microLife

Autotransporters are important diderm bacterial cell-surface proteins and are virulence factors enabling surface attachment and adhesion to other bacteria. These proteins are composed of a signal peptide, a {beta}-barrel that serves as an anchor in the outer membrane, and an extracellular passenger domain responsible for adhesion. Autotransporters rely on BamA for their insertion in the outer membrane (OM), but specific helper proteins, such as TpgA and SadB have been described to promote the surface exposure of trimeric autotransporters (TAAs), a specialized subclass of autotransporters forming homotrimer adhesins. To identify domains or proteins that could help TAAs secretion, we analyzed a recent dataset of all trimeric autotransporters found across the bacterial tree of life. While we did not find additional potential helper proteins for OM translocation, we found that the extended signal peptide (ESPR), sometimes found in TAAs, is associated with longer adhesins both for TAAs and type Va autotransporter adhesins. ESPRs are found in all bacteria but Fusobacteriia and Alphaproteobacteria. We also identified in Burkholderia, Veillonellales and Pasteurellales a DUF2827 domain proteins as potential glycosyltransferases constantly associated with TAAs. Finally, e describe the existence of extra periplasmic domains in some TAAs, featuring either a coiled-coil domain or a peptidoglycan-binding domain. Our research show that there is a strong phylogenetic separation between Terrabacteria, almost invariably displaying additional periplasmic domains, and Gracilicutes (represented by Proteobacteria) where they are largely absent. This suggests that the presence these domains might be correlated with specific Terrabacteria OM features. Using the diderm Firmicute Veillonella parvula as a model, we demonstrate that the absence of periplasmic domains in TAAs leads to a significant protein degradation, yet they are not essential for adhesin trimerization or secretion. Additionnaly, we show that the SLH domains of V. parvula TAAs excludes them from the septum during division, but that this exclusion is not crucial for adhesin function or stability in the tested conditions. Altogether, these results illuminate the genetic flexibility and modularity of autotransporters, enhancing our understanding of this important class of adhesins in diderm bacteria.

The worldwide rise in the prevalence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli is a major public health concern. In Europe, ESBL carriage frequency increased then stabilized at about 6-8 %. Past antibiotic use and travel in countries with high ESBL frequency, notably South-East Asia, have repeatedly been identified as risk factors of ESBL carriage. Yet, the relative contributions of these mechanisms to the observed maintenance of a stable low frequency of ESBL in Europe remains unknown. Here, we used comprehensive data on the risk factors for carriage of ESBL-producing E. coli in the French community, alongside detailed microbiological characterization of both resistant and overall E. coli, to develop a biologically plausible mathematical model of ESBL resistance spread in France. The model also includes several mechanisms previously showed to favor coexistence such as population structure, variability in carriage duration and within-host dynamics. The level of resistance in the community implies resistant strains transmit 14% less than sensitive (95% credible interval 0.6-38%), and are cleared at a +23% larger rate (0.9-62%). ESBL resistance is predicted to be strongly associated with factors prolonging residence in the gut. Both the rate of antibiotic treatment and transmission strongly impact the frequency of ESBL in the community. In contrast, travel has little impact on ESBL frequency. Whether reducing treatment or transmission is best to reduce resistance depends on community-specific parameters. Our study opens perspectives for the quantitative study of resistance evolution and argues for future work to improve the characterization of the duration of carriage of commensal bacterial strains.

Chronic wounds are difficult to treat because underlying medical conditions can impair the mechanical and physiological first-line innate immune defenses, leading to persistent microbial infections. We report here the isolation, molecular and phenotypic characterization of seven E. coli strains that were isolated concomitantly with Enterococcus faecalis after an open foot fracture caused by the 2004 tsunami resulting in a 10-year chronic bone and joint infection. Initially present antimicrobial resistant E. coli ST405 and ST940 isolates were followed by host adapted isolates of ubiquitous ST131 clone presumably acquired from the environment already upon initial foot fracture. The E. coli ST131 clade C1 strains showed genomic alterations associated with virulence and persistence including large chromosomal inversions and, subsequently, a large deletion to cause small colony variants and higher susceptibility to formaldehyde and other stress provoking In this context deletion of hemB catalyzing an early step in the pathway for heme biosynthesis was the major, but presumably not the only cause of small colony variant emergence. Surprisingly, ST131 isolates did not display pronounced biofilm formation in conventional biofilm assays suggesting unconventional modes of persistence. In summary, the genomes of ST131 clone members are highly plastic which enables their persistence in novel ecological niches. In individuals with underlying metabolic diseases such as diabetes wound infection can prepare for colonization with ST131 E. coli isolates. FundingThis work was partially funded by ALF, the Petrus and Augusta Hedlunds Foundation and the Karolinska Institutet.

The opportunistic pathogen Pseudomonas aeruginosa is highly adaptable to different environmental conditions due to its versatile sensing and metabolic capabilities. Both external temperature and metal availability have a strong influence on the virulence and pathogenicity of P. aeruginosa, but the coupling between these two factors is not well understood. While iron is recognized as major player in nutritional immunity, the role of cobalt and the cobalt-containing vitamin B12 (cobalamin) during host infection remains unclear. Here, we investigate the environmental isolate P. aeruginosa PA254 using high-resolution global proteomics and cellular cobalamin measurements over a temperature gradient spanning environmental, host-associated, and heat-stress conditions (22-42 {degrees}C). PA254 occupies a continuum between an ambient-temperature virulent state characterized by versatile secreted factors, exopolysaccharide-rich biofilms, and planktonic swimmers and surface swarmers; and a host-associated virulent state characterized by potent secretion effectors, alginate-dominated biofilms, and a strong proportion of surface twitching motility. Pathway analyses indicate a shift toward carbon sparing, energy conservation, redox control, and metabolic maintenance during a host-adapted lifestyle, along with the strong overexpression of alternative iron acquisition strategies relying on heme and siderophores. Proteins of the cobalamin biosynthetic pathway declined significantly above ambient temperatures, despite constant intracellular B12 concentrations across all conditions. This decoupling of biosynthesis from cellular pools implies prioritization and recycling within B12-dependent processes, while the lack of B12 production at human body temperatures creates avenues for therapeutics interfering with B12 supply. Altogether, this work highlights a gradual rather than stepwise reprogramming of the P. aeruginosa proteome in response to environmental cues, and highlights proteomics as a tool to investigate system level mechanisms of challenging pathogens.

BackgroundThe virulence-persistence trade-off is considered a fundamental organizing principle of Listeria monocytogenes population biology: hypervirulent clonal complexes dominate clinical cases but are rarely found in processing environments, while hypovirulent lineages dominate industrial niches but are underrepresented in severe disease. However, whether this dichotomy operates as an absolute paradigm has not been quantitatively evaluated at the population scale. Here we develop a multi-dimensional genomic scoring approach that simultaneously quantifies virulence potential (V), environmental persistence capacity (P), clonal epidemiological context (C), and antimicrobial resistance (R) across 903 genomes from four independent datasets spanning five countries, and apply it to test the universality of the trade-off and to characterize the ecological strategies of L. monocytogenes at the population level. MethodsThe scoring approach integrates four components into a composite 0-100 score through empirically calibrated weights (V: 30%, P: 40%, C: 20%, R: 10%). Validation employed 903 L. monocytogenes genomes from four public BioProjects: longitudinal industrial surveillance in Norway (Fagerlund et al. 2022, n = 513, PRJNA689484), retail environments in the United States (Stasiewicz et al. 2015, n = 191, PRJNA245909), clinical-environmental context in China (Wang et al. 2021, n = 151, PRJNA759341), and meat processing in Poland (Kurpas et al. 2020, n = 48, PRJNA629756). ResultsThe composite score achieved excellent discriminatory performance for identifying persistent clones (AUC = 0.933; 95% CI: 0.910-0.954) with perfect specificity (1.000; zero false positives). The inverse V-P correlation was statistically significant across all four datasets (Spearman {rho} from -0.144 to -0.713; p < 0.01), providing the first cross-dataset quantitative confirmation of the trade-off. However, simultaneous evaluation of V-P profiles at the population scale revealed that the species does not conform to a binary dichotomy but rather exhibits three quantitatively distinguishable ecological strategies, for which we propose a functional trophic taxonomy: nosotrophic lineages (22.7%; V > 65, P < 35), specialized in the pathogenic niche; saprotrophic lineages (5.8%; V < 30, P > 45), with irreversible virulence attenuation and industrial specialization; and, as the central finding, amphitrophic lineages (39.1%; V [&ge;] 35, P [&ge;] 40), which simultaneously retain functional inlA and stress tolerance determinants (SSI-1) without detectable genomic sacrifice. The three strategies differed significantly (Kruskal-Wallis H = 138.7; p = 7.6 x 10-3{superscript 1}). The correspondence between trophic strategy and CC was predominant but not absolute, demonstrating that this phenotypic classification captures intra-CC functional heterogeneity inaccessible through conventional typing. Furthermore, comparison between genome-based and surveillance-informed classifications revealed that 60 hypervirulent isolates (CC1/CC14), genetically classified as nosotrophic, persisted for up to 8 years in industrial facilities despite lacking any recognized persistence markers -- indicating that their prolonged survival reflects environmental opportunity rather than intrinsic genomic adaptation. ConclusionsMulti-dimensional genomic profiling reveals that the virulence-persistence trade-off, while statistically robust, does not operate as an absolute paradigm. The amphitrophic strategy -- documented here for the first time as a quantitatively distinguishable category encompassing 39.1% of the analyzed population -- challenges the prevailing dichotomous model and identifies a previously unrecognized combined ecological niche. The ability to discriminate between genome-encoded persistence capacity and environmentally facilitated persistence provides a biological framework for understanding the ecological determinants of L. monocytogenes population dynamics in anthropogenic environments.

The CenIR regulatory system of Clostridioides difficile comprises a predicted transcriptional repressor, CenI, and a predicted membrane metalloprotease, CenR. The physiological role of CenIR and activating signal(s) are not known. CenIR belongs to the BlaIR family of regulators that mediate resistance to {beta}-lactam antibiotics. In canonical BlaIR systems, binding of a {beta}-lactam to the extracellular transpeptidase domain of BlaR triggers proteolysis of BlaI and thus induction of a closely linked {beta}-lactamase gene. However, CenR lacks a {beta}-lactam-binding domain and transposon mutagenesis indicated CenI is essential for viability even when {beta}-lactams are not present. Here we confirmed essentiality of CenIR and determined its regulon contains [~]12 genes, including an exported protein of unknown function (CDR_0474) that is induced about 500-fold and a peptidoglycan hydrolase (Cwp6) that is induced about 7-fold when cells are depleted of CenIR. There are no essential genes or {beta}-lactamases in the regulon. Phenotypic characterization of CenIR-depletion strains revealed slower growth, mild elongation and cell lysis. Deletion of cdr_0474 corrected all three defects, while deletion of cwp6 only rescued the lysis phenotype. It was possible to delete cenIR in either a {Delta}cdr_0474 or {Delta}cwp6 background. We propose that CenIR is essential because its absence leads to lysis due to Cwp6 overproduction. Bioinformatic analyses revealed the predicted extracellular sensing domains in annotated "BlaR" proteins are diverse. Thus, BlaIR systems are not dedicated to defense against {beta}-lactams but probably enable bacteria to adapt to a variety of environmental stimuli. ImportanceMany of the regulatory systems for controlling cell envelope biogenesis and stress responses have yet to be studied. Here we characterize a Clostridioides difficile BlaIR-like regulatory system that we have named CenIR for cell envelope. Unlike canonical BlaIR systems, which bind {beta}-lactams and induce a {beta}-lactamase, CenIR lacks a {beta}-lactam binding domain and is essential for viability even in the absence of antibiotics. We identified the genes in the regulon and found that CenIR is essential because its absence leads to overproduction of the Cwp6 peptidoglycan hydrolase. We also show that most annotated BlaIR-like systems lack a {beta}-lactam-binding domain, from which we infer that these systems have much broader physiological roles than generally appreciated.

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

The phosphodiesterase (PDE) NbdA (NO-induced biofilm dispersion locus A) consists of a membrane-integrated MHYT domain, a degenerated diguanylate cyclase (DGC) AGDEF domain and an EAL domain. The integral membrane domain MHYT is proposed to sense a so far unknown extracellular signal and transfers the information to the cytosolic enzyme domains to modulate cellular c-di-GMP level. Here, we show that full length NbdA from Pseudomonas aeruginosa PAO1 is an active PDE in vivo. In line with its PDE activity, overexpression leads to slightly reduced global c-di-GMP levels, and reduced twitching motility. Surprisingly, overexpression of truncated cytosolic NbdA variants exhibited increased c-diGMP levels, suggesting previously uncharacterized DGC activity despite lacking a canonical GGDEF motif. While full-length NbdA overexpression resulted in only slight c-di-GMP reduction, cytosolic variants induced a significant increase, indicating a potential for nonenzymatic effects like protein-protein interactions. Further investigation revealed a connection between NbdA and type IV pilus (T4P) function. Overexpression of NbdA conferred resistance to the T4P-dependent phage DMS3vir, suggesting interference with T4P assembly or function. Microscopic analysis demonstrated dynamic localization of NbdA, partially co-localizing with T4P components, supporting a role in T4P regulation. However, no clear link was re-established with flagellar motor switching or chemotaxis signaling. These findings position NbdA in the complex signaling network of c-diGMP and T4P-mediated surface behavior in P. aeruginosa. Future work will focus on elucidating the precise mechanisms of NbdAs PDE activity and its interplay with other DGC/PDE networks. ImportanceIn this work, we show the in vivo activity of the membrane-bound phosphodiesterase NbdA of Pseudomonas aeruginosa, its role in c-di-GMP homeostasis, cellular localization and implications in surface behavior. Using strains overexpressing NbdA and truncated protein variants, we detected a strong defect in growth on solid surfaces and an altered phage susceptibility. Co-localization experiments supported further the hypothesis of interaction with the type IV pilus apparatus. We propose for NbdA to be part of the protein network responsible for c-di-GMP level modulation at the cell pole and thereby regulating the function of type IV pilus apparatus.

Listeria monocytogenes relies on a tightly controlled set of surface-associated and secreted proteins to mediate host interaction and infection. The correct localization and exposure of these proteins at the bacterial surface are critical for virulence, yet the role of cell wall components in organizing this process remains incompletely understood. In particular, wall teichoic acid (WTA) glycosylation has been implicated in anchoring and function of selected surface proteins, but its global impact on protein distribution across the bacterial cell envelope is unclear. Here, we performed a comprehensive proteomic analysis to investigate how WTA glycosylation influences protein distribution in L. monocytogenes. Using isogenic mutants lacking rhamnose ({Delta}rmlT) or GlcNAc ({Delta}lmo1079) WTA glycosylation, we compared the exoproteome, the surface-accessible proteome and the surface-exposed proteome. Loss of WTA glycosylation did not result in a global disruption of the surface proteome but instead induced a redistribution of proteins across extracellular and surface-associated fractions. This effect was dependent on protein anchoring mechanisms, with limited changes observed for LPXTG-anchored proteins, moderate effects on non-covalently associated proteins, and a marked enrichment of lipoproteins in the surface-exposed proteome, particularly in the {Delta}lmo1079 mutant. In parallel, virulence-associated proteins displayed altered accessibility and exposure, with a progressive shift towards increased surface localization and a combination of shared and mutant-specific responses. This global effect was supported by functional annotation, which revealed that the affected proteins were associated with similar biological processes across fractions, highlighting a broad rather than pathway-specific impact of WTA glycosylation loss Together, these findings indicate that WTA glycosylation plays a key role in organizing the bacterial surface by modulating protein retention, exposure and release. Rather than affecting specific proteins, WTA glycosylation broadly shapes the spatial distribution of proteins across the cell envelope, with potential consequences for host- pathogen interactions.

Plasmids play a key role in the spread of virulence and antimicrobial resistance genes to new genetic backgrounds. Genetic variation in the transfer operon, the genes responsible for conjugation, can lead to substantial differences in transfer potential even between closely related plasmids. However, it is not clear how much genetic diversity there is in transfer operons of natural bacterial populations. Here, we analyze the prevalence and transfer potential of F-like plasmids, a clinically important family of plasmids in Enterobacteriaceae. Using 1200 Escherichia coli genomes isolated from three livestock-associated environments, we find that the fraction of F-like transfer operons that are functionally complete was significantly higher in poultry than in bovine and swine associated bacteria. This difference was not captured in methods that use the presence of replication genes to estimate plasmid prevalence. Confounders such as the phylogenetic relatedness of E. coli or the presence of antibiotic resistance could not explain these significant differences in transfer potential. Instead, it seems the poultry environment selects for plasmids with high transfer potential, as it also contained more conjugative plasmid types per isolate. While we find environment specific differences in overall plasmid frequency, patterns of transfer gene presence/absence were similar across the three environments. Regulatory and exclusion genes are the exception to this pattern, suggesting environment specific modulation of transfer rates. This highlights the use of genomic data to uncover environment specific differences in plasmid prevalence and transfer potential, revealing the selection pressures shaping horizontal gene transfer in these environments.

The genus Staphylococcus contains important human commensals and pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), which is a frequent colonizer of humans and a leading cause of healthcare-associated and life-threatening infections. While its virulence and pathogenicity have been extensively studied, factors driving the colonization and distribution of MRSA as a pathobiont are less understood. Here, we report on a cst sulfide detoxification gene cluster located on SCCmec, the antibiotic resistance-mediating genetic element of MRSA. Bioinformatic analyses revealed a heterogeneous distribution of cst clusters in staphylococcal genomes and that many clinically relevant SCCmec types introduce an additional cst cluster (cst2) to MRSA. While the canonical cst cluster (cst1) consists of the five genes tauE, cstR, cstA, cstB, and sqr, most staphylococcal cst clusters, including the SCCmec-located cst2, lack the sqr gene, which encodes for a sulfide:quinone reductase responsible for the initial step of sulfide detoxification. Growth experiments with a diverse set of representative Staphylococcus strains, cst-deletion mutants, and complementation with cst-containing plasmids demonstrated that the cst cluster enables sqr-independent polysulfide-detoxification. Furthermore, the additional cst2 cluster confers high polysulfide tolerance to MRSA, providing the pathogen with a unique advantage in polysulfide-rich environments. Using serial passaging co-cultivation experiments with methicillin-sensitive S. aureus (MSSA) strains, we demonstrated that in the presence of polysulfides cst2-containing MRSA can invade an established MSSA population and outperform the occupying resident in direct competition. Overall, our findings indicate that polysulfides are critical stress factors for staphylococci, potentially contributing to the spread of cst2-containing SCCmec and MRSA. ImportanceMethicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent human pathogens responsible for millions of life-threatening infections worldwide. It acquires antibiotic resistance through the genetic element SCCmec, which contains the characteristic mecA gene that renders the organism resistant to most classes of {beta}-lactam antibiotics. Besides mecA and accessory gene complexes necessary for the transfer of SCCmec and phenotype manifestation, the genetic element also contains prominent gene clusters with unknown functions. Here, we report on a (poly-)sulfide-detoxification gene cluster (cst2) present on SCCmec that provides MRSA with a unique advantage in environments containing polysulfides - highly reactive intermediates of sulfide oxidation naturally occurring as microbial stressors on mucosal surfaces inside the human body. We demonstrate that in the presence of polysulfides, cst2 enables MRSA to outperform non-MRSA in direct competition, thus supporting the invasion and proliferation of this pathogen independent of its antibiotic resistance.

Copper is widely used as a fast-acting antimicrobial, yet the strategies that allow bacteria to survive copper stress remain incompletely understood. Here, we characterize the transcriptional responses of Escherichia coli MG1655 to excess ionic copper using RNA sequencing and a genome-wide GFP-based promoter library. We applied 2 mM copper, which slows growth, and 8 mM copper, a near-lethal concentration. RNA-seq revealed extensive transcriptome remodeling, with 487 genes upregulated at 2 mM and 364 at 8 mM. Both concentrations strongly induced canonical copper-responsive systems, oxidative stress defenses, histidine biosynthesis, and multiple iron acquisition pathways - including enterobactin biosynthesis and transport - despite external iron failing to reduce copper toxicity. At 2 mM copper, additional pathways were activated, including heat-shock and protein-folding functions as well as lipid A, methionine and arginine biosynthesis. Copper exposure also repressed large gene sets: 486 genes at 2 mM, enriched for biofilm formation and pH elevation, and 217 genes at 8 mM, enriched for anaerobic metabolism. In contrast to the robust RNA seq results, we investigated the Horizon Discovery E. coli genome-wide GFP based promoter library as an alternative screening tool. However, in our experiments it showed low signal to noise ratios, limiting its suitability for large scale gene expression screening.

Mycobacterium abscessus are non-tuberculous mycobacteria that are widespread in the environment and of increasing global clinical significance. Accumulating evidence shows that M. abscessus has emerged as an important pathogen, driven by highly drug-resistant lineages, enhanced transmissibility and the acquisition of specific virulence factors. In this study, we describe a previously uncharacterised ESX secretion system encoded on a 123-kbp plasmid identified in a clinical isolate of M. abscessus. This ESX system, termed ESX-pMA07, is distinct from ESX systems previously reported in M. abscessus in both sequence composition and locus organisation, characterised by a unique arrangement of core ESX components and low sequence identity to ESX-3, ESX-4 and plasmid-borne ESX-P systems. ESX-pMA07 was detected in geographically diverse clinical isolates but was restricted to particular genotypes within the global M. abscessus phylogeny. Transcriptional profiling revealed expression of ESX-pMA07 components in artificial cystic fibrosis media and during intracellular growth in macrophage cell lines. Using CRISPR interference, we show that inducible silencing of eccC, encoding the ATPase component of ESX-pMA07, significantly reduced intracellular survival of M. abscessus within macrophages. To our knowledge, this is the first characterisation of a functional, plasmid-borne ESX secretion system in M. abscessus, demonstrating that mobile genetic elements contribute to the pathogens intracellular persistence and may influence its evolving virulence. Author SummaryMycobacterium abscessus is a rapidly emerging, highly drug-resistant bacterium that causes chronic infections, particularly in people with underlying lung disease such as cystic fibrosis. The factors that enable certain M. abscessus strains to persist inside host cells are not fully understood. In this study, we identified a previously unrecognised type VII secretion system (ESX) encoded on a large plasmid in a clinical isolate of M. abscessus. This plasmid-borne ESX system, which we termed ESX-pMA07, is genetically distinct from the ESX systems normally found on the chromosome and was detected in geographically diverse clinical isolates, but restricted to specific lineages within the global M. abscessus population. We show that ESX-pMA07 genes are expressed under conditions relevant to lung infection and during intracellular growth in macrophages. Using inducible CRISPR interference to silence the ESX ATPase gene eccC, we demonstrate that ESX-pMA07 contributes to intracellular survival of M. abscessus in macrophages. These findings reveal that mobile genetic elements can encode functional secretion systems that enhance intracellular persistence, providing a mechanism for the emergence and spread of virulence traits in this important pathogen.

The probiotic E. coli Nissle 1917 (EcN) strain is known to promote intestinal homeostasis via flagellin, the protomer of its motility apparatus, the flagellum. The flagellin of EcN shows atypical features, namely a hypervariable region (HVR), whose structure and significance have remained elusive. We therefore determined the crystal structure of the E. coli Nissle 1917 flagellin FliC at a resolution of 1.2 [A] which revealed an unusual domain architecture: the canonical D1 domain was found connected by an extended linker to an extensive HVR whose D2, D3 and D4 domains form an outer domain (OD) which surrounds the filament core comprised of conserved domains D0-D1. Using both recombinant proteins and gene-edited EcN strains expressing mutant flagellins, the functional requirement for these unique features was subsequently studied for effects on immune recognition on intestinal epithelial and immune cells, as well as on flagellar protein expression, assembly and bacterial motility. While human and mouse TLR5 immune recognition of flagellar proteins or intact bacteria was unaffected by removal of linker, D4 or total HVR, linker removal reduced protein stability and bacterial motility in both soft agar and liquid media swimming assays. Interestingly, depending on the environment, D4 or HVR removal had different effects on motility and surface structure. Finally, a site-directed mutagenesis approach highlighted that loss of TLR5 recognition strictly entails loss of motility but not vice versa. Our data indicate that specific HVRs/OD might be relevant for motility of E. coli Nissle 1917 in specialized environments, but not for immune recognition. Moreover, we find mutational tolerance is greater for immune recognition than for motility, providing new insights into bacterial adaptation to the host environment.

Mycobacteria form rough and smooth colonies. The Mycobacterium marinum strain 1218S is a smooth colony forming variant isolated from the 1218R strain, which forms rough colonies and is more virulent than 1218S in infecting fish. Genes for the type VII secretion ESX-1 system, which includes mycobacterial virulence genes, have been partially duplicated in M. marinum and is refered to as ESX-6. We recently reported that several ESX-1 genes are missing in the 1218S strain. On the basis of the complete genomes of these two and three other M. marinum strains we provide insight into strain differences and similarities focusing on 1218R and 1218S, and ESX genes, selected virulence genes, and LOS genes, which are involved in lipooligosaccharide synthesis and smooth colony formation. We provide RNA-Seq data for 1218R and 1218S and two other well-characterized M. marinum strains suggesting that loss of ESX-1 genes in 1218S results in increased transcript levels of ESX-6 genes. Furthermore, while there is no difference in gene synteny and sequence of LOS genes comparing 1218R and 1218S, with the exception of duplication of lsr2, a regulator of LOS genes, in 1218S. Our RNA-Seq data show increased transcript levels of LOS genes in stationary 1218S cells relative to 1218R indicating that transcription and/or RNA degradation of LOS genes influence smooth and rough colony formation. We finally provide data suggesting that Ms1 RNA affect the transcription of LOS genes (and ESX-1 genes), and that loss of ESX-1 genes influence biofilm formation.

Horizontal gene transfer is an important evolutionary process by which DNA is exchanged between cells that are physically co-located but not direct evolutionary descendants. Horizontal transfer of highly divergent DNA is relatively easy to detect and can produce major phenotypic changes, exemplified by acquisition of antibiotic resistance determinants. However, transfer of high-identity DNA, for example between strains of the same species, is likely to be more frequent, harder to detect, and highly impactful in aggregate. In this work, we demonstrate that recombination between soil isolates of the alphaproteobacterium Novosphingobium aromaticivorans can exchange chromosomal DNA, leading to multiple unselected recombination events spanning approximately 10% of the chromosome. Chromosomal recombination was directional, more efficient near an integrative and conjugative element (ICE), and required a relaxase found in the ICE. Recombination could not be observed into strains from closely related Novosphingobium species. In combination, these results suggest that ICE-mediated recombination can efficiently recombine DNA within N. aromaticivorans, increasing the adaptive potential of the species while also enforcing species boundaries through preferential intraspecific recombination. ImportanceHorizontal gene transfer is a key process in bacterial evolution. Mechanisms for transfer of mobile genetic elements are well-characterized, but less is known about how chromosomal DNA is recombined. In this work, we demonstrate that integrative and conjugative elements can efficiently recombine chromosomal DNA between strains of Novosphingobium aromaticivorans but not between different Novosphingobium species. We conclude that ICE-mediated chromosomal recombination can be an important adaptive mechanism within a species, due to its ability to recombine nearby chromosomal alleles, but also serves to delineate species-specific gene pools as a result of its limited phylogenetic range.

Type IVa pili (T4aP) are bacterial surface appendages that perform various functions including twitching motility, surface attachment, cell-cell interactions, and DNA uptake for natural transformation. Pivotal to each of these functions is the ability of T4aP to be dynamically extended and retracted from the cell surface. However, the factors that regulate this dynamic activity remain poorly understood. To address this question, we employ the competence T4aP from Vibrio cholerae as a model system. T4aP are composed of major and minor pilin subunits, named based on their relative abundance in the pilus filament. Prior work has established that minor pilins form a complex that initiates T4aP assembly. This allows for the subsequent addition of major pilins to the filament, which promotes T4aP extension. Here, we uncover that the stoichiometry of minor-to-major pilins is a crucial determinant of T4aP dynamic activity. Specifically, we show that either (1) overexpressing minor pilins or (2) underexpressing the major pilin results is a dramatic increase in the frequency of T4aP dynamics. These results indicate that the stoichiometry of major-to-minor pilins, not their absolute abundance, is one mechanism that regulates T4aP dynamic activity. AUTHOR SUMMARYType IVa pili (T4aP) are a broadly conserved family of filamentous bacterial appendages that help bacteria colonize surfaces, move towards or away from stimuli, and gain new traits through a mechanism of horizontal gene transfer called natural transformation. T4aP are primarily composed of protein subunits called major and minor pilins, named based on their relative abundance in the pilus filament. Bacteria can dynamically extend and retract pilus filaments from their surface through polymerization and depolymerization of these pilins. This dynamic activity is critical for the activities that T4aP carry out. However, the factors that regulate this dynamic activity remain incompletely understood. Here, we find that the ratio of minor-to-major pilins is one factor that regulates the frequency of dynamic activity. Minor pilins are a universally conserved feature of T4aP. So, the minor-to-major pilin ratio may be a broadly conserved mechanism for controlling dynamic T4aP activity in diverse bacterial species.

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.

Yersinia pseudotuberculosis (Yptb) replicates in immune cell-encompassed microcolonies within tissues. Bacterial replication is controlled by protection against neutrophil attack and by macrophage-released antimicrobial factors, such as nitric oxide (NO). During these attacks, bacteria located on the microcolony periphery encounter extracellular signals that differ from those in the interior. To dissect individual microbial populations, {gamma} interferon-activated macrophages were used to challenge microdroplet-grown Yptb harboring an NO-responsive mCherry reporter. Subsequently, bacterial subpopulations that hyperactivated the reporter were isolated from droplets composed of a reversible polymer matrix. RNA-seq analysis indicated that induction of nitrosative stress-associated genes was the primary determinant distinguishing peripheral bacteria from the remaining population. In addition, a secondary stress response that induced prophage-associated genes was detected, which could not be traced to either DNA damage or nitrosative stress responses. Activated macrophages also induced the expression of the Yptb itaconate degradation enzyme-encoding transcript throughout the entire colony. To determine if itaconate production by the interferon-activated Irg1 protein played a role in restricting Yptb, bacteria harboring an itaconate-responsive reporter and Yptb mutants defective for itaconate degradation were analyzed during bacterial colonization of the murine spleen. Only a subset of colonies appeared to be exposed to itaconate, which may explain the very small defects exhibited by mutants unable to degrade the interferon-induced macrophage product. These results indicate that the primary response of bacteria to macrophage-elicited factors is likely associated with protection against NO-derived metabolites.

Some microbes externalize costly biosynthetic precursors in sufficient quantities to sustain a recipient population through cross-feeding. However, it is unclear whether metabolites are externalized purely for a reciprocal benefit or if metabolite externalization also plays a physiological role for the producer. Here we focus on adenine, a metabolite externalized by some strains of the phototrophic bacterium Rhodopseudomonas palustris at sufficient levels to support Escherichia coli growth. In 10 long-term monocultures and 22 cocultures pairing R. palustris with E. coli, extracellular adenine externalized by all 140 isolates screened was 1.7 - 3.4-fold higher than that by the ancestor, suggesting that there was selective pressure for adenine externalization. We hypothesized that adenine is toxic to R. palustris. The CGA0092 growth rate decreased by half in the presence of about 0.3 mM external adenine. This inhibitory effect increased by an order of magnitude when we over-expressed adenine phosphoribosyltransferase to overcome a bottleneck in the purine salvage pathway, suggesting that toxicity stems from a metabolite derived from adenine. To assess whether adenine tolerance is connected to adenine externalization, we surveyed 12 evolved isolates and 49 environmental strains that externalized different levels of adenine, revealing a significant positive correlation. Our data suggests a physiological role for externalization of costly-metabolites like adenine at the origin of cross-feeding. In addition to cross-feeding, resulting metabolic interactions could be negative, considering that even a biosynthetic precursor like adenine can be inhibitory.