserved cysteines carried by SigK serines on SigK activity were measured by luciferase under the control of mpt70p in M Error bars represent standard deviations of three independent experiments with 2 different clones each time We performed a similar experiment in E. coli by expressing the different versions of RskAenterocolitica, again with SigK where cysteine 181 was replaced by a serine (Figure 5B) SigK activity is minimal and no activation by the truncated RskA is observed This data supports a model where SigK requires the two cysteines to be fully activated by RskA This firmly suggests a strong ongoing adaptation of this Sigma factor/anti-Sigma factor partnership for several species that have members from these two families of ECF18 and ECF19 (as we first detected in M orygis) and particularly for those that do not have conserved these two cysteines Species with SigK that naturally evolved without cysteines have adapted RskA (A) Phylogenetic tree of 3717 ECF sequences done as described in the Materials and Methods section (C) Statistics on the SigK/RskA based on all representative sequences available and found in RefSeq as described in the section “Materials and Methods” To test whether the premature stop codon was a general feature of Nocardia sp. we sequenced this region in Nocardia asteroides 42007 we detected the same stop-codon mutation (and the absence of mpt83) the cysteines are absent in these species (whereas they are conserved in all the other mentioned species harboring a SigK and a non-truncated RskA) Figure 7. SigK that naturally evolved without cysteine is functional. (A) Schematic representation of sigK locus in Nocardia sp. and Rhodococcus equi. In Nocardia, the rskA gene encodes a truncated protein (1 to 81) and mpt83, a core member of the conserved mpt83-sigK-rskA cassette (Veyrier et al., 2008) The arrows indicate SigK binding sites and genes are named based on their M smegmatis with plasmid containing sigKnocardia or sigKnocardia and rskAnocardia (1 to 81) In the absence of the first 81 amino acids of RskA 348MFTsu5.1 harbors a full-length RskA (H290_RS0112295) and a SigK with the two cysteines (H290_RS0112290) indicating again that the these two cysteines in SigK are found concomitantly with the ancestral full-length RskA the equivalent domain of RskA is an inhibitor on its own we predict that the activating function of other anti-Sigma factors could be underappreciated tuberculosis does not appear to occur through SigK instability it could also be possible that SigK recognizes a signal through the cysteine bridge and that RskA is modulating this sensing via its interaction with SigK upon further characterization of the ECF18 and ECF19 we also shed light on the “recent” ongoing evolution of these family of Sigma factor/anti-Sigma factors in multiple instances The co-occurrence of the cysteines with full-length RskA suggests a to-be-determined role of these cysteines in the switching On/Off of SigK by full-length RskA We found that absence of the two cysteines is correlated with changes in the anti-Sigma factor (as compared to the RskA potential archetype) This could be the presence of another anti-Sigma from a different family than RskA but also we found that the majority of Nocardia spp have evolved a Sigma-factor without cysteine and a truncated RskA (around 80 amino acids) unlike when we artificially mutated the cysteines in SigK naturally evolved cysteine-less SigK are highly active suggesting of compensatory amino-acid changes is still “functional” and serves as an inhibitor of SigK The species-specific evolution of this family is complexifying the study of the SigK/RskA partnership In the light of these examples of species-specific evolution (M Nocardia spp.) the study of the partnership between SigK and RskA could be complicated and imply some species-specific characteristics these species have all lost or altered the part of RskA that is supposed to sense the signal this may argue for the possibility that SigK would also sense a specific/alternative signal our results emphasize a broader and complex function for anti-Sigma factors as modulators (inhibitor/activator) of the Sigma factor the nature of the signal which initiates this cascade is still unknown this knowledge can serve to engineer differential expression of the SigK regulon in the same bacterial background and generate tools to decipher in detail the transcriptional activation and the function of this regulon using natural and engineered anti-Sigma factor mutations represents an attractive approach to control the activity of specific Sigma factors which serves to understand the function of their regulons This fragment was treated with T4 polymerase to generate blunt ends and ligated with pMV306-Hyg::dyad pMV::dyadbovis or pMV::dyadoryx digested by BsrDI pMV::dyadbovis-Lx70 and pMV::dyadoryx-Lx70 have been respectively Oligonucleotide primers used in this study (2) Plasmids coding for truncated versions of RskA: To construct all these plasmids a fragment of rskA from pMV::dyadbovis-Lx70 was removed after XhoI-HindIII digestion (Figure 1A), replaced by PCR products obtained using Pfu cloned polymerase (Stratagen Inc.) and digested with the same enzymes. These PCR products were obtained using Rv0444cOF and a specific reverse primer (Table 2) this was done using RskA1/121R which contained a HindIII site designed to introduce a stop codon in rskA after the 121 amino acids of RskA the ligation of the PCR product obtained using M tuberculosis gDNA template has generated pMV::dyad1/121Rv whereas the ligation of the PCR product obtained using the M bovis gDNA template has generated pMV::dyad1/121bovis pMV::dyadΔrskA was obtained by self ligation of pMV::dyadbovis-Lx70 digested with XhoI-HindIII and treated with T4 polymerase All the other versions of RskA were reintroduced in this backbone plasmid as described above using XhoI-HindIII (4) Plasmids expressing SigK with serines instead of cysteines: To generate these constructs, site-directed mutagenesis on cysteine amino acids residues position 133 and 183 on SigK was performed, using three fragments PCR assembly method and specific primers SigK133F, SigK133R, and SigK183R (Table 2) PCR product was digested by MunI-HindIII and cloned into pOE-Lx plasmid digested by the same enzymes to generate pOE-Lx SigKaloneSer were cloned into the generated pOE-Lx::DyadSerM.tb pOE-Lx::DyadSerM.bo and pOE-Lx::DyadSerM.or (5) FLAG-tagged SigK: To generate the FLAG versions of SigK, we introduced a 1X FLAG-tag at the N-terminal part of sigK in both cysteine and serine version. Both versions of sigK were amplified from plasmids pOE-Lx::SigKalone and pOE-Lx::SigKaloneSer by PCR using Phusion HF DNA polymerase (New England, BioLabs) and primers FlagSigKF and SigKR (Table 2) PCR products and pOE-Lx plasmids were digested by MunI-HindIII and ligated together orygis were amplified from previous pOE-Lx plasmids digested by XhoI-HindIII and ligated to pOE-Lx::SigKalone and pOE-Lx::SigKaloneSer to generate pOE-Lx::SigKaloneFlag with Dyadtuberculosis versus pOE-Lx::SigKaloneSerFlag with Dyadtuberculosis (6) Nocardia version of sigK-rskA: The locus from Nocardia asteroides 42007 abscessus 91107 was amplified by the AccuPrimeTM Taq DNA polymerase High Fidelity (Invitrogen) These PCR products were cloned into TOPO using TOPO® TA Cloning® Kit (Invitrogen) The fragments were sequenced at the McGill University and Genome Quebec Innovation Center asteroides was deposited in GenBank (accession numbers: FJ935781) ligated with pMV::dyadRv-Lx70 digested with HindIII-ApaLI (which removed the dyad from M tuberculosis) and treated with T4 polymerase to obtain pMV::dyadN42-Lx70 In a derivate plasmid called pMV::dyadN42stop-Lx70 a premature stop codon was inserted in rskAnocardia by ligation of pre-annealed primers (PpuMIStopF and PpuMIStopR) with PpuMI digested pMV::dyadN42-Lx70 All plasmids used in Mycobacteria (Table 1) were derived from pMV306 and were therefore integrated into the genomes of the specified mycobacteria. Plasmids were electroporated into M. smegmatis, M. tuberculosis H37Ra and H37Rv, and M. marinum as previously described (Belley et al., 2004) Hygromycin resistant clones were verified by PCR and selected for further experiments As described before (Veyrier et al., 2008) bacteria were grown in 7H9 with hygromycin until the cultures reached an OD600 of 0.5 cultures were resuspended in Phosphate-Buffered Saline (PBS) and 10 μl of 1% n-decyl aldehyde (Sigma) in ethanol was added to 90 μl (9 × 106 bacteria) in 96 well plates (Becton Dickinson labware) Light was immediately measured in the Victor 3TM Wallac 1420 Multilabel Counter (PerkinElmer) over a 10 s period 2 transformants have been measured two times and luminescence output was expressed in RLU (Relative Light Unit) To test expression of luciferase in bacteria harboring the transposon bacteria were grown overnight in 1 ml of 7H9 with hygromycin and kanamycin cultures were resuspended in Phosphate-Buffered Saline (PBS) and tested as described above we normalized the amount of light with the OD600 measured in the 96 well plates with the Victor 3TM Wallac 1420 Multilabel Counter (PerkinElmer) 10 ml of culture supernatants of indicated M tuberculosis strains were concentrated using a Millipore Ultra-10 Centrifugal Filter Unit (10 000 MW cut-off) and filtered through a 0.22 μm membrane For membrane associated proteins (MPT70 and MPT83) culture pellet of a 10 ml culture was resuspended in 1 ml of water and samples were subjected to FastPrep (BIO 101 Savant) for 15 s at 6.0 rpm 3 times as well as its mutated versions (C133S and C183S) were picked inoculated into 2 mL of 7H9 medium supplemented with 50 μg mL–1 hygromycin and incubated 48 h at 37°C Bacteria harvesting was done by centrifugation of 1 mL of culture at 8,000 × g Cell pellets were resuspended in 200 μL 1X PBS Lysis was performed by addition of 65 μL of 4X Laemmli sample buffer followed by boiling at 100°C for 10 min Samples were run on a 12% SDS−PAGE gel for 1 h at 200V protein transfer was performed on PVDF membranes (BioRad) at 80 V for 2 h Membrane blocking was carried out for 1 h at room temperature using TBS-Tween 0.1% containing 5% BSA (TBS-TB) Incubation with rabbit polyclonal antibodies against MPT70 and MPT83 (gifts of Harald Wiker) (1:500 dilution) mouse monoclonal antibodies against HSP65 (Abcam) Mouse monoclonal DYKDDDDK Tag Antibody (FG4R) and Mouse RpoB Monoclonal Antibody (8RB13) were blotted onto the PVDF membrane overnight at 4°C followed by 1h incubation with anti-rabbit IgG − horseradish peroxidase conjugate (1:15 000 dilution) or anti-mouse IgG-horseradish peroxidase conjugate (1:15 000 dilution) Three washing steps with TBS-Tween 0.1% of 10 min each were added after incubation with both antibodies using the substrate SuperSignal West Pico (Thermo Fisher Scientific) SigK-RskA179 and SigK-RskA85 were amplified using specific primers Restriction sites for BspHI (forward primer) and XbaI (reverse primer) were used to clone into pBAD digested with NcoI and XbaI sigK was amplified using the forward primer mentioned above Different variants of rskA were amplified using the reverse primers already described allowing the fusion with sigKC181S and generating the variants sigKC181SrskA The PCR products were digested with BspHI and XbaI and cloned into pBAD-Km vector digested with NcoI and XbaI (4) pET28a::SigK: SigK was cloned into pET28a(+) vector using primers SigKYeNdeIF and SigKYeBamHIR Both PCR product and vector were digested by NdeI and BamHI for introduction of a N-terminal 6xHis coli DH5α and then transformed in E bacteria co-transformed with pBAD-Km vector containing the different SigK-RskA constructions and pKO5′luc in LB medium containing 50 μg mL–1 kanamycin and 25 μg mL–1 chloramphenicol 100 μL were inoculated into 3 mL LB with the antibiotics already mentioned 67.5 μL of each fresh culture were dispensed in triplicate considering two conditions: induction and non-induction the plate included bacteria expressing the different versions of SigK-RskA (wild-type and mutant) as well as bacteria expressing the empty vector (pBAD-Km both in absence and presence of L-arabinose The induction was carried out for the indicated time at 30°C 75 μL of the Luciferase Assay Reagent (Promega) were added in each well and the luminescence was measured using a Wallac Victor 3 (Perkin Elmer) luminometer Luminescence was normalized by the bacterial OD determined by absorbance at 600 nm 10 mL were inoculated into 1 L of LB medium supplemented with 50 μg mL–1 kanamycin and grown at 37°C Induction was performed with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 4 h Bacteria harvesting was carried out by centrifugation at 10,000 rpm Cell pellets were resuspended in 20 mL native binding buffer (50 mM NaH2PO4 treated with 1 mg mL–1 lysozyme and 1 mM PMSF for 30 min Lysates were clarified by centrifugation at 15,000 rpm and purified using HisPurTM Ni-NTA Resin (Thermo Fisher Scientific) Lysates were loaded onto the purification column (previously equilibrated with native binding buffer) unspecific binding was removed by washing (50 mM NaH2PO4 Protein identity was confirmed by western blotting using a monoclonal Anti-His IgG1 − from mouse (Sigma) inoculated into 5 mL of LB supplemented with 50 μg mL–1 kanamycin and incubated overnight at 37°C The overnight cultures were diluted into 500 mL LB supplemented with 50 μg mL–1 kanamycin Induction was performed as described before Bacteria harvesting was done by centrifugation at 10,000 rpm Cell pellets were resuspended in 10 mL 1X PBS containing 0.5% SDS Lysates were clarified as previously indicated 5 μL of each sample were diluted into 7 μL H2O and 4 μL of 4X Laemmli sample buffer (0.2 M Tris pH 6.8 Samples were run on a 12% SDS−PAGE gel for 1 h at 200 V protein transfer was performed on PVDF membranes (BioRad) at 90 V for 1 h 30 min Incubation with 1:500 in TBS-TB of primary antibody (monoclonal Anti-His IgG1 − from mouse) was performed overnight at 4°C An anti-mouse IgG − HRP conjugate − from rabbit was used as a secondary antibody (1:10000 in TBS-TB) RpoB detection [by incubation with an RNA polymerase beta Monoclonal Antibody (8RB13) – from mouse] The 4157 SigK sequences were then aligned by MAFFT with 25 iterations The amino acids at positions 133 and 183 according to the sequence of M tuberculosis were extracted in order to verify the conservation of the cysteines CXXC (zinc finger) and length were determined using a custom python script the nucleotide sequences of the RskAs encoded as pseudogenes in RefSeq for the genus Nocardia have been downloaded translated into amino acids and their length calculated with an in-house script The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material and AV performed and analyzed the experiments All authors have read and approved the manuscript This work was supported by the FV Natural Sciences and Engineering Research Council of Canada (NSERC) under Grant RGPIN-2016-04940 and a CIHR Foundation Grant FDN–148362 AV received a Postdoctoral Fellowship from the NSERC FV was a research scholar of the Fonds de Recherche du Québec-Santé MB was supported by a Tier 1 Canada Research Chair The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.558166/full#supplementary-material FIGURE S1 | Alignment (A) and schematic representations (B) of RskA versions used in this study (natural or engineered variants) FIGURE S2 | (A) Effect of FLAG-tag linked to the N-terminus part of SigK was measured by luciferase under the control of mpt70p in M smegmatis (B) Effect of His-tag linked to the N-terminus part of SigK was measured by luciferase under the control of mpt83p in E Error bars represent standard deviations of 3 independent experiments with 2 different clones each time FIGURE S3 | Expression of SigK regulon through MPT70 antigen production was detected by immunoblot of M TABLE S1 | Table presenting the in-silico analyses of SigK homologues using RefSeq database Self-cleavage of the Pseudomonas aeruginosa cell-surface signaling anti-sigma factor FoxR occurs through an N-O Acyl rearrangement Impact of methoxymycolic acid production by Mycobacterium bovis BCG vaccines A new phylogenetic framework for the animal-adapted Mycobacterium tuberculosis complex Signaling mechanisms for activation of extracytoplasmic function (ECF) sigma factors Reduced expression of antigenic 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Frédéric J. Veyrier, ZnJlZGVyaWMudmV5cmllckBpbnJzLmNh; Marcel A. Behr, bWFyY2VsLmJlaHJAbWNnaWxsLmNh Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish And you're so warm and moist – an ambulatory habitat for trillions of tiny, microscopic bacteria. Your skin, your follicles, your innards, are all crawling with little organisms too small to see or feel Shhh, it's OK. It's better this way. In fact, it is highly possible that you wouldn't be you without them But some of those little microbes are pretty strange – evolving and adapting to the unique environments provided by the human body One of those, scientists have found, is a bacteria that lives in your mouth. Neisseriaceae is a family of microbes that includes caterpillar-shaped genera and new research suggests that they evolved this body shape because it's better suited to the environment in the human oral cavity and gives us some valuable information on the biodiversity in your mouth It also has implications for the study of bacterial adaptability – which is really important for understanding how to develop more effective antibacterial agents to rid the body of infections "Our work sheds light on the evolution of multicellularity and longitudinal division in bacteria," write the team co-led by cell biologist Silvia Bulgheresi of the University of Vienna in Austria and microbial geneticist Frédéric Veyrier from the French National Institute of Scientific Research (INRS) "[It] suggests that members of the Neisseriaceae family may be good models to study these processes due to their morphological plasticity and genetic traceability." Although your mouth might seem like a pretty nice place for microbes to live it isn't exactly the most welcoming environment The cells lining the inner surface of your mouth are constantly being shed as new cells take their place and your saliva makes things very slippery This makes it more difficult for any crawly things in there to find purchase (which hasn't seemed to slow them down – there are around 700 species that live in the human mouth) and their colleagues believe that this might be why some Neisseriaceae species have developed an interesting way to multiply the team used electron microscopy to study the bacteria shape in detail using fluorescence to understand cellular growth they took rod-shaped Neisseriaceae and introduced genetic changes to see if they could reproduce the evolution from a rod-shaped organism to the caterpillar-shaped types of clusters that can be found wriggling around in the human mouth Their research suggests that the organisms did the bacteria divide along their longitudinal axis But, rather than separating, the individual bacteria then remain attached to each other, resulting in a segmented cluster wrapped in a shared outer membrane, a bit like a bacterial version of the rolly-polly mascot, Bibendum Some of the microbes in this tiny collective also take on different shapes This might be an example of how an organism evolves from a single-celled one into a multicellular one "Multicellularity makes cooperation between cells possible, for example, in the form of division of labor, and may therefore help bacteria to survive nutritional stress," the researchers write in their paper The team was unable to replicate the clustered shape of multicellular species such as Conchiformibius steedae or Simonsiella muelleri perhaps because they could not introduce all the genetic events that led to the current caterpillar-shaped form "We speculate that in the course of evolution, through a reworking of the elongation and division processes, the cell shape changed, perhaps to better thrive in the oral cavity," Veyrier says will be required to explore the bacteria in greater detail the evolutionary approach could be a complementary way to study these tiny organisms and how they work in addition to a better understanding of the mechanisms underlying the symbiotic relationship they have with their (comparatively) giant The research has been published in Nature Communications SeaBubbles has now reached a new goal as it becomes a commercial service open to public It has already embarked its first passengers at the pier in Veyrier-du-Lac and crossed towards Annecy les Marquisats in early July 2023 the Agglomération du Grand Annecy and SeaBubbles Mobility Solutions are teaming up to carry out and look further into the for the first zero-emission experimental electric hydrofoil as public transportation in the city There will be 8 round trips per day between 9am and 7pm over the next several weeks as the operation of the electric flying water taxi progresses transporting passengers between Veyrier-du-Lac and Annecy les Marquisats ‘Our eco-designed boats are the perfect setting for unique events and out-of-the-ordinary meetings all while enjoying a breathtaking view of the water with no noise and no pollution,’ SeaBubbles states.  By the looks of its design, the passengers of SeaBubbles’ electric flying water taxi with hydrofoils can have scenic landscapes through the series of design elements that allow the doors and windows to be opened at varying angles The doors can open sideways first before they can be lifted upwards projecting the image of a winged watercraft The overhead roof can also be flapped open to enable more airflow and provide passengers with unobstructed views of the outside the company is now gearing up to develop SmartBubbles a bigger sibling of the electric flying water taxi but this setup is now retractable plus the interior can accommodate between eight to 12 people at once The wider water vehicle is currently being tested and validated on Lake Annecy with SeaBubbles’ hopes of making its first flights in the coming months A second version of SeaBubbles is also underway whose upgrade points out its fast-charging feature getting its battery fully powered in just four minutes it has already embarked its first passengers between Veyrier-du-Lac and Annecy les Marquisats in early July 2023 The ranges of electric flying water taxis from SeaBubbles are mounted on inverted T-shape foils which are operated by a dynamic control system and the actuators automatically alter the flying height and counteract the water vehicle’s rolling The side sensors assess the flying height and send flight stability data to the onboard computer designed specifically for the boat’s hull uses an inertial device to regulate the flaps on the foils in real-time both in terms of sensory pleasure and carbon footprint zero-emission’ transportation solution is both passenger- and ecologically friendly making it ideal for usage in cities and protected regions The SeaBubble is as subtle as a bubble gliding over water and mirrors what might be the thrusting movement of a flying fish drawing inspiration from nature’s richness and offering an alternative technical and eco-friendly mobility experience to the public there will be 8 round trips per day between 9am and 7pm over the next several weeks from the time of publishing this story a five-seater electric flying water taxi with hydrofoils has begun operating its commercial shuttle service in France the electric flying water taxi with hydrofoils operates between Veyrier-du-Lac and Annecy les Marquisats for now name: SeaBubbles electric flying water taxi company: SeaBubbles AXOR presents three bathroom concepts that are not merely places of function but destinations in themselves — sanctuaries of style Metrics details Site-specific endonucleases that exclusively cut single-stranded DNA have hitherto never been described and constitute a barrier to the development of ssDNA-based technologies We identify and characterize one such family of widely distributed site-specific single-stranded nucleases (Ssn) exhibiting unique ssDNA cleavage properties By first comprehensively studying the Ssn homolog from Neisseria meningitidis we demonstrate that it interacts specifically with a sequence (called NTS) present in hundreds of copies and surrounding important genes in pathogenic Neisseria NTS/Ssn interactions modulate natural transformation and thus constitute an additional mechanism shaping genome dynamics We further identify thousands of Ssn homologs and demonstrate a range of Ssn nuclease specificities for their corresponding sequence We demonstrate proofs of concept for applications including ssDNA detection and digestion of ssDNA from RCA This discovery and its applications set the stage for the development of innovative ssDNA-based molecular tools and technologies unlike the modular proteins from this superfamily single-domain GIY-YIG small proteins such as the cd10448 family remain uncharacterized with thousands of homologs across the entire bacterial domain termed Neisseria Transformation Sequence (NTS) that encompasses the shorter dRS3 sequence The NTS likely forms a hairpin structure and is an order of magnitude more frequent in the pathogenic Neisseria genomes relative to commensal species We show that it is present in variable numbers in the genomes of selected Neisseriaceae and that these sequences cluster non-randomly around certain important genes in N we demonstrate here that these repeated NTSs are also spatially associated with a specific gene (NMV_0044 or NMB0047 in the Nm 8013 and MC58 genomes respectively) across broad phylogenetic distance in bacteria These homologs encode small hypothetical proteins and encompass the cd10448 (NCBI Conserved Domain Database): GIY-YIG_unchar_3 protein family and are abundant in Pseudomonadati is a site-specific single-stranded endonuclease capable of binding and cutting the NTS only as ssDNA We demonstrate that this ssDNase activity regulates natural competence and modulates the integration of transforming DNA We also show that SsnA belongs to a vast family of small single-domain endonucleases (Ssn) with unique specificities for ssDNA sequences We then demonstrate potential applications including ssDNA detection and digestion of ssDNA from Rolling Circle Amplification (RCA) Because of their diverse set of ssDNA cleavage specificities we anticipate that the widely distributed Ssn nuclease family may be harnessed as molecular tools to selectively modify single-stranded DNA in multiple biotechnological applications a Logos and predicted secondary structures of repeated sequences from N the different colors represent the base-pair probability b Core-genome phylogeny of Neisseria species with their associated repeated elements The four color gradients represent the relative abundance of the different enumerated elements meningitidis 8013 genome with its DUS (green) and NTS (red) repeats depicted Major clusters of NTS are highlighted with black arrows pointing to the corresponding genomic landscape Those major clusters are emphasized with schematic representations of the loci of interest (not drawn to the scale) d Logos and predicted structures and (e) whole-genome distribution of SRM repeats in R a Distribution of homologous-protein encoding genes of the Nm proteome and the fraction of such homologs with NTS or SRM repeats flanking their genes at both sides Each point represents one protein in the Nm proteome and the plot relates the number of homologs found in the NCBI reference / representative genome database to the fraction of those homologs flanked by SRMs b Distribution of the NTS repeats (red) near selected ssnA homologs (blue) c Sequence alignment of ssnA-associated NTS repeats from selected species Darker blue indicates better conserved nucleotides This clearly demonstrates the strong association of Ssn encoding genes with their SRM motif in multiple genomes from a diversity of phyla This observation suggests that recognition is not solely mediated by the structure but also by the sequence All experiments were performed at least three times (or more) Complementing this analysis to focus on the taxonomic distribution of Ssn proteins, we used the full NCBI CDD database to generate an extensive list of thousands of presumed homologous single-domain cd10448-containing GIY-YIG proteins of similar size. All sequences were aligned and used to infer a maximum-likelihood phylogeny (Fig. 5b) While most of the SsnA homologs are found in gram-negative bacteria (mainly Pseudomonadati) they are widely distributed throughout across bacterial phyla these results confirm that members of the widespread Ssn family (cd10448: GIY-YIG_unchar_3 protein family) are specific single-stranded endonucleases hence supporting the identification of SsnA proteins as a distinct functional enzyme family with a diverse set of cleavage specificities These genes may be less effectively repaired leading to increased sequence variability and thus antigenic drift it is also possible that alternative configurations of NTS elements relative to the position of gene(s) in the transforming DNA could have other modulating effects on recombination These intriguing similarities with SsnA and the NTS repeats raise the hypothesis that this genetic element (NTS-ssnA-NTS) was domesticated by Neisseria or other symbiotic species to perform important biological functions linked with genome dynamics (such as controlling HGT) This scenario may have led to the massive expansion of NTS repeats in some species The few enzymes capable of cutting single-stranded nucleic acids also act on dsDNA or RNA or show no sequence specificity SsnA is unique in the sense that no binding nor cutting could be detected with both dsDNA and RNA the NTS is directional as its ssDNA reverse strand is not cut by SsnA We have demonstrated in vitro that this enzyme possesses a complex binding and cutting specificity where it binds to NTS repeats to cleave ssDNA upstream Another advantage of the Ssn/NTS system is that cleavage can also happen in the presence of two ssDNA oligos that harbor each half the NTS sequence Although this programable cleavage mechanism is comparable to other commercially available nucleic acid-guided nuclease systems (such as Prokaryotic Argonaut or CRISPR/cas) the Ssn/NTS system offers the advantage of a vast array of potential paired sequence/enzyme specificities that could be used combinatorically for direct ssDNA cleavage or DNA-guided cleavage the coupling of RCA technique with the specific cleavage of SsnA could enable the development of particularly sensitive ssDNA detection methods we anticipate that these unique and flexible properties will form the basis of a suite of molecular tools that may offer alternatives to existing technologies or open the door to future ssDNA-based applications we report important insights on the evolution of the pathogenic Neisseria species the discovery and characterization of the SsnA protein and its family of endonucleases provide an important class of tools for molecular biology and biotechnology research ssDNA has numerous applications in basic research Once the sequence specificities of a broader set of Ssn nucleases are characterized this unique enzyme family may serve as versatile molecular tools to manipulate ssDNA NTS and NTSvar were queried with one mismatch permitted and dRS3 with no mismatches permitted the number of species containing homologs found in the genome database and the fraction of those organisms containing homologs for which at least one NTS element was found in both the 5′ and 3′ 5 kb flanking regions were tabulated and plotted To examine the relationship between NTS repeats and the ssnA gene their association was then investigated at the genome level in the NCBI reference/representative database described above the number of NTS elements was determined using fuzznuc searching in both strands with one further position mismatches permitted the presence or absence of a homolog of the SsnA protein was determined by using tblastn and an in-house script to query the Nm SsnA protein sequence (CAX49033.1) against each translated genome: a homolog was considered present if the search yielded an alignment of > 75% of the query sequence length and an E-value of <1e−10 A stricter criterion of 60% amino acid identity over the aligned length was also used To confirm these results without biasing the analysis by using the SsnA protein from N the analysis was repeated using the CDD database psiblast was used to query with the position-specific scoring matrix (PSSM) that defines the GIY-YIG_unchar_3 protein family (cd10448) in the NCBI CDD database to find homologs of the GIY-YIG_unchar_3 subfamily those potential GIY-YIG_unchar_3 family proteins were used as queries in a rpsblast to search of the NCBI CDD database and a GIY-YIG_unchar_3 subfamily protein was considered present in the genome if the rpsblast score was >100 this time only on the 1 kb 5′ and 3′ of the gene These shuffled genomes were queried for SRM elements which form a genome-specific empiric estimate of the number of expected SRM elements due to chance were then compared to the SRM counts in the original genome yielding a fold enrichment in SRM elements for each genome Using the rpsblast blast hits described above and a custom script each protein was assigned a top CDD family based on the top rpsblast hit Where the superfamily PSSM was the top rpsblast hit the next best hit (by bitscore) was reported if one was found the distribution of SRMs in the 500 bp flanking the genomic region encoding each protein was also examined using a custom script and proteins were identified as being flanked if at least one SRM was detected in both the 5′ and 3′ flanking regions the resulting phylogeny and annotations were examined and a phylogenetic clade representing the proposed Ssn nuclease family was identified by the overlapping distributions of SsnA blastp hits oligos were annealed by mixing equimolar amounts in annealing buffer (10 mM Tris-HCl pH8 heating them to 95 °C and letting them cool down For assessment of the sequence specificity of SsnA EMSA and nuclease assays were performed as described on mutated versions of NTS75 (oligos NTS75mut) where single-nucleotide mutations were introduced along the NTS repeat Each mutated position was substituted for any other nucleotide (A to C/G/T) Nuclease assays involving oligonucleotide hybridization with a quencher were conducted post-hybridization (equimolar amounts in annealing buffer as above heated to 95 °C and cooled down gradually) The enzyme buffers and concentrations remained consistent with the previous experiments Fluorescent oligonucleotides were used at a concentration of 1 µM while non-fluorescent ones were used at varying concentrations (see legend of relevant figures) Measurements were carried out using a Victor3V 1420 multilabel counter (PerkinElmer) with P490/F535 filters for top-reading and an exposure time of 1 sec Similarly to75 single-stranded ssDNA oligonucleotides were hybridized at an equimolar concentration (5 µM) with short partially complementary oligonucleotides (T7F/R) by heating at 95 °C followed by gradual cooling These hybrids were then circularized using T4 ligase (NEB) according to the supplier’s protocol This ligation product (1 µM) was used to perform rolling circle amplification (RCA) The RCA was carried out with phi29 DNA polymerase (NEB) for 16 h at 30 °C followed by enzyme inactivation for 10 min at 65 °C c) using a TBE-acrylamide gel with 8 M urea that was incubated with an intercalating dye (ssGreen for ssDNA In the case of fluorescence quenching experiments (Fig. 6e) different concentrations of oligonucleotides (as indicated in the figure) were circularized and amplified as described above Fluorescent oligonucleotides bearing a 6-FAM and a quencher (BHQ1) were used at a final concentration of 0.2 µM for hybridization with the RCA were conducted in 96-well black-bottom microplates (fluotrac The enzyme buffers were similar to previous experiments except that the enzyme SsnA was used at a final concentration of 1.5 µM Measurements were carried out with a Cytation 3 image reader from Biotek using wavelengths of 490/520 for bottom reading and automatic gain adjustment of Nm 8013 and mutants using pKONMV_0504::Cm of Nm 8013 and mutants using gDNA of Nm 8013 rpsLK43R yebN::Cm d of Nm 8013 and mutants using pKOlgtFrfaK::Cm of Nm 8013 and mutants using gDNA of Nm rpsLK43R f of Nm 8013 and mutants using gDNA of Nm 8013 NalR DNA exchange between Nm 8013 rpsLK43R yebN::CmStop co-cultured with Nm 8013 rpsLK43R yebN::Cm donor strain which were conducted twice with independent purifications Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article and CRISPR/Cas-based methods for genome engineering CRISPR-Cas guides the future of genetic engineering Phylogenomic analysis of the GIY-YIG nuclease superfamily The I-TevI nuclease and linker domains contribute to the specificity of monomeric TALENs Monomeric site-specific nucleases for genome editing Compact designer TALENs for efficient genome engineering Transforming activities and base contents of deoxyribo-nucleate preparations from various Neisseriae Functional analysis of the interdependence between DNA Uptake sequence and its cognate ComP receptor during natural transformation in Neisseria species Dialects of the DNA uptake sequence in Neisseriaceae Natural transformation facilitates transfer of transposons integrons and gene cassettes between bacterial species Competence for natural transformation in Neisseria gonorrhoeae: a model system for studies of horizontal gene transfer Transformation-mediated exchange of virulence determinants by co-cultivation of pathogenic Neisseriae Virulence evolution of the human pathogen neisseria meningitidis by recombination in the core and accessory genome Genome sequencing reveals widespread virulence gene exchange among human Neisseria species Horizontal transfer of antibiotic resistance genes in clinical environments Various pathways leading to the acquisition of antibiotic resistance by natural transformation Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance Factors affecting genetic transformation of Neisseria gonorrhoeae Genetic manipulation of Neisseria gonorrhoeae DNA uptake sequence-mediated enhancement of transformation in Neisseria gonorrhoeae is strain dependent New functional identity for the DNA uptake sequence in transformation and its presence in transcriptional terminators Sequence-specific DNA uptake in Haemophilus transformation Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae Whole-genome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis A chromosomally integrated bacteriophage in invasive meningococci Genome comparison in silico in Neisseria suggests integration of filamentous bacteriophages by their own transposase Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18 The repertoire of silent pilus genes in Neisseria gonorrhoeae: evidence for gene conversion Veyrier, F. et al. Discovery of the widespread site-specific single-stranded nuclease family Ssn. https://doi.org/10.5281/zenodo.14623445 (2025) Endonucleases that selectively cleave single-stranded nucleic acids and uses thereof Prokaryotic Argonautes for in vivo biotechnology and molecular diagnostics Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491 Specific DNA recognition mediated by a type IV pilin Functional analysis of the interdependence between DNA uptake sequence and its cognate ComP receptor during natural transformation in Neisseria species Biased distribution of DNA uptake sequences towards genome maintenance genes Commensal Neisseria Kill Neisseria gonorrhoeae through a DNA-Dependent Mechanism repair and replication in the pathogenic Neisseriae: the 3 R′s of molecular genetics of two human-specific bacterial pathogens Genetic interactions of DNA repair pathways in the pathogen Neisseria meningitidis Natural transformation of Neisseria gonorrhoeae: from DNA donation to homologous recombination Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases He, S. et al. The IS200/IS605 Family and “Peel and Paste” Single-strand Transposition Mechanism. Microbiol. Spectr. 3, https://doi.org/10.1128/microbiolspec.MDNA3-0039-2014 (2015) Identification and characterization of repetitive extragenic palindromes (REP)-associated tyrosine transposases: implications for REP evolution and dynamics in bacterial genomes Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences The GIY‐YIG endonuclease domain of Arabidopsis MutS homolog 1 specifically binds to branched DNA structures 0: leveraging the creation of Circos plot with enhanced usability and advanced features Automated generation of heuristics for biological sequence comparison BEDTools: a flexible suite of utilities for comparing genomic features Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences R: A Language and Environment for Statistical Computing (R Foundation for Statistical computing Discovering sequence motifs with arbitrary insertions and deletions SeqKit2: a Swiss army knife for sequence and alignment processing Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries Veyrier, F. et al. Discovery of the widespread site-specific single-stranded nuclease family Ssn-sourcedata. https://doi.org/10.5281/zenodo.14630455 (2025) Download references Muhamed-Kheir Taha for providing LNP Neisseria isolates Myriam Letourneau and Mounir Bouyakoub for insights and technical assistance This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant (RGPIN-2023-05657) and Canadian Institute of Health Research (CIHR) project grant (450862) (F.V.) Fellowship from Fonds de Recherche du Québec – Santé (FRQS) L.B.H received funding from the FRQS/Québec Ministère de la Santé et des Services sociaux Clinician-Scientist Training Program Fellowship from Fonds de Recherche du Québec – Nature et Technologies (FRQNT) This research was enabled in part by support provided by Calcul Québec and the Digital Research Alliance of Canada (alliancecan.ca) These authors contributed equally: Martin Chenal declare the following competing interests: co-inventors on the international patent application no The remaining authors declare no competing interests Download citation DOI: https://doi.org/10.1038/s41467-025-57514-1 Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology Please enable JS and disable any ad blocker Switzerland - Louis Deletraz was looking forward to combining a season of Formula 2 racing with a reserve role for the Haas F1 team this year but now the 22-year-old Swiss is worried about what the future might hold The COVID-19 pandemic has thrown a spanner in the works for motor sports in general with racing stalled and fears about the damage done confirmed less than a month ago in his F1 position that means more obstacles on what is already a tough career path "I am definitely worried for the sport in general because we rely on a lot of sponsors and on TV rights," Deletraz told Reuters while training at his home outside Geneva because there is no racing and sponsors also are not so keen anymore to do motor sports "There are worse things happening in the world but the sport will suffer from it and I hope we can get back on our feet quite quickly." Formula 1 has yet to get started after a failed attempt to race in Australia last month with the glamorous Monaco highlight also canceled and a string of other races waiting to be rescheduled Haas is privately owned and operates on the smallest budget in Formula 1 with most of its British-based staff furloughed and the factory shut The smaller Formula 2 teams are also reliant on sponsorship broadcast revenues and prize money to stay in business who is due to race for the Czech-based Charouz team in F2 this year after finishing eighth overall in 2019 with three podiums for Carlin said he had not lost funding or sponsors but the situation was not easy "In F2 it is obviously less budget than Formula 1 we also have sponsors but the sponsors right now are not in the best shape," he said "No one wants to spend money outside of themselves but maybe it will be more tough for the next few years "In motor sports you always have to do things early Contract talks would already start right now for the next season but we have not even yet started the 2020 season e-sports and exercise fills the time for a man used to spending his days on the road The son of former F1 and sportscar racer Jean-Denis Deletraz is active on several platforms and represents Haas in official F1 virtual races The Swiss finished seventh in a virtual Chinese Grand Prix on Sunday with five of those ahead of him either current or past Formula 1 drivers Within the framework of the Tallinn Mechanism the project "Building Cybersecurity Capabilities for Ukraine" (CCBU) was launched with the participation of Deputy Minister of Digital Transformation for European Integration Valeria Ionan and French Ambassador Gael Veyrier "The Tallinn Mechanism is a powerful example of international cooperation that helps Ukraine counter cyber threats and strengthen digital resilience We are grateful to our partners for their continued support which not only contributes to protecting critical infrastructure but also enhances the expertise of Ukrainian specialists The development of knowledge and skills of civil servants and cybersecurity professionals is a key element of Ukraine’s national security and an inseparable part of our cooperation with the international community," said Valeria Ionan which France will conduct for Ukrainian partners with the support of Expertise France until December 2025 These cybersecurity training sessions will strengthen Ukraine’s cyber resilience and promote the implementation of best international practices and standards in the field "This project illustrates our commitment to supporting Ukraine on a long-term basis and promoting the exchange of best practices in cybersecurity," emphasized Ambassador Veyrier The event brought together a wide audience of participants representing Ukraine's Ministry of Digital Transformation and international partners of the Tallinn Mechanism participants learned about the best international practices in cybersecurity and identified ways to enhance the protection of Ukraine’s critical infrastructure The project is funded by the French Ministry of Foreign Affairs through the mAIDan Facility Ukraine program and is being implemented within the framework of the Tallinn Mechanism Metrics details Global warming has been associated with increased episodes of mass mortality events in invertebrates Although the spread of pathogens is one of multiple factors that contribute to such mass mortality events we don’t fully understand the pathophysiological consequences of sea warming on invertebrates we show that in temperature stress conditions circulating hemocytes in mussels leave the hemolymph to gain access to the intervalvar fluid before being released in seawater External hemocytes can survive for several hours in seawater before entering other mussels externally-infected hemocytes can enter naive mussels and promote bacterial dissemination in the host These results reveal the existence of a new opportunistic mechanism used by pathogens to disseminate in marine ecosystems Such mechanisms may explain how thermal anomalies triggered by global warming can favor episodic mass mortality observed in recent years in marine ecosystem there has been increasing interest in studying the physiology of their immune system which has until recently received considerably less attention compared to other Metazoans these studies show that movement of hemocytes in bivalves plays a central role in the infection process It is thus critical to better understand how environmental stress factors impact on the relationship between marine bacteria and invertebrates in marine coastal ecosystems and how such interplay may affect the community structure we report that hemocytes from mussels are released in seawater following temperature stress Hemocytes can survive for several hours in seawater while remaining functionally competent before entering other mussels We further show that opportunistic bacteria can infect hemocytes in seawater and take advantage of the entry of hemocytes into other mussels to spread into naive populations Release of hemocytes in the intervalvar fluid (A) Number of viable hemocytes in the intervalvar fluid (IVF) of M ater (AA) immediately an acute temperature stress at 30 °C (B) IVF volume collected from MD and AA immediately after the temperature stress The results represent the pool of two independent experiments (C) Viability of hemocytes found in IVF after the temperature stress as measured by trypan blue dye exclusion The results represent the pool of five independent experiments (D) Viability of hemolymphatic hemocytes collected immediately after the temperature stress (black bars) Viability was measured by trypan blue dye exclusion test Phagocytic activity of hemocytes released in seawater (A) Percentage of phagocytic cells and (B) number of beads phagocytosed by hemocytes released in seawater The data represent the pool of measures (n = 17) taken between 0.5–6 h The percentages of phagocytic cells and phagocytosed beads did not varied significantly within this interval of time for both mussel species (C) Representative flow cytometric histograms showing phagocytic activity of hemocytes from A ater released in seawater 3 and 5 h after thermal stress IVF and hemolymph were collected and analyzed for the presence of CFSE-positive cells (A) A representative contour plot showing hemocytes collected from IVF of mussels incubated without (left) and with CFSE-positive hemocytes (right) In the upper corner of the left contour plot a representative overlay histogram of unstained (control) hemocytes in blue and CFSE stained hemocytes in green (B) Effect of temperature on entry of CFSE-positive cells in IVF of mussels (C) Percentage of CFSE-positive cells recovered in IVF of mussels at different temperatures 90 min after addition of CFSE-stained cells The autofluorescence controls are shown in red Its shows the autofluorescence as measured in (A) generated by unstained hemocytes recovered in IVF of mussels incubated in the same conditions (D) Percentages of CFSE-positive cells present in hemolymph of Mytilus spp maintained at 23 °C for 90 min after addition of CFSE-positive cells The dotted line represents the average of CFSE-positive cells in hemolymph The data are representative of two independent experiments Infection of hemocytes by M. marinum. (A,B) Hemocytes from Mytilus spp. were infected with mCherry-expressing M. marinum. Cells were incubated with the CFSE and H33342 prior to analysis by confocal microscopy. (C) Uninfected hemocytes (Control). (D) Transmission electron microscopy showing a representative hemocyte infected with M. marinum. The insert on the upper right shows an enlarged area of the infected cell. The data are representative of two independent experiments. Hemocytes can serve as Trojan horses to infect mussels marinum or free bacteria were added to mussels the number of bacteria in IVF (A) and hemolymph (B) were measured following CFU counts Each point represents individual CFU counts our findings in mussels indicate the existence of a new opportunistic mechanism used by external bacteria to spread into naive hosts in marine ecosystems This mechanism is initiated by a temperature- and time-dependent release of circulating hemocytes in IVF Once in the IVF (also called “shell cavity”) hemocytes are released in seawater where they can survive for several hours while remaining functionally competent a mussel species only found in the Southern hemisphere and in blue mussels (Mytilus spp.) present in both Southern and Northern hemispheres We further showed that hemocytes in seawater can enter other mussels before gaining access to their hemolymphatic system we found that hemocytes in seawater can be infected by M marinum thereby providing an opportunity for the bacteria to enter and disseminate in naive mussels while being protected from the humoral and cellular components of the innate immune system these results suggest that temperature stress may play a critical role in spatial dynamics of pathogen propagation in marine ecosystems Although we still ignore if the release of hemocytes in seawater and their re-entry into other individuals also occur in other bivalves we do know that it occurs with Mytilus spp. suggesting that this migratory behavior of hemocytes is not restricted to mussel populations in Subantarctic regions but rather present in the Northern hemisphere as well Future studies will be needed to determine how hemocytes proceed from the hemolymphatic circulation to IVF Based on previous studies in humans and animals it is logical to hypothesize that elevation of temperature may initiate a cascade of events that include adhesion of hemocytes to activated vascular cells before extravasation through a more permeable vascular system It is logical to assume that global warming will favor external release of normal and leukemic hemocytes thereby accelerating horizontal transmission of cancer in mussels our findings reveal the existence of a new mechanism triggered by elevated temperatures that may have significant impact on the dissemination of pathogens Such mechanisms could explain how thermal anomalies triggered by global warming may favor episodic mass mortality in mussel beds observed in recent years Adult specimens (55–70 mm length) of Mytilus edulis desolationis (M ater) were collected on the intertidal rocky shore of Port-aux-Français (049°21.235S 070°13.490E) at Kerguelen Islands in December 2018 Mussels were sampled in the intertidal zone and kept in a container containing seawater from the sampling site The water was oxygenated by aeration and organisms were maintained throughout the transport phase at a water temperature closed to that measured on field (+ 7.5 °C) Mussels were immediately transferred to the marine laboratory of Port-aux-Français and placed in temperature-controlled (8 °C salinity ~ 34 ‰) aerated 50 l tanks containing filtered recirculating seawater maintained on a 12 h:12 h light/dark cycle 55–70 mm length) were obtained from a commercial supplier (PEI Mussel King Inc. Canada) and placed in a temperature-controlled (4 °C) aerated tank containing 20 l of 32‰ artificial saline water (Reef Crystal artificial marine salt individuals shell lengths and weight were measured hemocyte samples were collected from single individuals The intervalvar fluid (IVF) was first carefully and rapidly removed with the tip of a knife to minimize contamination with extrapallial fluids and collected into 15 ml sterile Falcon tubes Hemolymph was withdrawn from the adductor muscle using a syringe fitted with a 25 gauge needle The number of viable cells present in cell suspensions was determined by the standard trypan blue exclusion method and/or by flow cytometry using propidium iodide (PI) mussels (n = 6) of similar length were transferred into 1L tanks containing oxygenated seawater at 30 °C or To measure the release of hemocytes in seawater at different times (minutes) post-transfer aliquots of 0.5 ml (triplicates) of seawater were collected centrifuged at 280×g for 10 min at room temperature and resuspended in 50 μl of seawater This cell suspension was used for measuring the number of viable cells present in cell suspensions by by light microscopy using standard trypan blue exclusion method and to measure their phagocytic activity as described above Hemocytes were collected from 60 to 100 blue mussels (Mytilus spp.) and subsequently washed in seawater by low speed centrifugation at room temperature To measure survival of hemocytes in seawater at different temperatures monodispersed cell suspensions were obtained and cells added to 1L tanks (final cell concentration: 0.5 × 106 cells/ml) This cell suspension was used for measuring the number of viable cells present in cell suspensions by light microscopy using standard trypan blue exclusion method and for measuring phagocytic activity by flow cytometry For measuring entry of hemocytes into mussels hemocytes were stained with 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE OR) at a final concentration of 106 cells/ml following the manufacturer’s instructions resuspended and added to 1L tanks containing oxygenated seawater at the indicated temperatures obtain a final concentration of 0.5 × 106 cells/ml A total of 15–20 adult mussels (Mytilus spp.) were then added to each tank mussels (n = 5) were removed and their IVF and hemolymphatic cells collected and resuspended in seawater for analysis by flow cytometry as described above A total of 1000–5000 events was acquired for each sample and stored in the list mode data format for analysis using the CellQuestPro software Cells were lysed with one ml of Triton 0.1% (v/v) and detached using a sterile cell-scraper Bacteria were counted using serial dilution on 7H10 agar plate containing hygromycin (50 μg/ml) Colonies on plate were counted after 2 weeks of growth marinum genetically engineered to express mCherry constitutively were stained with CFSE and Hoescht 33342 (LifeTechnologies) and fixed with paraformaldehyde 2% (v/v) Fixed cells were mounted on microscope glass slides using ProLong Gold antifade reagent (Invitrogen) Images were acquired in sequential scanning mode on an LSM780 confocal microscope (Carl Zeiss Microimaging) every 370 nm The 3D images adjustments were as follow: zoom of 3,16 Pixel (0.04 mµ) and a pinhole of 1 airy Cells were fixed in 2.5% glutaraldehyde in cacodylate buffer at pH 7.4 with 0.2 M sucrose overnight and then post-fixated in 1.33% osmium tetroxide in Collidine buffer pH 7.4 for 1 h at room temperature After dehydration by successive passages through 25 95% and 100% (twice) solutions of acetone in water (for 15–30 min each) samples were immersed for 16–18 h in Spurr:acetone (1:1 v/v) Samples were then embedded in BEEM capsules using Spurr’s resin (TedPella) before incubation at 60–65 °C for 20–30 h samples were sliced using an ultramicrotome (LKB Brooma—2128 Ultratome) and were put onto Formvar and carbon coated-copper 200-mesh grids Samples were contrasted with 5% uranyl acetate in 50% ethanol (v/v) for 15 min followed by lead citrate for five minutes Cells were visualized using a Hitachi H-7100 transmission electron microscope with AMT XR-111 camera Mussels were acclimated for 1 week before the start of experiment hemocytes were isolated and infected with M Infected hemocytes were then added to a reservoir containing mussels A second (control) group of mussels were challenged with M marinum previously treated with amikacin for 24 h at room temperature washed in seawater and re-cultured for 8 days at room temperature Another control group included mussels not infected with M hemolymph and intervalvar fluids were collected centrifuged to collect cell pellets and processed for CFU counts as described above Statistical significance of the experiments was evaluated using the unpaired Student’s t-test or the Fisher’s exact test Results were considered statistically significant at P ≤ 0.05 The non-parametric Mann–Whitney U test was used for comparison of data that were not normally distributed n represents the number of experiments on individual animals Blue mussels (Mytilus edulis spp.) as sentinel organisms in coastal pollution monitoring: a review Horizontal transmission of clonal cancer cells causes leukemia in soft-shell clams Widespread transmission of independent cancer lineages within multiple bivalve species The significance of viruses to mortality in aquatic microbial communities Immune responses to infectious diseases in bivalves In Mucosal Health in Aquaculture (eds Beck Characterization of hemocytes from different body fluids of the eastern oyster Crassostrea virginica Transepithelial migration of mucosal hemocytes in Crassostrea virginica and potential role in Perkinsus marinus pathogenesis Bivalve immunity and response to infections: are we looking at the right place? Regulation of oyster (Crassostrea virginica) hemocyte motility by the intracellular parasite Perkinsus marinus: a possible mechanism for host infection Variation in abundance of Pacific blue mussel (Mytilus trossulus) in the Northern Gulf of Alaska Caza, F., Cledon, M. & St-Pierre, Y. Biomonitoring climate change and pollution in marine ecosystems: a review on Aulacomya ater. J. Mar. Biol. https://doi.org/10.1155/2016/183813 (2016) Seasonal changes in mRNA encoding for cell stress markers in the oyster Crassostrea gigas exposed to radioactive discharges in their natural environment Thermal stress and cellular signaling processes in hemocytes of native (Mytilus californianus) and invasive (M galloprovincialis) mussels: cell cycle regulation and DNA repair Negri, A. et al. Transcriptional response of the mussel Mytilus galloprovincialis (Lam.) following exposure to heat stress and copper. PLoS ONE 8, e66802. https://doi.org/10.1371/journal.pone/0066802 (2013) Heare, J. E., White, S. J., Vadopalas, B. & Roberts, S. B. Differential response to stress in Ostrea lurida as measured by gene expression. 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SOS Pinna nobilis: a mass mortality event in western Mediterranean Sea. Front. Mar. Sci. 4, 220. https://doi.org/10.3389/fmars.2017.00220 (2017) Rivetti, I., Fraschetti, S., Lionello, P., Zambianchi, E. & Boero, F. Global warming and mass mortalities of benthic invertebrates in the Mediterranean Sea. PLoS ONE 9, e115655. https://doi.org/10.1371/journal.pone.0115655 (2014) Allograft inflammatory factor-1 stimulates hemocyte immune activation by enhancing phagocytosis and expression of inflammatory cytokines in Crassostrea gigas Specific expression of antimicrobial peptide and HSP70 genes in response to heat-shock and several bacterial challenges in mussels Novoa, B. et al. Immune tolerance in Mytilus galloprovincialis haemocytes after repeated contact with Vibrio splendidus. Front. Immunol. 10, 1894. https://doi.org/10.3389/fimmu.2019.01894 (2019) Yonemitsu, M. A. et al. A single clonal lineage of transmissible cancer identified in two marine mussel species in South America and Europe. 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Physiol. 9, 839. https://doi.org/10.3389/fphys.2018.00839 (2018) Download references Part of this research project was performed at Port-aux-Français Station (Kerguelen Islands) and supported by the French Polar Institute Paul-Emile-Victor (IPEV) The authors would like to thank all the personnel from the IPEV and the Terres Australes et Antarctiques Françaises (TAAF) for their help and hospitality during our stay in the Kerguelen archipelago Marlène Fortier for their excellent technical help This work was funded in part by the National Science and Engineering Research Council of Canada (YSP) and IPEV (SB) UMR-I 02 SEBIO Stress Environnementaux et Biosurveillance des Milieux Aquatiques All authors were responsible for interpretation of data and critical appraisal drafted the manuscript with input from all authors All authors discussed the results and implications and commented on the manuscript at all stages Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-020-76677-z Feature Films Database Southern Mediterranean films database Scriptwriters European Film Schools Production Companies Distributors International Sales Submit a Film Industry Reports Co-Production Podcast Online Screenwriting Training Course Guided Course for Feature Film Writing Script Analysis Analysis of the potential of your series Cineuropa's Training Catalogue Film Festival Photographs Newsletter Photogalleries EUFCN Location Award Euro Film Fest 27 Times Cinema GoCritic! Advertise on Cineuropa Logos and Banners PRODUCTION / FUNDING France by Fabien Lemercier Charlie Anson and Annabelle Lengronne all star in Laura Piani’s first feature film produced by Les Films du Veyrier and Sciapode the story for this romantic comedy revolves around Agathe who realises that her life doesn’t live up to the promises of literature finds herself forced to fulfil her dreams of becoming a writer in order to stop messing up her love life Please subscribe to our newsletter to receive more stories like this directly in your inbox 02/05/2025Production / Funding – Italy Shooting begins on Walter Fasano’s Nino, a portrait of scoring maestro Nino Rota 02/05/2025Production / Funding – Belgium Wallimage is backing Michaël R Roskam's Le Faux Soir 30/04/2025Production / Funding – Italy The final clapperboard slams on Il falsario, starring Pietro Castellitto 30/04/2025Production / Funding – UK/France/Germany Sally Potter’s Alma to star Pamela Anderson and Dakota Fanning 29/04/2025Production / Funding – Spain Claudia Pinto finishes filming Morir no siempre sale bien 29/04/2025Production / Funding – Latvia The National Film Centre of Latvia unveils the recipients of its latest round of funding Subscribe to our newsletter to receive the most important daily or weekly news on European cinema Crossing Europe 2025 Review: Callas, Darling Cannes 2025 Marché du Film The Party’s Over! leads France TV Distribution’s Cannes slate CPH:DOX 2025 CPH:DOX Industry Europa Distribution explores the release of documentaries at CPH:DOX Cannes 2025 Marché du Film AFCI runs its second annual Global Film Commission Network Summit at Marché du Film Festivals / Awards Czech Republic Czech Republic’s Anifilm goes sci-fi Distribution / Releases / Exhibitors Europe European Arthouse Cinema Day set to return on 23 November Cannes 2025 Marché du Film Indie Sales presents a three-star line-up at Cannes HOFF 2025 The Shadow and U Are the Universe win at Estonia’s Haapsalu Horror and Fantasy Film Festival Crossing Europe 2025 Awards The New Year That Never Came and The Flats crowned at Crossing Europe Cannes 2025 Marché du Film Be For Films to sell Love Me Tender in Cannes Cannes 2025/Sponsored Latvia set to shine bright at Cannes, led by Sergei Loznitsa’s competition entry Two Prosecutors Las Palmas 2025 MECAS/Awards Manuel Muñoz Rivas and Joana Carro win awards at the eighth MECAS Market TrendsFOCUSA busy spring festival season awaits the European film industry. Cineuropa will continue to keep its readers up to date with the latest news and market insights, covering the buzziest events, including Cannes, Kraków, Karlovy Vary, Tribeca, Hot Docs, Annecy, Brussels, Munich and many others Distribution, Exhibition and Streaming – 06/05/2025Europa Distribution explores the release of documentaries at CPH:DOXThe network has held a case study workshop as part of its brand-new partnership with the Copenhagen-based festival Distribution, Exhibition and Streaming – 02/05/2025Slovak crime-thriller Černák becomes the highest-grossing film in domestic cinemasThe second film in the saga about a local mafia boss, directed by Jakub Króner, outgrossed its first part, which dominated Slovak cinemas last year Jaśmina Wójcik • Director of King Matt the First The Polish director discusses her approach to taking on a 1920s children’s literary classic in an unexpected way Želimir Žilnik • Director of Eighty Plus The Serbian director discusses his deep suspicion of ideologies in relation to his irresistibly charming latest feature, which follows a man whose life spans three political systems Paulina Jaroszewicz • Distribution and marketing manager, New Horizons Association Cineuropa sat down with the Polish distributor to discuss her company’s strategy as well as the connection between its distribution line-up and BNP Paribas New Horizons Festival’s programme Lorcan Finnegan • Director of The Surfer The Irish filmmaker discusses his mystery-thriller, how he created the character with Nicolas Cage and his approach to the use of colours in the film Privacy Policy The images used on this website have been provided by journalists and are believed to be free of rights if you are the owner of an image used on this website and believe that its use infringes on your copyright We will remove the image in question as soon as possible We have made reasonable efforts to ensure that all images used on this website are used legally and in accordance with copyright laws About us | Contact us | Logos and Banners Mission | Partners | Team | Donations | Terms and conditions 1: Confocal microscope image of the caterpillar-like bacterium Conchiformibius steedae incubated with fluorescently labelled cell wall precursors to follow its cell growth (CC BY 4.0 Philipp Weber and Silvia Bulgheresi) 2: Scanning electron micrograph of the caterpillar-like bactrioid Simonsiella muelleri up to 4 µm long (CC BY 4.0 Sammy Nyongesa and Frédéric Veyrier) Evolution of longitudinal division and multicellularity in oral bacteria bacteria evolved to divide along their longitudinal axis without parting from one another A research team co-led by environmental cell biologist Silvia Bulgheresi from the University of Vienna and microbial geneticist Frédéric Veyrier from the Institut national de la recherche scientifique (INRS) just published their new insights in Nature Communications they described the division mode of these caterpillar-like bacteria and their evolution from a rod-shaped ancestor They propose to establish Neisseriaceae oral bacteria as new model organisms that could help pinpoint new antimicrobial targets Although our mouth houses over 700 species of bacteria and its microbiota is not much is known about how oral bacteria grow and divide The mouth is a tough place to live in for bacteria The epithelial cells lining the inner surface of the oral cavity are constantly shed and organisms that inhabit this surface will therefore struggle for attachment It is perhaps to better stick to our mouth that bacteria of the family Neisseriaceae have evolved a new way to multiply Whereas typical rods split transversally and then detach from each other some commensal Neisseriaceae that live in our mouths attach to the substrate with their tips and divide longitudinally – along their long axis they remain attached to one another forming caterpillar-like filaments Some cells in the resulting filament also adopt different shapes possibly to perform specific functions to the benefit of the whole filament The researchers explain: "Multicellularity makes cooperation between cells possible for example in the form of division of labor and may therefore help bacteria to survive nutritional stress." The team of researchers first employed electron microscopy to survey bacterial cell shape across the Neisseriaceae family that include the two standard cell shapes (rod and coccus) in addition to the caterpillar-like filaments By comparing their cell shapes and genomes throughout the Neisseriaceae family longitudinally dividing bacteria evolved out of rod-shaped they could pinpoint which genes were likely responsible for the unusual multiplication strategy They then used fluorescence labelling techniques to visualize the progression of cell growth in the multicellular bacteria and finally compared the genetic make-up of these with 'classic' they tried to recreate that evolution by introducing the genetic changes into rod-shaped Neisseriaceae Although they could not force rod-shaped bacteria to become multicellular genetic manipulation resulted into longer and thinner cells "We speculate that in the course of evolution through a reworking of the elongation and division processes perhaps to better thrive in the oral cavity" "Apart from helping us to understand how cell shape evolved multicellular Neisseriaceae may be useful to study how bacteria learned to live attached to the surface of animals the only place they have been found to occur so far explains Silvia Bulgheresi from the Department of Functional and Evolutionary Ecology at the University of Vienna Philipp Weber from the University of Vienna highlights that "expanding the cell biology field to additional morphologies and symbiotic species is also crucial to increase the pool of protein targets (e.g. antibiotic targets) for biopharmaceutical applications." Sammy Nyongesa such as that undertaken here for the Neisseriaceae Veyrier F.J.*2: Evolution of longitudinal division in multicellular bacteria of the Neisseriaceae family *1authors contributed equally *2authors contributed equally In: Nature Communications DOI: 10.1038/s41467-022-32260-w More research content on the topic of cells can be found in the University of Vienna's science magazine Rudolphina Sitemap | Impressum | Barrierefreiheit | Datenschutz­erklärung | Cookie-Hinweis | Druckversion Maria Crawford a village on the eastern shore of Lake Annecy Chambéry Savoie Mont Blanc airport and Geneva airport are both about 50 minutes’ drive away What A contemporary house with more than 440 sq m of living space one of which is part of a studio with its own kitchen landscaped gardens and a heated infinity pool Why The house has uninterrupted views across Lake Annecy that can be seen from most of the rooms as well as the various outdoor seating areas and pool Who Savills €3mnWhere About 10 minutes’ walk from the centre of Chamonix and just over an hour’s drive south-east of Geneva airport including a master suite with a wood-burning stove two garages and a hot tub on one of the terraces A guest chalet houses a further en-suite bedroom with an additional wood-burning stove Why The property has direct views of Mont Blanc Who Barnes International less than 10km from the border with Switzerland Les Gets is about 30 minutes by car; Morzine is 40 minutes What An 18th-century farmhouse and barn that have been renovated by the current owners to create 900 sq m of living space between the two buildings including a master suite that fills the top floor Why The property is on 3,000 sq m of walled grounds the ground floor of the barn has been designed for use as a yoga or exercise studio Who Savills The nearest international airport is Geneva What A four-bedroom chalet with an open-plan There are open and sheltered terraces for outdoor dining and for taking in the views The property also comes with an insulated garage hiking and mountain biking opportunities nearby; in winter Who Knight Frank about midway between Annecy to the south and Geneva to the north What An architect-designed house built in 2018 It has 168 sq m of living space including three bedrooms and there is a large garage and garden shed Why The property’s floor-to-ceiling windows and long terrace with a swimming pool offer stunning views of the surrounding mountains Who Sotheby’s International Realty Find out about our latest stories first — follow @FTProperty on Twitter or @ft_houseandhome on Instagram The Local Europe ABVästmannagatan 43113 25 StockholmSweden Please log in here to leave a comment When Londoners Jackie and Paul Herbert started house-hunting on Lake Annecy in the Haute-Savoie region of France they were seeking a “little house on the lake” who have three adult children and lived in the Middle East for many years loved the convenience of Annecy from Geneva across the border in Switzerland (45 minutes) and its proximity to the pretty ski resort of La Clusaz The modest holiday home they planned in 2010 turned out to be neither modest nor a holiday home. Instead, they acquired an old French farmhouse and barn that they have transformed into a nine-bedroom luxurious retreat. It is on the market for €13.75 million (£12.66 million) La compo des Amazones Reserve face à l'ASM Romagnat Découvrez la composition de l'équipe Crabos pour cette nouvelle journée de championnat Rendez-vous sur le terrain annexe du Stade Lesdiguières demain dès 14h00 (Alamercery) et 16h00 pour les CRABOS