MicrobiologyOpen

From a One Health perspective, identifying environmental reservoirs of antibiotic resistance determinants with potential clinical relevance is increasingly important. Among resistance mechanisms, {beta}-lactamases are of particular concern because they compromise the efficacy of {beta}-lactam antibiotics, the most widely used class in clinical practice. Antarctic soils harbor thousands of putative {beta}-lactamase genes, yet the functional properties and resistance contributions of these enzymes remain largely unexplored. Here, we investigated the distribution, diversity, and functional impact of intrinsic class C {beta}-lactamases (AmpC-type) in Antarctic members of the Pseudomonas fluorescens species complex, a lineage widely distributed in Antarctic soils and increasingly recognized as an opportunistic pathogen of humans, animals, and plants. Genome-scale analyses revealed that class C {beta}-lactamases are intrinsic, widespread, and highly diverse within this lineage. Phenotypic assays demonstrated that Antarctic isolates exhibit elevated resistance to {beta}-lactam antibiotics, particularly oxyimino-cephalosporins such as cefotaxime and ceftazidime, and that this phenotype is largely attenuated by {beta}-lactamase inhibition. Heterologous expression of selected Antarctic AmpC variants in a susceptible Escherichia coli host confirmed their ability to increase minimum inhibitory concentrations to oxyimino-cephalosporins. Notably, several Antarctic {beta}-lactamases harbor amino acid substitutions previously associated with extended-spectrum AmpC (ESAC) variants, including M174L and N346I, while others display broader substrate profiles despite lacking known ESAC-associated signatures. Comparative analyses further showed that the sequence diversity of Antarctic class C {beta}-lactamases exceeds that reported for clinical PDC variants from Pseudomonas aeruginosa. Together, these findings provide functional evidence that pristine Antarctic environments constitute reservoirs of naturally occurring class C {beta}-lactamases with extended-spectrum potential. Our results highlight the evolutionary depth and functional diversity of environmental {beta}-lactamases and underscore the importance of incorporating remote ecosystems into One Health-oriented antimicrobial resistance surveillance frameworks.

The use of antimicrobial compounds in humans, animals and in agriculture leads to environmental antimicrobial contamination through domestic, industrial and agricultural wastewater. Efforts have been made to perform environmental risk assessments based on the potential of these compounds to select for antimicrobial resistance (AMR) at certain concentrations in bacteria. This has resulted in predicted no effect concentrations (PNEC) which determine the minimum thresholds required to select for resistance. However, the effects of these compounds on intracellular transposition within bacterial isolates, a major driver of AMR, have not been previously assessed. Here, we assess the effect of differing sub-inhibitory concentrations of the third-generation cephalosporin, ceftriaxone, on the rate of intracellular transposition in combination with colistin and kanamycin. Two triple replicons systems (RS1 and RS2) were developed to assess this, each containing a chromosome, plasmid and entrapment vector. We show that sub-inhibitory concentrations of ceftriaxone exert hormetic effects on the intracellular transposition rate in RS1 and a steady linear increase in RS2. This defines a predicted no effect concentration for transposition (PNECT) for ceftriaxone as 320 ng/L in RS1 and 3200 ng/L in RS2. This provides a minimum threshold for the environmental impact of ceftriaxone on biological systems at the sub-cellular scale, which is applicable to industrial standards of waste management, where consideration of ecological impact is central.

ABSTRACT: The bacterial small RNA (sRNA) OxyS is expressed in Escherichia coli during oxidative stress. The sRNA OxyS enhances cell survival by controlling genes involved in the regulation of hydrogen peroxide (H2O2) and iron-sulfur (Fe-S) cluster formation. Here, we used the MS2 affinity purification coupled with RNA sequencing (MAPS) technique to identify new target mRNAs of the sRNA OxyS. Our analysis revealed a significant enrichment of mepS mRNA, which encodes a peptidoglycan endopeptidase that promotes cell growth. Our results confirm a previous report on the sRNA OxyS repressing the translation of mepS. We also found that an {Delta}oxyS background facilitates the emergence of mutations, conferring increased resistance to the last-resort antibiotics polymyxin B and E (colistin), but only in the presence of the target mepS gene. This suggests that the translation repression of mepS by OxyS could prevent mutations in bacterial DNA during H2O2-induced oxidative stress. Moreover, we show that adding the antioxidant thiourea or sequestering iron in the {Delta}oxyS background effectively reduces the emergence of resistance against both polymyxin B and colistin. These results suggest that reactive oxygen species (ROS), in conjunction with intracellular iron, play a key role in driving the emergence of antibiotic resistance. Overall, our work underlines a mechanism of antimicrobial emergence implicating oxidative stress, intracellular Fe, and cell wall remodeling in E. coli. IMPORTANCE: This study uncovers an underexplored link between peptidoglycan remodeling and oxidative stress responses during exposure to antibiotics. By elucidating how MepS and the sRNA OxyS interact in the presence of polymyxins and oxidative stress, our study suggests that MepS may exert an anti-mutator function. The repression of mepS translation by OxyS seems to limit the emergence of antibiotic resistance driven by DNA mutations. Together, these findings suggest cell wall remodeling and oxidative stress response pathways as promising targets to enhance antibiotic efficacy and limit the emergence of resistance.

Life on Earth is strictly dependent on liquid water. In polar terrestrial environments, water exists in solid state during almost the entire year. Polar microorganisms have not only to adjust their metabolism to survive at subzero temperatures, but also need to cope with extremely dry conditions. We investigated the presence of desiccation-adapted bacteria in Arctic permafrost and Antarctic surface soils and characterized their survivability to dryness. We selected desiccation tolerant cells by treating the soils with chloroform prior to cultivation, in order to mimic the stress of low water activity for long periods. From over 1000 colonies from different samples, 23 unique strains were selected and identified as members of phyla Firmicutes, Proteobacteria and Actinobacteria. About 60% of the strains survived after 50 days in anhydrobiosis. The competence to withstand desiccation varied between close related strains isolated from different locations, bringing the question if environmental conditions may play a role in the observed desiccation tolerance. Survivability was also affected by the solution in which the cells were suspended before drying; R2B medium being more protective than water. This is the first time that chloroform was used to select desiccation tolerant microorganisms from polar soils. The collection of polar microorganisms described herein opens the possibility of further experiments aiming to investigate the resistance mechanisms of polar anhydrobionts. Desiccation tolerance is fundamental to the survivability of microorganisms to the space environment and at the surface of thin-atmosphere planets like Mars. Therefore, the selected strains may open a road to better understand the limits of cold adapted life on Earth and beyond, and compare mechanisms of resistance with anhydrobionts from divergent extreme environments.

The genetic engineering of thermophilic bacteria is constrained by limited availability of thermostable antibiotic resistance markers for selection. Clostridium thermocellum, a promising candidate for consolidated bioprocessing of lignocellulosic biomass, requires reliable selection systems that function at elevated temperatures. Here, we systematically evaluated antibiotic susceptibility profiles and identified novel resistance markers for this thermophile through bioinformatic screening and experimental validation. We screened 823 thermophilic genomes against the Comprehensive Antibiotic Resistance Database, identifying 1,115 antibiotic resistance genes. From these, we selected candidates with highest homology to resistance determinants for rifampicin, tetracycline, erythromycin, thiamphenicol, and neomycin. We identified three novel antibiotic resistance systems that function in this organism: tetracycline/tet(45), erythromycin/cmeC, and rifampicin/rbpA. Of these, the rifampicin/rbpA provided the highest selection range , > 10,000-fold. Our results establish rbpA as an outstanding selectable marker for thermophilic genetic engineering and provide a validated workflow for discovering thermostable resistance determinants in high-temperature microorganisms. ImportanceThermophilic bacteria like Clostridium thermocellum hold tremendous potential for sustainable biofuel production from plant biomass, but their genetic manipulation has been severely limited by the lack of selection markers that work at high temperatures. Many existing antibiotic resistance systems do not function at thermophilic temperatures, and many approaches to genetic manipulation require multiple antibiotic resistance markers. Currently only two markers are available for C. thermocellum, and only one (cat) functions well. The newly-developed rbpA marker functions well in C. thermocellum and is likely to provide dramatic new opportunities for engineering thermophilic host organisms.

Polymicrobial infections are small communities of multiple interacting bacterial species. Interactions among constituent species may modify the growth of community members in the presence of antibiotics, for example via degradation of the antibiotic or induction of specific resistance mechanisms. However, for most polymicrobial infections the nature of such interactions is opaque, while they may affect both treatment efficacy and the evolution of antibiotic resistance. Here, we describe that past interaction of enterococci with Pseudomonas aeruginosa creates legacy effects that substantially alter their antibiotic tolerance and resistance evolution. Specifically, we find that the temporary exposure to pyocyanin, a secondary metabolite produced by P. aeruginosa, increases the efflux in enterococci. These tolerance legacy effects promote the evolution of antibiotic resistance of enterococci. This work shows that transient interactions in polymicrobial communities can alter the evolutionary fate of community members.

The gut microbiome plays a vital role in host homeostasis and understanding of its biology is essential for a better comprehension of the etiology of disorders such as foetal alcohol spectrum disorders. Here we assessed the effectiveness of targeted and untargeted (metagenomic) nanopore sequencing approaches to profile the gut microbiota of infant mice exposed to ethanol in utero. DNA extracts from the gut content of 12 infant mice exposed to ethanol in utero were analysed using one untargeted and two targeted (full-length 16S rRNA gene and the 16S-ITS-23S region of the ribosomal RNA operon) nanopore sequencing approaches. The targeting of the full-length 16S rRNA gene provided the most comprehensive analysis of the mouse gut microbiota. The differences in diversity between approaches were accounted by the sequencing target (p-value < 0.001). Faecalibaculum rodentium and Duncaniella sp. were the two most prevalent taxa detected using targeted sequencing approaches, while bacterial taxa were more evenly represented when using the metagenomic approach. Full-length 16S rRNA gene nanopore sequencing provides the most discriminatory microbiota compositional analysis of mice faecal samples. However, using nanopore sequencing approaches targeting the metagenome or different taxonomically-informative DNA region appears to introduce significant target-related biases. ImportanceCurrent nanopore approaches have not been standardized which may confound the biological interpretations of hight-throughput sequencing datasets. Additionally, nanopore sequencing still present a high error-rate compared to other more mature sequencing technologies, such as Illumina sequencing. These technological handicaps create the need to study and optimize nanopore sequencing approaches to answer biological questions, such as interrogations of the microbial composition and abundance of clinical and environmental samples. In this work, three nanopore sequencing approaches were designed and attempted to optimize fungal and bacterial profiling sequencing methodologies. Two targeted methods based on the bacterial 16S rRNA gene, and 16S-ITS-23S rrn operon region, and one untargeted shotgun/metagenomic approach were tested. Despite potential experimental and/or bioinformatical biases were found, the 16S rRNA gene-targeted nanopore sequencing was the most comprehensive approach to study the microbial composition of the infant mice gut microbiotas.

Trans-translation is a crucial bacterial process and a target for new antibiotics. We developed two Pseudomonas aeruginosa biosensor strains that detect trans-translation inhibitors by exploiting the bacteriums natural red fluorescence, linked to protoporphyrin IX accumulation. The first biosensor monitors tmRNA-SmpB-mediated tagging, while the second serves as control for biosensor 1 by keeping track of ClpP1-related proteolysis and porphyrin biosynthesis. Validation through gene deletions and complementation confirmed their specificity. These biosensors were effective in screening antibiotics and designed inhibitors, demonstrating their potential for high-throughput identification of trans-translation inhibitors in drug-resistant P. aeruginosa.

Plasmids can impact the evolution of their hosts, e.g. due to carriage of mutagenic genes, through cross-talk with host genes or as result of SOS induction during transfer. Here we demonstrate that plasmids can cause microindel mutations in the host genome. These mutations are driven by the production of single-stranded DNA molecules that invade replication forks at microhomologies and subsequently get integrated into the genome. Using the gammaproteobacterial model organism Acinetobacter baylyi, we show that carriage of broad host range plasmids from different incompatibility groups can cause microindel mutations directly or indirectly. The plasmid pQLICE belonging to the incompatibility group Q (IncQ) and replicating by a characteristic strand displacement mechanism can generate chromosomal microindel mutations directly with short stretches of DNA originating from pQLICE. In addition, the presence of plasmids can increase microindel mutation frequencies indirectly (i.e., with chromosomal ectopic DNA) as shown with the IncP plasmid vector pRK415 (theta replication mechanism), presumably through plasmid-chromosome interactions that lead to DNA damages. These results provide new mechanistic insights into the microindel mutation mechanism, suggesting that single-stranded DNA repair intermediates are the causing agents. By contrast, the IncN plasmid RN3 appears to suppress host microindel mutations. The suppression mechanism remains unknown. Other plasmids in this study confer ambiguous or no quantifiable mutagenic effects.

Plasmids are genetic elements that disperse horizontally between different strains and species of bacteria and a major factor in the dissemination of virulence factors and antibiotic resistance. Understanding the ecology of plasmids has a notable anthropocentric value and therefore the interactions between bacterial hosts and individual plasmids have been studied in detail. However, bacterial systems often carry multiple genetically distinct plasmids, but dynamics of these multiplasmid "clashes" has remained unstudied. Here, we set to investigate the survival of 11 mobilizable or conjugative plasmids in five different ecological settings. The key incentive was to determine whether plasmid dynamics are reproducible and whether there are trade-offs in plasmid fitness that stem from the ecological situation of their initial hosts. Growth rates and maximum population densities increased in all communities and treatments over the 42-day evolution experiment although plasmid contents at the end varied notably. We show that large multiresistance conferring plasmids are unfit when the community also contains smaller plasmids with fewer resistance genes. This suggests that restraining the use to few antibiotics can make bacterial communities sensitive to others. The hosts also appear to react to the presence of multiple genetically different plasmids by enhancing fimbriae production instead of alleviating costs of individual plasmids. In general, the survivors of the here-studied multi-plasmid clash are significantly affected by the presence or absence of antibiotic selection and plasmid-free hosts of varying fitness. Therefore, these trade-offs in different settings can explain for example why some resistance plasmids have an advantage during a rapid proliferation of antibiotic sensitive pathogen whereas others dominate in alternative situations.

Toxin-antitoxin (TA) systems are important for stress adaptation in prokaryotes, including persistence, antibiotic resistance, pathogenicity, and biofilm formation. Toxins can cause cell death, reversible growth stasis, and direct inhibition of crucial cellular processes through various mechanisms, while antitoxins neutralize the effects of toxins. In bacteria, these systems have been studied in detail, whereas their function in archaea remains elusive. During heat stress, the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibited an increase in the expression of several bicistronic type II vapBC TA systems, with the highest expression observed in the vapBC4 system. In the current study, we performed a comprehensive biochemical characterization of the VapBC4 TA system, establishing it as a bonafide type II toxin-antitoxin system. The VapC4 toxin is shown to have high-temperature catalyzed RNase activity specific for mRNA and rRNA, while the VapB4 antitoxin inhibits the toxic activity of VapC4 by interacting with it. VapC4 toxin expression led to heat-induced persister-like cell formation, allowing the cell to cope with the stress. Furthermore, this study explored the impact of vapBC4 deletion on biofilm formation, whereby deletion of vapC4 led to increased biofilm formation, suggesting its role in regulating biofilm formation. Thus, during heat stress, the liberated VapC4 toxin in cells could potentially signal a preference for persister cell formation over biofilm growth. Thus, our findings shed light on the diverse roles of the VapC4 toxin in inhibiting translation, inducing persister cell formation, and regulating biofilm formation in S. acidocaldarius, enhancing our understanding of TA systems in archaea. IMPORTANCEThis research enhances our knowledge of Toxin-antitoxin (TA) systems in archaea, specifically in the thermoacidophilic archaeon Sulfolobus acidocaldarius. TA systems are widespread in both bacterial and archaeal genomes, indicating their evolutionary importance. However, their exact functions in archaeal cellular physiology are still not well understood. This study sheds light on the complex roles of TA systems and their critical involvement in archaeal stress adaptation, including persistence and biofilm formation. By focusing on S. acidocaldarius, which lives in habitats with fluctuating temperatures that can reach up to 90, the study reveals the unique challenges and survival mechanisms of this organism. The detailed biochemical analysis of the VapBC4 TA system, and its crucial role during heat stress, provides insights into how extremophiles can survive in harsh conditions. The findings of this study show the various functions of the VapC4 toxin, including inhibiting translation, inducing persister-like cell formation, and regulating biofilm formation. This knowledge improves our understanding of TA systems in thermoacidophiles and has broader implications for understanding how microorganisms adapt to extreme environments.

Plasmid-encoded mobile colistin resistance (mcr) genes have raised concern due to dissemination potential. While mcr variants are reported across One Health compartments, they remain unreported in grass. This study characterises a novel mcr variant (mcr-13.1), detected in Escherichia coli isolated from the grass phylosphere within an agricultural grassland. The two mcr-positive isolates were clonal copies isolated at timepoints eight weeks apart. They belonged to the serotype O17:H18 and were of the sequence type ST394. The E. coli were phenotypically susceptible to {beta}-lactams, aminoglycosides, quinolones, sulphonamides, phenicols, tetracyclines, diaminopyrimidine and colistin (Minimum Inhibitory Concentration (MIC) = 0.5 {micro}g/mL). The mcr-13.1 gene was encoded on an IncFIB plasmid. This plasmid was transferable by conjugation but the colistin MIC of the E. coli J53 transconjugant did not change (0.5 {micro}g/mL). Further, cloned pUC19::mcr-13.1 did not alter the colistin MIC for E. coli DH5 (0.25 {micro}g/mL). The translated amino acid sequence showed highest homology (82 %) to MCR-10.2 and MCR-10.4. Our findings identify grass as a previously unrecognised reservoir for E. coli carrying mobile mcr genes, reports the identification of the novel mcr-13.1 variant from this niche and demonstrates the importance of genomic screening in identifying mcr genes that would otherwise remain undetected.

Biocides are widely used to control and prevent healthcare-associated infections and understanding how nosocomial pathogens respond to biocidal agents is key to improving infection prevention and control products and practices. An evolution model was used to study how Staphylococcus aureus and Enterococcus faecalis responded after repeated exposure to sub-lethal concentrations of chlorhexidine digluconate (CHX) and octenidine dihydrochloride (OCT) when grown planktonically and as biofilms. Both pathogens were able to adapt to grow at concentrations above the MIC of both biocides with planktonic lineages surviving at higher concentrations of both agents than biofilm lineages. Exposure to CHX was linked with lower biofilm biomass production in E. faecalis although biofilm biomass increased for S. aureus isolates after exposure to both agents. Evolved isolates had no major fitness deficit and only low-level changes to susceptibility to antibiotics were observed after biocide exposure. Sequencing of biocide adapted mutants repeatedly identified mutations within fakA encoding a fatty acid kinase in independent lineages of S. aureus after exposure to both biocides in all conditions. Analogous changes were observed within the homologous gene in parallel experiments with Enterococcus faecalis. Further assays to study the mechanistic basis and relationship to phospholipid production showed that evolved isolates with fakA mutations accumulated less ethidium bromide than parent strains, exhibited altered cell envelope morphology and decreased susceptibility to daptomycin. This data shows important pathogens can evolve limited tolerance to two common biocides but that this has collateral impacts on biofilm formation, colony morphology and fitness. FakA appears to play an important role in biocide tolerance.

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify non-neutral mutations by selection or counter- selection, neutral mutations usually require DNA sequencing to be identified in a population, which are normally expensive and time-consuming. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counter- selection. We propose a novel methodology to quantify DNA using Sanger sequencing, that we validated experimentally with specially-engineered plasmids both in vitro and in co-transformed E. coli by and assessing our predictions with qPCR and fluorescence quantifications. The method relies on the alignment of the electropherograms from the query and reference samples, where we quantify the DNA concentration from the amplitude ratio of aligned electropherogram peaks. Our DNA quantification will allow quantifying genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with consistent reduction of costs compared to canonical approaches such as qPCR.

Understanding diverse microbial communities is important due to their ecological and medical significance. Bacterial cells are genetically and phenotypically heterogeneous, making their interactions in the communities complex. The heterogeneity and interactions of cells contribute to the formation of specific spatial structures, such as biofilms, and the spread of antibiotic resistance. Here, we describe a novel single-cell approach for studying the cellular heterogeneity and spatial interactions in microbial communities that combines polyacrylamide bead encapsulation of cells and split-pool-barcoding. We demonstrate the method by determining artificially imposed interactions and connecting the taxonomic information in a mock three-species bacterial community with a species-specific genomic target. The method can be utilised for the spatial analysis of microbial communities as well as, once fully optimised for single-cell resolution, linking genetic traits to single cells.

Marine bacteria that have adapted to thrive in extreme environments, such as Pseudoalteromonas haloplanktis TAC125 (PhTAC125), offer a unique biotechnological potential. The discovery of an endogenous megaplasmid (pMEGA) raised questions about its metabolic impact and functional role in this strain. This study aimed at streamlining the host genetic background by curing PhTAC125 from the pMEGA plasmid using a sequential genetic approach. We combined homologous recombination by exploiting a suicide vector with the PTasRNA gene silencing technology to interfere with pMEGA replication machinery. This approach led to the construction of the novel PhTAC125 KrPL2 strain, cured from the pMEGA plasmid, which exhibited no significant differences in the growth behaviour, though showcasing enhanced resistance to oxidative stress and a reduced capability of biofilm formation. These findings represent a significant achievement for understanding of the role of pMEGA plasmid and for the biotechnological applications of PhTAC125 in recombinant protein production. This opens up the possibility to exploit pMEGA valuable genetic elements and further advancing the genetic tools for PhTAC125.

The emergence and proliferation of multidrug-resistant bacteria pose a major threat to global public health. To address an imminent crisis, it is essential to identify and characterize new antibacterial molecules. With that in mind, we developed the ADP-MoA platform, that facilitates the discovery of new antibiotics and provides preliminary insights into their mechanisms of action. The basic idea is to simultaneously visualize antibiotic activity - growth inhibition, along with one of the three classic antibiotics mechanisms of action: DNA damage/inhibition of DNA replication, protein synthesis inhibition and cell wall damage. The platform consists of three different chromosomal fusions between the promoters of recA, ampC or armZ and the luxCDABE operon. The platform was constructed and hitherto tested in the pathogenic opportunistic bacterium Pseudomonas aeruginosa. As a proof of concept we showed that the promoter fusions were each activated by the expected antibiotics with known mechanisms of action. The armZ::luxCDABE fusion responded to antibiotics that inhibit protein synthesis (macrolides, chloramphenicol, tetracyclines and aminoglycosides), ampC::luxCDABE was induced by {beta}-lactams and recA::luxCDABE was induced by quinolones. Interestingly, ciprofloxacin induced PampC and ParmZ as well, albeit at a lower level. The ADP-MoA platform offers a readily implementable, low-cost approach with significant potential for high-throughput screening of antimicrobials against P. aeruginosa and other bacterial species.

Thermophilic anaerobic organisms, particularly species that can naturally degrade lignocellulosic biomass, show great promise for next generation bioprocessing. This has led to the development of nascent genetic systems to metabolically engineer these non-model organisms. However, a major challenge remains a lack of reliable reporter systems compatible with the combination of thermophilic and anaerobic growth conditions. Additionally, native glycoside hydrolases in these organisms limit the usefulness of traditional glycosidic enzyme reporters (e.g. LacZ) because of the native background activity present on para-nitrophenyl glucoside substrates. Here we describe the development of a straightforward and robust enzymatic reporter system that overcomes these challenges in Anaerocellum (f. Caldicellulosiruptor) bescii, an anaerobic, extremely thermophilic (Topt [~]78 {degrees}C), lignocellulolytic bacterium. Our method is based on heterologous expression of hyperthermophilic archaeal galactosidases: an -galactosidase from Pyroccous furiosus (Pfgal), and a {beta}-galactosidase from Caldivirga maquilingensis (Cm{beta}gal). We show that these reporters produce strong, orthogonal signals on colorimetric substrates at high temperatures ([&ge;]90{degrees}C) that eliminate background activity from endogenous galactosidases. We then demonstrate the capability of Cm{beta}gal, the stronger of the two reporters, to distinguish differences in levels of expression between A. bescii promoter sequences, which we verify through qRT-PCR. With its high signal to noise ratio and ease of use, this reporter system offers a reliable method for assessing protein expression in anaerobic thermophilic organisms, opening doors to improved genetic tools and metabolic engineering applications for industrial biotechnology.

The homology of the archaeal and eukaryotic ribosome provides one of the key pieces of evidence that underpins the idea that eukaryotes acquired their core information processing machinery from archaea. Since this discovery, reverse genetics has been used to study the functions of many archaeal proteins with eukaryotic homologues. Yet, our general understanding of archaeal growth and division remains unclear, in part because of difficulties of carrying out unbiased genetic screens in archaea. Here, by overcoming several technical hurdles we have used a screen of temperature sensitive mutants in Sulfolobus acidocaldarius to identify core regulators of cell growth and division. First, flow cytometry was used to define DNA content, identifying a set of mutants defective in cell cycle progression at elevated growth temperatures. Using genome sequencing and plasmid rescue, we then identified a point mutation in the large ribosomal subunit that inhibits translation and prevents entry into division following a shift to the restrictive temperature. This study reveals a link between translation and cell cycle control, and opens up the future possibility of using forward genetic screens in archaea to further our understanding of the similarities and differences in the cell biology of archaea, bacteria and eukaryotes. Significance statementO_LICurrent knowledge of archaeal cell biology is limited by the lack of forward genetics. C_LIO_LIWhole genome sequencing and plasmid rescue identifies causative mutation in a temperature sensitive mutant strain. C_LIO_LIA mutation in a ribosomal subunit blocks translation to prevent entry into division. C_LI

Microbial preference for elevated hydrostatic pressure (HP) is a recognized key feature of environmental and industrial processes. HP effects on macromolecules and, consequently, cell functionality has been accurately described in the last decades. While there is little debate about the importance of HP in shaping microbial life, a systematic definition of microbial preference for increased HP is missing. The lack of a consensus about true piezophiles, and low or high HP levels, has deleterious repercussions on microbiology and biotechnology. As certain levels are considered low they are not applied to assess microbial activity. Most microorganisms collected in deep waters or sediments have not been tested (nor isolated) using the corresponding HP at which they were captured. Microbial response to HP is notoriously dependent on other environmental parameters, most notably temperature, but also on availability of nutrients, growth substrate, pH and salinity. This implies that countless isolates retrieved from ambient pressure conditions may very well require increased HP to grow optimally, as already demonstrated in both Archaea and Bacteria. In the present study, I collected the data from described piezophilic isolates and used the fundamental correlation existing between HP and temperature, as first suggested in seminal works by Yayanos, to update the definition of piezophiles. Thanks to the numerous new piezophilic isolates available since such seminal studies, the present analysis brings forward updated definitions which concern 1) the actual beginning of the piezosphere, the area in the deep sea where piezophiles thrive; 2) the HP thresholds which should be considered low, medium and high HP, and their implications for experimental design in Microbiology; and 3) the nature of obligate piezophiles and their location in the deep sea.