microLife

The cell envelope of bacteria and fungi is a major source of microbial-associated molecular patterns (MAMPs), which are recognised by multiple host pattern recognition receptors (PRRs) such as TLRs and NODs. The key role of this recognition in protecting against infection and regulating diverse physiological processes has been studied for decades. In contrast, although the archaea are growingly recognised as an important component of the animal and human microbiome, their interaction with the innate immune system has been largely understudied. Here, we asked whether the archaeal cell envelope acts as MAMP. We selected 15 archaeal species from different environments, including the human microbiome, and harbouring diverse envelope characteristics, and we tested their ability to activate innate immune responses in human and murine immune cells. We first show that archaeal cells activate the innate immune transcription factor NF-kB and induce IL-6 secretion. Using whole archaeal cells, supernatants, and purified envelope components, we then find that none of the main PRR receptors which recognise the bacterial envelope (TLR4, TLR5, NOD1 and NOD2) are involved in this activation. We only detected activation of TLR2, regardless of the environmental origins of the archaea tested, although with overall lower activity compared to bacteria. We further demonstrate that different TLR2 heterodimers are involved, depending on the species, although the archaeal agonist remains unknown. Altogether, our results highlight a surprising paucity of interaction of archaeal cell envelope components with host PPRs, in contrast to bacteria, and open new avenues of research toward further investigation of archaea/host interplay.

Adherent Invasive Escherichia coli (AIEC) colonize the ileal mucosa of Crohn's disease patients. Among bacterial virulence factors potentially implicated, type 6 secretion systems (T6SS) are molecular machineries sending effectors in prokaryotic or eukaryotic target cells. Two T6SS have been reported in AIEC LF82 reference strain. Here, we show that T6SS genes are more prevalent in Crohn's disease patients' microbiota compared to healthy controls, and that T6SS are more frequent in AIEC pathovar compared to E. coli in general and associated with FimH pathoadaptive polymorphism. The use of an in vitro gastrointestinal system coupled with T6SS promoter reporter assay highlighted expression of LF82 T6SS under gastrointestinal conditions, with a major role for bile salts. In mice infected with AIEC LF82 versus T6SS-deleted mutants, T6SS showed a functional impact in modulating the composition of mucosa-associated gut microbiota. Thus, AIEC may use their T6SS to compete with endogenous microbiota to colonize host mucosa.

The endonuclear symbiont Holospora obtusa invades the macronucleus of Paramecium caudatum using an infectious-form invasion-tip containing a major 89-kDa periplasmic protein implicated in escape from the host digestive vacuole and macronuclear invasion. Sequence-based analyses placed this protein within a Holospora-specific family and predicted an N-terminal membrane-anchoring module and a long alpha-helical C-terminal region. AlphaFold2 modeling followed by molecular dynamics indicated that the C-terminal region forms an antiparallel, spectrin-repeat-like helical solenoid containing a localized three-helix coiled-coil. The protein exhibits a large pH-dependent charge difference, becoming 55 units more positive at pH 4 than at pH 7. Independent simulations at these pH values differed mainly in the relative orientation of the N-terminal core and C-terminal rod, described by DynDom as an approximately 35 degrees rigid-body rotation about a hinge centered on Gly291. However, none of the nearby glutamates (Glu273, Glu275, and Glu302) formed a pH-dependent salt bridge, and no specific protonation-dependent interaction responsible for the conformational difference was identified. We nonetheless propose, as a testable hypothesis, that acidification of the digestive vacuole may promote this reorientation, thereby repositioning the N-terminal membrane-anchoring module at the bacterial surface as an early step toward escape from the vacuole.

Uropathogenic Escherichia coli (UPEC) is the predominant causative agent of urinary tract infections. The UPEC strain, CFT073, has a mosaic genome containing 60 genomic islands enriched in virulence genes. In this study, we explore the small proteome of CFT073 using ribosome profiling (Ribo-seq) coupled with RNA sequencing (RNA-seq), and reveal 338 translated, previously unannotated, putative small open reading frames (sORFs) with less than 100 codons. Among them, 78 sORFs are located in intergenic regions. Fifteen intergenic sORFs encode a predicted transmembrane helix (smemORFs), of which seven are hypothetical or have not been characterized in other E. coli strains. The majority of the remaining putative sORFs encodes uncharacterized or novel soluble proteins (ssolORFs). Considering the evolution of many sORFs is likely to be recent, functional sORFs are more likely to have conserved gene neighborhood in other genomes. Searching into the genomes of 10 other E. coli strains and four enterobacteria species, synteny analysis reveals that 10 of 15 putative smemORFs, and 27 of 63 putative ssolORFs, have a homologous sequence in a similar genomic context in at least one other genome, supporting their likelihood of encoding functional proteins. Further validation of putative sORFs using proteomics, cell-free protein synthesis, and mNeonGreen tagging provide strong evidence for the translation of nine predicted intergenic sORFs. Notably, four previously uncharacterized small membrane proteins have increased abundance under oxidative stress, suggesting a potential role in the stress response. Together, our findings reveal the small proteome of an extraintestinal pathogenic E. coli and provide a catalog of putative sORFs for further investigation.

A subgroup of membrane-anchored bacterial Alkaline Phosphatase superfamily members transfers phospholipid headgroups from phospholipids to diverse acceptor molecules. As part of type II and hybrid type I/II cellulose biosynthesis gene clusters BcsG transfers phosphorylethanolamine pEtN from phospholipid to the emerging 1,4 {beta}-D-glucan chain synthesized by the BcsABC cellulose biosynthesis nanomachine. BcsG homologs throughout the phylogenetic tree possess a uniquely structured tripartite linker that connects the fifth transmembrane domain with the periplasmic catalytic domain. A N-terminal unstructured sequence of highly variable length and biased amino acid composition, the N-linker, is followed by an -helix and an unstructured sequence of high similarity and constant length leading into the first {beta}-strand of the catalytic domain. With BcsG from Salmonella typhimurium synthesizing pEtN cellulose as a model, linkers were shown to be promiscuous and linker length positively correlated with transfer efficiency of the pEtN headgroup and protein stability. As the donor substrate, the phosphatidylethanolamine content of the membranes affects pEtN transfer efficiency. With Alkaline Phosphatase superfamily members to be promiscuous enzymes the catalytic domain of OpgE transfers pEtN to osmoregulated periplasmic glucan. BcsG-OpgE hybrid proteins cause colony morphology alterations thus potentially transferring pEtN to the BcsA synthesized glucan chain. With BcsG with shortest linkers not associated with a cellulose biosynthesis gene cluster, multiple hypotheses can be proposed that select for different length of the N-linker in BcsG homologs located within a cellulose biosynthesis gene cluster in other Pseudomonadati.

To persist during infection Klebsiella pneumoniae must overcome nutrient iron and zinc limitation imposed by the host immune system through a process called nutritional immunity. Secreted small molecule siderophores are a major virulence determinant of Klebsiella pneumoniae pathogenesis and are presumed to overcome nutritional immunity by binding iron for bacterial acquisition. In this work, we set out to identify how a multi-drug resistant K. pneumoniae grows in zinc limited environments. Using unbiased transcriptomics, proteomics, and an arrayed transposon screen, we identified that synthesis and uptake of the siderophore enterobactin is required to allow for growth in low zinc conditions. Iron-specific chelators did not replicate this phenotype and addition of supplemental iron through heme in growth media could not complement severe growth defects of enterobactin mutant K. pneumoniae experiencing zinc limitation. Finally, zinc starvation induced enterobactin production independent of the canonical zinc uptake regulator (Zur) transcription factor suggesting an unidentified regulatory mechanism by which Gram-negative pathogens may respond to zinc stress. Together, these studies expand the role of enterobactin beyond iron regulation and highlight a previously unreported link between iron and zinc homeostasis in Klebsiella pneumoniae.

Bacterial cultures entering stationary phase (SP) undergo complex physiological alterations, helping the cells to survive when medium resources are exhausted. The onset of various stress responses underlying SP physiology is generally believed to be due to reactions of individual cells to the environmental cues such as starvation, toxic metabolites, etc. The SP physiological state makes bacteria unsuitable for the replication of most bacteriophages; however, some phages are able to infect and multiply in them. We investigated Escherichia coli phage DH23 growth in SP cultures of E. coli MG1655 infected at different hours post inoculation (ages). Under our conditions the cells enter SP at about 6 h, but the phage replication was possible till 11h and then dropped abruptly by 13h of culture aging, indicating an abrupt physiological change to deeper dormancy during SP. The onset of this phage-inhibiting middle-SP dormancy (MSPD) turned out to be mediated by intercellular communication mediated by the middle-SP signal particles (MSP) which are larger than 100 kDa and contain both RNA and DNA. These MSP are inactivated by RNAse or by DNAse, enabling phage growth in 14h-old cultures and lifts the tolerance of such cultures to kanamycin. Moreover, the RNAse or DNAse treatment makes 24h culture spent media suitable for additional cell growth, and cultures initiated in fresh LB supplemented with RNAse reach an OD600 about 1.5 times higher compared to the untreated control. This indicates that MSP signaling influences cell physiology well before the MSPD onset and helps the population cease growth pre-emptively to save about 1/3 of medium resources to support the viability during SP.

Metals are essential for all forms of life and are therefore the subject of tight competition at the host-pathogen interface. Manganese (Mn) is an important cofactor for enzymes involved in various processes in bacteria, including the response to oxidative stress, and the ability to scavenge Mn through the expression of high-affinity Mn transporters has been associated with virulence of many extracellular and vacuolar pathogens. While the cytosol has been established to be restrictive for growth of non-adapted pathogens, the extent to which metal limitation contributes to this is not well understood. Here, we investigate whether high-affinity Mn acquisition is important for intracellular adaptation and virulence of the cytosolic pathogen Listeria monocytogenes (Lmo). To do so, we generated a strain of Lmo in which both of its predicted high-affinity Mn transporters, MntH and MntABC, are disrupted. We found that this strain was severely defective in growth in vitro under Mn-limited conditions and had lower intracellular Mn content. Additionally, we observed Mn-dependent transcriptional regulation of the mntH and mntA genes. Together, these data strongly support that MntH and MntABC are high-affinity Mn transporters and are likely to be the only such dedicated Mn transporters expressed in Lmo. Using the {Delta}mntH {Delta}mntA strain, we provide evidence that Mn limitation sensitizes Lmo to oxidative stress in vitro, consistent with the known role of Mn in mitigating reactive oxygen species. Ultimately, we demonstrate that expression of high-affinity Mn transporters is essential for intracellular replication of Lmo in macrophages, plaquing on fibroblast monolayers, and for virulence in an animal model of systemic infection. Our work, therefore, suggests that Lmo is challenged by Mn limitation in the cytosol and requires Mn scavenging to adapt to its intracellular niche.

L-form bacteria are wall-deficient variants that proliferate without an intact cell wall and independently of the canonical cell division machinery. Despite increasing insights into how L-forms survive and proliferate without a cell wall, fundamental questions regarding how cell cycle processes, including DNA and RNA synthesis, are coordinated in the wall-less state remain poorly understood. Here, we demonstrate that fluorescence-activated cell sorting (FACS) provides a robust approach for quantitative analysis of Escherichia coli L-forms. Flow cytometry revealed two reversible subpopulations differing in intracellular nucleic acid content, which rapidly re-established their heterogeneous distribution following sorting. Remarkably, cells with initially low nucleic acid content underwent a synchronized, population-wide increase in nucleic acid synthesis within 24 hours, a phenomenon that was independently confirmed by time-lapse fluorescence microscopy. Analysis of liquid cultures stained with SYTO and DAPI dyes showed that this transient increase occurred during early exponential growth and was driven predominantly by RNA rather than DNA synthesis. Consistent with observations in walled E. coli, RNA levels peaked before declining as cultures transitioned towards nutrient limitation. Together, these findings establish FACS as a powerful tool for studying L-form biology and reveal that, despite the absence of an intact cell wall and canonical cell division, L-forms retain coordinated, population-wide regulation of nucleic acid synthesis.

The type VI secretion system (T6SS) is a molecular harpoon used by 19% of bacteria to inject toxic proteins into microbial or host cells. Despite its widespread distribution, many T6SS effector (T6Es) genes remain unidentified. Here, we developed an XGBoost classifier integrating 42 genetic and biochemical features to identify novel T6Es. Applying our model to 2,559 T6SS-encoding bacterial genomes revealed 19,958 T6E candidates. We validated our computational predictions and confirmed that three effector candidates, having new 3D structures, demonstrated cytotoxicity to bacteria and yeast, mostly in the bacterial periplasm. For two of these, cognate immunity proteins neutralizing toxicity have been validated. T6EC11, a new peptidoglycan degrading effector, quickly leads to spherical E. coli, with inner membrane separation, and gradual peptidoglycan degradation in a unipolar manner, demonstrating an intriguing moon-like structural phenotype. T6EC12, an effector toxic to bacteria and yeast, carries a conserved zinc-binding motif. Mutations the motif markedly reduced toxicity, suggesting that T6EC12 represents a novel metalloprotease. Our study expands the known T6E repertoire, and provides thousands of T6E predictions in all T6SS-encoding bacteria.

Bacteria encode diverse antiviral defense systems, yet the evolutionary reservoirs that generate this immune innovation remain poorly defined. Although genomic context has been instrumental in discovering defense systems, genomic islands (GIs), variable chromosomal regions enriched in defense genes and proteins of unknown function, have not been systematically explored as sources of new bacterial immunity. Here, using comparative genomics of Streptococcus thermophilus, we identified 30 defense-associated GIs displaying extensive accessory gene diversity. Investigation of candidate loci from three GIs uncovered 15 previously uncharacterized antiviral systems, including 10 with novel architectures or features, revealing the potential within even a small subset of GIs. Several systems were not located near known defense genes, highlighting that GI-based discovery complements the traditional guilt-by-association approach, revealing systems that would otherwise remain undetected. Together, our findings establish GIs as reservoirs of antiviral innovation and provide a framework for uncovering the diversity of bacterial immune systems.

Reliable constitutive promoters for non-model bacteria are lacking, resulting in a bottleneck for genetic tool development. Existing computational promoter predictors are trained on model Gammaproteobacteria and degrade on phylogenetically distant or compositionally atypical genomes. Here we present ProFinder, a computational method that integrates three biologically informed criteria for promoter prediction: a conserved 98-gene constitutive marker panel, an empirically derived intergenic search window, and a 72-motif {sigma}70 motif catalogue built from diverse lineages. From an input bacterial assembly, ProFinder returns a ranked shortlist of putative constitutive promoters. It matches the precision of full-genome classifiers and is more robust to GC extremes. Applied to 56,742 bacterial genomes spanning 149 phyla, ProFinder returned candidate promoters for 99.76%, delivering a pan-bacterial catalogue of pre-computed constitutive promoter parts to accelerate chassis development across the bacterial tree. ProFinder can be accessed at: https://plabase.cs.uni-tuebingen.de/profinder/.

Patient movement within and between hospitals creates a complex hospital network that facilitates the spread of antimicrobial-resistant bacteria and contributes to healthcare-associated infections. Yet most national-scale studies infer spread from aggregated hospital-to-hospital patient sharing, leaving patient-to-patient contact pathways and the role of routine patient mobility in shaping colonization risk poorly characterized. Using Frances 2023-2024 national hospital-discharge data, we reconstructed a temporal patient-based network of 10.4 million patients across 1,408 acute-care hospitals, comprising 326.6 million overnight ward co-location interactions. We simulated colonization over 365 days on the observed day-by-day temporal network using a stochastic susceptible-colonized-susceptible model, with a mean colonization duration of 180 days. We used national carbapenemase-producing Enterobacterales (CPE) surveillance data to calibrate differences in transmission risk across clinical settings. Across 2,000 simulations initiated on randomly selected dates in 2023, we calculated patient-level colonization probabilities and summarized risk across patients residential areas, wards, and hospitals. We further assessed hospital-level consistency between simulated risk and recorded CPE occurrence. Simulated risk was highly heterogeneous across patients and care settings. When mapped by place of residence, hospital-associated colonization risk showed strong spatial clustering in both urban and rural areas. Multivariable regression analyses identified patient- and hospital-level characteristics associated with heterogeneity in simulated colonization risk. Sensitivity analyses varying initial seeding and colonization duration showed that relative risk patterns remained highly stable. This nationwide framework shows how patient mobility and ward co-location patterns can reveal patterns of hospital-associated colonization risk and could support pathogen-specific surveillance and intervention evaluation. Significance StatementAsymptomatic carriage allows antimicrobial-resistant bacteria to spread through healthcare systems while colonized patients may remain unrecognized in the absence of active screening. We reconstructed a nationwide, time-resolved network of ward co-location among hospitalized patients in France. We accounted for differences in transmission risk across clinical settings using national surveillance data on carbapenemase-producing Enterobacterales. Using a transmission model, we characterized colonization-risk patterns and identified patient- and hospital-level features associated with variation in risk. This framework offers health authorities a way to examine where patient movement and hospital care may contribute to concentrations of colonization risk, although pathogen- and context-specific adaptation and evaluation remain necessary to guide surveillance or interventions. Author contributionsS.B., L.C., L.T., A.K., T.D., V.C., and P.C. designed research; S.B. and P.C. performed research; S.B. and P.C. analyzed data; S.B. and P.C. wrote the paper; all authors reviewed and edited the final manuscript.

Pre-prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyzes the first step in the lipoprotein modification pathway and is essential for growth and viability of Helicobacter pylori. We explored the fate of H. pylori cells under Lgt depletion conditions to obtain insight into Lgt function on morphology and physiology. Downregulation of lgt expression led to growth arrest and a heterogenous population of small rod-shaped cells and small round cells with spiky protrusions, a loose outer membrane and disintegrated flagella. H. pylori overcomes a defect in lipoprotein modification through alteration of its gene expression profile resulting in so-called revertants. Outer membrane protein synthesis, carbon metabolism and nutrient uptake was reduced, and protein translation and flagellar biogenesis increased. Lgt specific regulation was observed in phospholipid biosynthesis resulting in an initial increase in cardiolipin followed by an increase in lysophospholipids at the time of adaptation. Suppressors of {Delta}lgt that were selected on solid media under restrictive conditions either lacked chromosomal mutations or had mutations in the cagUT-tetR regulon controlling the expression of lgt. A suppressor without mutations had an altered expression profile that led to downregulation of a DNA methyltransferase and upregulation of HP_0014, a motility associated glycosyltransferase family protein. Transmission electron microscopy and single cell video microscopy showed that a subpopulation of cells is motile in the revertant while the suppressor regained full motility with higher average speed compared to the wild type strain. Overall, our data show that lipoprotein modification by Lgt is important for membrane and flagellar integrity and that the heterogenous character of H. pylori cultures allows individual cells to overcome this cell envelope stress and survive. Our findings are important for antibacterial development strategies targeting essential integral membrane enzymes involved in lipoprotein modification and stimulate exploration of synergetic activity between known antibiotics and novel antibacterial agents targeting novel cellular pathways.

Bacteria utilize specialized protein secretion systems, particularly the type VI secretion system (T6SS), to dominate interbacterial competitions and kill the prey bacteria in a contact-dependent manner. Here, we report that disruption of the thioesterase gene encoded in the gramibactin (a bacterial siderophore) biosynthetic gene cluster activates the T6SS in the Burkholderia gladioli strain NGJ1, even in the absence of prey. The thioesterase mutant exhibits enhanced antibacterial activity, and ectopic expression of the gene complements the phenotype. Notably, thio mutant as well as thioesterase-complement strains show enhanced sensitivity to oxidative stress. The qRT-PCR analysis showed that the thio mutant had upregulated expression of RpoN1, a sigma factor, which positively regulates T6SS functions. We demonstrate that rpoN1 mutant is hypersensitive to oxidative stress and defective in T6SS activation, whereas overexpression of RpoN1 enhances oxidative stress tolerance in NGJ1 and activates T6SS. We propose that, during interbacterial conflicts encountered during plant colonization, NGJ1 faces an oxidative-stress-enriched environment that modulates thioesterase expression, induces RpoN1, and activates T6SS. Overall, our study exemplifies the broader physiological role of Thioesterase in maintaining intracellular ROS homeostasis in bacteria and stimulating antibacterial activity.

Adherent Invasive Escherichia coli (AIEC) strains are associated with ileal Crohn's disease and may contribute to intestinal dysbiosis through antibacterial competition. The AIEC reference strain LF82 encodes two type VI secretion systems (T6SSs), a contractile machine that injects an effector-loaded needle made of Hcp, VgrG and PAAR into neighbouring rivals, favoring niche colonization. LF82 T6SSs harbour several putative antibacterial toxin-immunity modules. Here we characterize the T6SS-1 encoded TmeAIEC, a PAAR-fused T6SS effector carrying a C-terminal Tme domain, hallmark of Tme (Type VI membrane disrupting effector) toxins, and its DUF1240-like immunity protein TmiAIEC. When heterologously produced in E. coli K-12 as well as in E. coli and Citrobacter clinical isolates, TmeAIEC is toxic only upon periplasmic targeting, and its toxicity is neutralized by TmiAIEC. Periplasmically-targeted TmeAIEC inserts into the inner membrane, collapses the proton motive force and increases cell permeability, consistent with a membrane-disrupting activity. Mutational analyses coupled to functional assays identified a predicted hydrophobic transmembrane segment and a conserved aspartate residue within the Tme domain as key determinants for TmeAIEC toxicity. These findings establish TmeAIEC/TmiAIEC as a functional T6SS toxin-immunity pair and reveal conserved determinants underlying the activity of Tme-family membrane-disrupting effectors.

Translation initiation is a critical stage of protein synthesis in prokaryotes, where ribosomes assemble on mRNA to begin decoding genetic information. The interactions between ribosomal RNA (rRNA) and mRNA significantly influence this process, with features like ribosomal binding site (RBS) affecting ribosomal dynamics. However, direct experimental validation of these types of interaction is yet to be provided. In this study, we developed an optimized small sub-unit sequencing (SSU-seq) protocol for Escherichia coli, enabling the detailed examination of ribosomal complexes and subunits dynamics. Unlike Ribo-seq, which primarily maps fully assembled ribosomes, SSU-seq captures both ribosomal subunits and intact ribosomes, offering a comprehensive view of translation initiation and elongation events. By integrating experimental data with predictive models, our study advances the understanding of translation regulation in prokaryotes and underscores the utility of SSU-seq in decoding the complexities of ribosome-mRNA interactions. Our analysis reveals distinct patterns of small subunit distribution across the entire mRNAs with higher density near positions with stronger rRNA-mRNA hybridization. The results may suggest that rRNA-mRNA hybridization positions along the entire transcript facilitate efficient start codon recognition by maintaining proximity between the small subunit and the mRNA.

The resurgence of phage therapy has renewed interest in the interplay between phage resistance and antibiotic susceptibility and has stimulated research in the emergent field of non-canonical cellular functions carried out by defence systems. Yet it remains largely unknown whether intracellular antiphage defence systems influence bacterial physiology, resistance or persistence to antibiotics. Here we discovered that besides its canonical antiphage defence function, the type I restriction modification (RM) system profoundly affects the physiology of the opportunistic pathogen Pseudomonas aeruginosa. Deletion of the type I RM methyltransferase HsdM reduces the size of the bacterial nucleoid and delays DNA replication initiation and exit from lag phase in P. aeruginosa PAO1. Crucially, P. aeruginosa strains isolated from patients with cystic fibrosis (CF) and lacking the RM type I system also display slower growth compared to strains isolated from other sites of infections and encoding this system. Deletion of HsdM selectively increases the levels of persisters that survive treatment with fluoroquinolones by displaying enhanced SOS response but without acquiring resistance. Importantly, we measured elevated persistence to fluoroquinolones also in P. aeruginosa CF isolates lacking the type I RM system, providing a functional link between RM systems, slow growth and persistence to fluoroquinolones. Together these findings open a new way of approaching bacterial susceptibility to antibiotics, bringing antiphage defence systems in a forward-facing position in this field.

The opportunistic pathogen Pseudomonas aeruginosa can store carbon by polymerizing (R)-3-hydroxy fatty acids into polyhydroxyalkanoates (PHA) and forming large intracellular carbonosomes. These structures primarily function as stores of excess carbon that can be employed during nutrient imbalances, though they have been increasingly reported to have secondary functions in stress responses. A range of Proteobacteria can form carbonosomes, and they have been well characterised in strains used in bioplastic production and wastewater treatment. However, the occurrence, regulation and functions of carbonosomes in pathogenic bacteria remain poorly understood. Using flow cytometry and fluorescence microscopy following staining of carbonosomes with BODIPY 493/503, we characterised carbonosome abundance and dynamics in growth-arrested P. aeruginosa starved of nitrogen but provided with excess carbon. We found that carbonosomes substantially impact the energetic and biosynthetic output of growth-arrested P. aeruginosa, affecting protein biosynthesis, flagellar motility, and ATP/NADPH pools. The production and use of carbonosomes was found to confer a strong fitness advantage to growth-arrested P. aeruginosa during fluctuating nutrient conditions. Small carbonosomes were observed during the lag phase of growth in rich media and the ability to produce these structures was conserved across several different laboratory and clinical strains, although with different dynamics and maximal amounts. For example, the Liverpool epidemic strain LESB58 showed very little BODIPY staining while the closely related LESB65 strain showed high levels of staining. Crucially, we found that growth-arrested cells depolymerize carbonosomes upon tobramycin exposure and that carbonosome depolymerization significantly increases P. aeruginosa tobramycin sensitivity. Together, our observations suggest that carbonosomes significantly impact the physiology, metabolism, and antibiotic susceptibility of P. aeruginosa.

The presence of metaparalogs (related genes diverging in function) has been proven to be extensive in the pangenomes of streamlined Pelagibacterales genomes. Here by leveraging a large number of complete genomes from an Arctic population we have been able to establish their universality and mechanistic origins. The diversity of some metaparalog families was as high within a single population than comparing with another (Mediterranean) population/species, which points to an ancient diversification of metaparalog families predating genomospecies separation. Metaparalog families provided diversity for transport and envelope diversity which would provide vast capacities to degrade substrates within DOM and interact with other cells or viruses or other subcellular entities (like EVs). The pool does not saturate at fifteen genomes. Our results highlight the immense importance of the pangenome in understanding the biology of bacteria in natural environments and how critical is the study of population genomics for the future of microbiology.