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.

Syngas fermentation by carbon monoxide (CO)-utilising acetogens offers a sustainable route for converting gasified waste materials into value-added chemicals. In this study, we isolated a novel thermophilic CO-utilising bacterium, strain AZ2, from marine hydrothermal sediment collected on the island of Sao Miguel, Azores, Portugal. Strain AZ2 is an obligately anaerobic, spore-forming bacterium. Average nucleotide identity (ANI; 78.4-86.7%) and digital DNA-DNA hybridization (dDDH; 23.4-32.5 %) analyses indicate that strain AZ2 represents a novel species within a previously uncharacterised lineage represented by the GTDB placeholder genus UBA2545 in the Neomoorellaceae family. Strain AZ2 was able to grow fermentatively on CO, producing acetate. We further demonstrated that its closest isolated relatives - Thermanaeromonas toyohensis, T. burensis, and Thermanaeromonas sp. strain 9S - are capable of growing on CO, producing either acetate or hydrogen gas (H2). Additionally, we unveiled the genomic potential for CO utilisation within other members of the GTDB placeholder class DSM-521 (previously Moorellia) to which our isolate belongs, expanding the list of possible thermophilic CO-utilising acetogens. We propose that strain AZ2T represents the type strain of a novel genus and species, named Thermobium azorense gen. nov., sp. nov. (= DSM 121889T = JCM 39698T).

Aminopeptidases are widely distributed in bacteria, but outside of a few model strains, their function is largely unexplored. Focussing on beta-alanine aminopeptidase activity, a new series of selectively-activatable, caged fluorescent probes were designed and synthesised. A beta alanine amino acid was coupled to resorufin or 7-hydroxycoumarin via a self-imolative linker, such that amino acid removal led to gain of fluorescence. These were used to probe selectivity and specificity of probe activation, against a range of priority drug-resistant pathogens. When added to bacterial growth curves run in Muller Hinton broth, these probes allowed essentially real time fluorescence measurement of activation by bacterial species, modelled on the standard microbroth dilution method. Activation was observed for all Pseudomonas aeruginosa and Burkholderia spp strains tested. Selective activation was seen for Ochrabactrum species, with the probe activated by O.anthropii (2/4 strains) but not O.intermedium and strain-specific activation was seen for some isolates of Serratia marcescens (2/4 strains). No activation was observed in any isolates of Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii or Staphylococcus aureus or Eneterocccus faecium/faecalis PAO1 transposon mutants in the putative beta-alanine aminopeptidase gene (annotated as bapF or dmpA; PW3678) showed no activation of the probe in growth assays, confirming the specificity of the probe for beta-alanine aminopeptidase. Transposon mutants in other aminopeptidase genes, including those encoded by pepN, PepP and the prolyl aminopeptidase gene had no effect on probe activation in PAO1. Based on the operon structure in PA01, transposon mutants in two adjacent genes were also tested for probe activation. Mutants in both a putative transcriptional regulator (PW3674) and a predicted amino acid permease (PW3676) retained their ability to activate the beta-alanine probes with activation significantly higher than the wild type, when assessed by the total fluorescence yield after 10 hours growth. This points to both redundancy in permease function and perhaps the presence of a feedback regulatory mechanism controlling beta alanine aminopeptidase activity in P.aeruginosa. Given that the operon structure is conserved in other species, this may point to a common mechanism of beta alanine aminopeptidase function, perhaps related to exploiting beta-alanine containing peptides in certain environmental niches.

Plasmids are extrachromosomal mobile genetic elements that can facilitate rapid bacterial adaptation by transferring genes between individuals. While plasmids are known to exist in diverse habitats and encode a range of traits, most of our knowledge about plasmids comes from clinically-associated antimicrobial resistance (AMR) plasmids that have already been recruited as vectors of drug resistance and have likely been shaped by strong selection for plasmid-encoded resistance. Here, we investigated 26 plasmids from the pQBR collection -- a set of large, co-existing mercury resistance environmental plasmids isolated in Pseudomonas spp. from a field in Oxfordshire in the 1990s -- and explored the ability of pQBR plasmids to mobilise novel chromosomally-encoded traits. New whole genome sequences for 25 plasmids confirmed that these soil-isolated plasmids are generally very large (140-588 kb), constitute at least five distinct genetic groups, and have relatives in various other Pseudomonas species and habitats. Despite significant nucleotide-level divergence, Groups I (pQBR103-like, [~]406 kb) and IV (pQBR57-like, [~]328 kb) showed remarkable ancient similarities in synteny and gene content both with one other, and with the PInc-2 family of plasmids known to mobilise clinically significant drug resistance in Pseudomonas aeruginosa. None of the pQBR plasmids sequenced to date harboured known AMR determinants, but putative phage defence systems and metal resistances were evident. Transposable elements, including the Tn5042 mercury resistance transposon, were responsible for significant structural variation within plasmid groups, consistent with a predominant role of transposons in rapidly remodelling plasmids. To experimentally test the ability of pQBR plasmids to spread new traits, we developed a novel transposon mobilisation assay which showed that certain Group IV pQBR plasmids were especially effective at acquiring the chromosomally-encoded transposon Tn6291, and that this mobilisation was likely due to specific plasmid factors rather than generic conjugation rate. Our work presents a tractable set of sequenced plasmids suitable for exploring the evolution and dynamics of gene acquisition by pre-AMR plasmids, and provides a key case study highlighting the pervasive interplay between plasmids and transposable elements that can drive microbial genome evolution. Repositories: github.com/jpjh/PQBR_PLASMIDS Impact statementPlasmids can drive microbial evolution by acting as vectors for horizontal gene transfer. Because of their central role in disseminating antimicrobial resistance (AMR), plasmids are mainly explored as vehicles for AMR traits, meaning that our knowledge of the diversity and evolutionary dynamics of non-AMR plasmids is more limited. Here, we explore sequences from a set of mercury resistance plasmids isolated in Pseudomonas spp. from pristine agricultural land that lack AMR determinants. By providing new whole genome sequencing analyses we expand the set of sequenced pQBR plasmids to 26, finding globally dispersed relatives from clinical, environmental, and industrial settings, and identifying an ancient plasmid backbone shared amongst divergent modern environmental and clinical AMR plasmids. We experimentally verify the role of pQBR plasmids in readily mobilising chromosomal traits using a novel transposon mobilisation assay, which suggests that specific plasmid-transposon interactions may drive trait spread. Overall, our work expands our understanding of the role of environmental plasmids in mobilising and disseminating adaptive traits.

Myxococcus faecalis was recently described from human fecal isolates, although subsequent evidence indicates an environmental distribution for this lineage. Here, we report the isolation and genomic characterization of two M. faecalis strains (BRX-014 and BRX-032) recovered from mangrove ecosystems along the southeastern coast of Brazil, representing the first record of the species in a marine-coastal biome. Phylogenomic reconstruction based on 120 conserved bacterial marker genes, together with Average Nucleotide Identity (ANI >97.6%) and digital DNA-DNA hybridization (dDDH 77.7-90.4%) analyses, confirmed their assignment to M. faecalis and demonstrated high genomic relatedness to strains previously recovered from soil and human feces samples. Pangenome analysis of five available genomes revealed a total repertoire of 9,827 genes, with a large core genome comprising 7,499 genes (76.3%), consistent with a highly conserved and nearly closed pangenome structure. Functional classification based on COG categories showed uniform distributions across all isolates. Comparative analysis of the degradome further revealed strong conservation of proteolytic and carbohydrate-active enzyme repertoires, dominated by serine and metallopeptidases and diverse glycoside hydrolases. The extensive genomic and functional similarity among isolates from geographically distant and ecologically distinct environments supports a broad ecological distribution of M. faecalis and suggests that its large and conserved genomic repertoire underpins its persistence across contrasting habitats. These findings expand the known ecological range of the species and provide a comparative genomic framework for future investigations into its distribution and functional potential across different habitats.

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.

Corals harbor a diverse bacterial community that facilitates adaptation and sustains their health. In coral holobiont research, culture-independent approaches have transformed the existing paradigm. Molecular techniques, such as metabarcoding, revealed a high diversity of previously unrecognized bacterial symbionts. Coral microbiota characterization has relied on these techniques over the last decade, but relying solely on them does not provide a detailed understanding of the dynamics of the coral holobiont complex. Returning to classic microbiological methods and in vitro experimentation can yield novel insights into symbiont roles, physiology, and interactions within the holobiont. Under this premise, we aimed to isolate and culture bacteria from four Mediterranean corals. The recovery of 84 pure bacterial isolates and their initial classification based on the 16S rRNA gene revealed substantial diversity among symbionts amenable to culture. Several isolates represent novel species within relevant genera, such as Vibrio, underscoring the value of culture-based studies. All cultures were cryopreserved to guarantee long-term accessibility for future projects. This represents a key step towards describing the roles of bacteria within the coral holobiont, as cultures enable in-depth morphological and physiological characterization of the symbionts and experimental ecology studies.

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.

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.

The Bacillus genus comprises physiologically versatile, endospore-forming bacteria widely distributed in natural environments. In this study, we report the isolation and genomic characterization of strain Bva_UNVM-123, recovered from agricultural soil in Pergamino, Argentina. Whole-genome sequencing using Illumina technology yielded a 5.1 Mbp draft genome assembled in 67 contigs with a GC content of 36%. Comparative genomic analyses using the TYGS server and digital DNADNA hybridization (dDDH) values supported its classification as a potentially novel species within the Bacillus sensu lato (s.l.) group. Genome annotation revealed 4,866 protein-coding genes, including multiple determinants conferring resistance to antibiotics (e.g., fosfomycin, tetracycline, beta-lactams) and toxic heavy metals (e.g., arsenic, cadmium, mercury), supporting its potential application in bioremediation. Additionally, PathogenFinder predicted a low probability of human pathogenicity (0.207), reinforcing its safety for environmental use. Functional classification based on Swiss-Prot further supported a metabolically versatile profile and revealed the presence of resistance-related categories associated with environmental adaptation. This study adds to the growing knowledge of environmental Bacillus species and their biotechnological potential

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.

A facultatively anaerobic, hydrogen-oxidizing, thermophilic bacterium (strain KUI-RBT) was isolated from a geothermal spring biofilm in Rotorua, New Zealand. Strain KUI-RBT is a motile, straight rod, measuring approximately 0.7 {micro}m by 1.0 to 1.5 {micro}m with a diderm cell wall. Growth of KUI-RBT occurred from 39 to 74 {degrees}C (Topt 64.5 {degrees}C), pH 5.0 to 7.5 (pHopt 6.5), and 0 to 1% (w/v) NaCl (NaClopt 0.4-0.7%, w/v). KUI-RBT utilizes carbon dioxide and various organic carbon substrates as carbon sources and hydrogen as an electron donor. KUI-RBT can use oxygen (0-21%, v/v), elemental sulfur, thiosulfate, sulfite, nitrate, arsenate, and selenate as terminal electron acceptors. Major fatty acids of strain KUI-RBT include C20:1, C18:1, and C18:0 and the primary quinone is MTK-7. The whole genome G+C content is 34.23 mol%. Phylogenetic analyses indicate KUI-RBT to be a member of the family Hydrogenothermaceae, with Sulfurihydrogenibium azorense Az-Fu1T its closest characterised relative (94.51% 16S rRNA gene sequence similarity, 78.01% whole genome ANI, 61.34% whole genome AAI). Based on phylogenetic and phenotypic analyses, we propose KUI-RBT represents a novel genus and species within the family Hydrogenothermaceae, for which we propose the name Reysenbachia aerophila gen. nov., sp. nov. The type strain is KUI-RBT (=KCTC accession =JCM accession). The GenBank accession number for the 16S rRNA gene sequence of strain KUI-RBT is PZ052650. The GenBank accession number for the whole genome of strain KUI-RBT is JBVODP000000000.