microLife

Archaea have several cell surface structures that belong to the type 4 filament (TFF) superfamily. What all have in common is the presence of core minimal assembly systems consisting of an ATPase, a platform protein, a filament forming protein and a class III signal peptidase. Here we generated novel MacSyFinder2 models to identify and classify archaeal TFF systems. Our analysis revealed a vast diversity of archaeal TFF with several members harboring one or more TFF assembly machineries. Structure-based phylogenetic analyses revealed that the variable N-terminal domain of TFF-related ATPases reflects the subsystem clustering. This indicates a diversification of the core machinery components within the archaeal secretion ATPase family driven through structural innovation. Genome-wide screening of SP-III containing proteins revealed the widespread presence of substrate binding proteins with SPIII. We hypothesize that these binding proteins with canonical SPIII cleavage sites are used to functionalize TFF machineries for efficient substrate scavenging, expanding the functional repertoire of archaeal TFF systems beyond currently characterized roles. Author SummaryThe type IV filament superfamily (TFF) comprises a broad group of surface structures that are widespread across both Archaea and Bacteria. While most well-characterized members originate from bacteria, only a limited number of archaeal TFF systems have been experimentally studied so far. Here, we expand the known diversity of archaeal TFF loci through bioinformatic and comparative genomic analyses. Our results show that these systems are far more diverse and versatile than previously appreciated, often exhibiting specialized functions. The structural diversification of the ATPase machinery likely played a key role in driving the functional diversification of TFF systems in Archaea. Overall, these findings deepen our understanding of how archaea adapt and persist in diverse environments, highlighting their surface structures as essential tools for communication, adhesion, and nutrient acquisition.

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI

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.

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (cas) genes provide adaptive immunity to bacteria and archaea. CRISPR-Cas systems acquire short DNA fragments from the genomes of infecting plasmids and viruses, which are inserted into the CRISPR locus as a "spacer" sequence in between repeats. Spacers constitute a memory of infection that is used to recognize and attack invading genetic elements in future infections. Despite the evolutionarily divergent genetic backgrounds of bacteria and archaea, the same CRISPR-Cas systems are functional in both of these prokaryotic domains. In bacteria, efficient spacer acquisition requires the DNA repair nucleases RecBCD/AddAB. These nucleases, however, are not present in archaea. Here we investigated the importance of the DNA repair systems in the Haloferax volcanii Type I-B CRISPR-Cas response. We found that elimination of the DNA repair nuclease Mre11-Rad50, but not Fen1, substantially reduces spacer acquisition. CRISPR immunity against H. volcanii pleomorphic virus 1 (HFPV-1), on the other hand, was not affected by these deletions. Our results describe how CRISPR-Cas systems have adapted to provide anti-viral defense to hosts from different domains of life.

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.

BackgroundStaphylococcus aureus (S. aureus) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. The mechanisms underlying these differences remain poorly understood. This study investigates how distinct intracellular S. aureus isolates influence host signaling programs and infection outcomes by modulating cell death pathways and TNF-R1 dependent regulation of host cell fates across different human cell lines. MethodsFour S. aureus isolates were analyzed for intracellular localization using transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block-face scanning electron microscopy (SBF-SEM), and imaging flow cytometry. Transcriptional reprogramming of infected U937 monocytes was examined by mRNA sequencing. Infection outcomes were characterized and compared to A549 and SaOS-2 cell lines employing Luminex cytokine assays, flow cytometry and Western blot analysis to characterize host cell death mechanisms in both wild-type and TNF-R1 deficient backgrounds. ResultsAll S. aureus isolates localized to endolysosomal and cytosolic compartments but also peri and putatively intranuclearly, revealing an unexpected intracellular niche. In U937 monocytes, infection induced a conserved stress signature alongside isolatespecific transcriptional programs divergently affecting inflammation, metabolism, and cell fate, which was markedly attenuated in response to the chronicinfection isolate EDCC 5464. Cell death outcomes were likewise isolatedependent, involving intrinsic and extrinsic apoptosis, mitochondrial depolarization, and caspase-1 activation at distinct temporal dynamics. TNFR1 loss initially delayed but exacerbated late, isolate-independent cytotoxicity, identifying TNFR1 as a key regulator of U937 infection outcome. SaOS2 and A549 cell death was far less affected by isolate or TNF-R1 deficiency. ConclusionsThese results highlight the multilayered determinants governing intracellular S. aureus survival, non-canonical intracellular localization, and host cell susceptibility. The TNF/TNF-R1 axis is identified to critically determine regulated host defense during early infection stages in a tissue-specific manner. Together with distinct isolate-driven gene expression profiles, infection risks under TNF-targeted therapies and the contribution of S. aureus heterogeneity should be considered in the design of future host-directed treatment strategies. Plain English summaryThe bacterium Staphylococcus aureus (S. aureus) often lives harmlessly in humans but can cause severe or recurrent infections when the skin barrier is broken or the immune system is weakened. A major reason for its persistence is its ability to hide inside human cells, where it is shielded from immune attacks and antibiotics. To effectively target such bacteria, it is crucial to understand that infections vary depending on both the bacterial strain and the infected cell type. Many reasons behind these differences are still puzzling. We explored how different types of S. aureus (collected from different disease types) change how human cells respond to infection. We focused on how the different strains influence the way immune cells adjust their gene activity during infection, and how a receptor called TNF-R1 is involved in managing cell death responses. Bacteria were found not only in compartments meant to destroy them but also near and even inside the cell nucleus, an unexpected location. All strains triggered a similar stress response but also distinct patterns influencing inflammation, metabolism, and cell survival. A strain linked to chronic infection caused weaker responses, suggesting greater stealth. Cells lacking TNF-R1 initially survived longer but later showed greater damage, indicating this receptors role in infection control. In lung and bone cells, these effects were less pronounced. Concludingly, S. aureus occupies unexpected niches inside human cells and uses varying survival strategies. TNF-R1 is a key regulator of host infection responses in the analyzed immune cells, highlighting that both bacterial diversity and host factors must be considered when developing targeted treatments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/723175v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1b4214org.highwire.dtl.DTLVardef@18f4ee6org.highwire.dtl.DTLVardef@1851742org.highwire.dtl.DTLVardef@ba0359_HPS_FORMAT_FIGEXP M_FIG Peri- and intranuclear localization early after S. aureus uptake across host cell lines, with isolate-specific modulation of host fates and a critical role for TNF-R1 to mediate regulated death responses of U937 cells. At 2 hpi, intracellular S. aureus not only localizes in (LAMP-1 decorated) membrane-enclosed compartments or directly in the cytosol, but within invaginations of the nuclear surface and intranuclearly with or without being surrounded by a vesicular membrane in U937wt, SaOS-2wt, and A549wt cells. At 4 hpi, S. aureus triggers differential gene expression in (A) U937wt cells to an isolate-specific extent, with both unique and shared transcriptomic signatures across the four isolates, that is muted for the chronic infection isolate EDCC 5464. Apoptotic cell death is induced to an isolate-dependent extent involving extrinsic initiator caspase-8, intrinsic initiator caspase-9 (EDCC 5055 only), and variable effector caspase-3/-7 activity in the earlier stages of infection (6 hpi), which then barely increases (24 hpi) in U937wt cells. S. aureus-induced cell death and caspase activation is abolished in (B) U937{Delta}TNF-R1 at 6 hpi, but is significantly reinforced at 24 hpi with diminished isolate-specificity. Correspondingly, mitochondrial trans-membrane potential ({Delta}{Psi}m) is disrupted for all isolates upon TNF-R1 knockout, as well as caspase-1 activity, suggesting pyroptotic pathway activation at later stages of infection. (C) SaOS-2 wt cells show moderate caspase-3/-7 and -1 activation, while infection induces detachment of (D) A549wt cells with minimal caspase activation. Infection induces an isolate- and cell line-dependent cytokine release. Coloured arrows indicate the mean proportion of effector-positive cells ({uparrow} [~]20-40%, {uparrow} {uparrow} 40-60%, {uparrow} {uparrow} {uparrow} >60%) representing each S. aureus isolate. Grayed signaling arrows indicate the hypothesis by which TNF-R1 activation and internalization is required to kill lysosomal S. aureus via activation of anti-microbial enzymes and downstream regulated death pathway activation. Created with BioRender.com. C_FIG

In 2003, Suhre and Claverie demonstrated that the difference between the fraction of charged amino acids and the fraction of polar uncharged amino acids in a proteome (the CvP-bias) was the single genomic feature that most strongly discriminates hyperthermophilic microorganisms from their mesophilic and thermophilic counterparts. The original analysis was based on 71 completely sequenced genomes available at the time. Here, using modern genome databases -- specifically the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) for curated optimal growth temperature (OGT) metadata and the NCBI RefSeq FTP archive for sequence data -- the same analysis is repeated at approximately 28-fold larger scale, covering 1,963 bacterial and archaeal genomes (103 hyperthermophiles, 409 thermophiles, 1,451 mesophiles). The original finding is confirmed with high statistical confidence: CvP-bias is elevated in hyperthermophiles (mean 11.1 {+/-} 3.1%) relative to mesophiles (5.0 {+/-} 2.0%; ANOVA F = 496, p < 10-170), with a large effect size (Cohens d = 2.32) and area under the receiver operating characteristic curve of 0.94 for binary hyper/meso classification. Principal component analysis confirms that the first principal component, explaining 47% of variance, is loaded by CvP-bias as a major contributor, separating hyperthermophiles from other organisms. These results establish that the CvP-bias signal identified in 2003 is not an artifact of small sample size but a genuine, robust property of hyperthermophilic proteomes.

Intestinal inflammation increases the abundance of Enterobacteriaceae in the gastrointestinal tract by several orders of magnitude. These population expansions, or blooms, are associated with disease progression and have been suggested to exacerbate intestinal pathologies in some settings. Murine studies have shown that during the early stages of Escherichia coli colonization, i.e., during engraftment, inflammation enhances fitness through processes that depend on Moco, an enzyme cofactor found in a variety of oxidoreductases that consists of molybdenum coordinated by a pterin molecule. Using a murine commensal E. coli isolate and a DSS-induced colitis model in mice, we investigated whether Moco is also important for blooms of E. coli that are part of the resident microbiota, that is, for E. coli that have engrafted well before the onset of inflammation. We show that resident wild-type and Moco- E. coli exhibit comparable expansions in response to inflammation, indicating that, in this context, Moco-dependent processes such as nitrate respiration or formate oxidation were not important for inflammation-induced blooms. We find that Moco is important, however, for E. coli colonization in the absence of inflammation, suggesting that alternative respiratory pathways or other Moco-dependent processes are necessary for E. coli colonization of a healthy murine gut. Our findings demonstrate that the mechanisms underlying inflammation-induced blooms can depend on the temporal relationship between engraftment and inflammation, and also highlight the importance of considering colonization stage in identifying and interpreting the factors that affect the fitness of microbes colonizing the intestine.

The facultatively anaerobic bacterium Shewanella oneidensis MR-1 contains in its genome two operons, so_3056-3058 and so_3299-3301, each including genes for putative periplasmic flavocytochrome c and ammonia-lyase of aromatic amino acids. To determine their role in anaerobic respiration, we produced the encoded ammonia-lyases SO_3057 and SO_3299 in Escherichia coli and determined their substrate specificities. SO_3057 was found to cleave trimethylammonium group from ergothioneine to yield thiourocanic acid, whereas SO_3299 catalyzed a similar conversion of N({pi})-methyl histidine betaine to yield N({pi})-methyl urocanate. The catalytic efficiencies (kcat/Km values) were (3-4) x 106 M-1 s-1, and the pH optima of activity were between 8 and 9. Ergothioneine induced SO_3057 synthesis in anaerobic S. oneidensis cells and their growth, and thiourocanate stimulated respiration as an alternative terminal electron acceptor. The predicted 3D structures of the genetically coupled flavocytochromes c (SO_3056/58 and SO_3300/3301) are consistent with their use of thiourocanate and N({pi})-methyl urocanate, respectively, as electron acceptors. We therefore conclude that the periplasmic lyases encoded by the so_3057 and so_3299 genes contribute to anaerobic respiration in S. oneidensis by producing terminal electron acceptors for the genetically coupled flavocytochromes c.

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.

Bacterial pathogens must adapt to dynamic host tissue environments to proliferate. Accordingly, elegant regulatory systems evolved to overcome challenges presented by the host and satisfy nutritional requirements. Sulfur is an essential macronutrient and Gram-positive bacteria such as Staphylococcus aureus balance this nutritional requirement by employing the transcriptional repressor, CymR. Previous investigations defined the S. aureus CymR regulon by comparing transcripts generated in a cymR mutant cultured in cystine replete, rich medium to wild type cells. This study defines the S. aureus CymR-dependent and -independent sulfur-starvation response in chemically defined growth conditions. Results demonstrate that the sulfur starvation and sulfur replete CymR regulons exhibit considerable overlap, including previously noted connections between iron acquisition, oxidative stress, and sulfur metabolism. The link between iron acquisition, oxidative stress, and sulfur metabolism is validated further by the finding that sulfur-containing glutathione (GSH) mitigates heme and peroxide toxicity. In addition to GSH, Cys and thiosulfate fulfill the S. aureus sulfur requirement. Transcriptional responses to organic (cysteine, cystine, reduced and oxidized GSH) or inorganic thiosulfate were quantified, revealing sulfur source-specific expression patterns. Thiosulfate induced the largest number of differentially expressed genes. Consequently, the thiosulfate transporter (SAUSA300_RS10985) has been confirmed as essential for S. aureus growth when thiosulfate is the sulfur source. Furthermore, we demonstrate that a hypothetical protein operonic with SAUSA300_RS10985, SAUSA300_RS10980, supports maximal growth on thiosulfate. Collectively, a resourceful transcriptomics framework is provided which underscores the dynamic nature of S. aureus sulfur metabolism.

Anaerobic methanotrophic archaea are key members of the biological methane filter, thereby preventing emissions of this strong greenhouse gas into the atmosphere. Previous studies on freshwater anaerobic methanotrophs targeted the activity of these microorganisms at circumneutral pH whereas molecular ecology studies identified this phylotype also in acidic environments such as peatlands; it is currently unknown whether they can adapt to low pH and remain effective in the biological methane filter in low pH environments. Here we show that a granular enrichment culture of the freshwater methanotroph Ca. M. vercellensis loses activity when experiencing pH stress but remains metabolically active down to pH 5.65 with appropriate adaptation time, indicating that adaptive changes are necessary to accommodate anaerobic methane oxidation at lower pH. Analyses of archaeal lipids revealed an increase in zwitterionic intact polar lipids over anionic lipids as an adaptation. This coincided with a change in granule structure while methane oxidation rate and enrichment state of Ca. M. vercellensis remained stable. We show that Ca. M. vercellensis remains metabolically active at lower pH values, despite increased maintenance energy demands and the need for cytoplasmic pH homeostasis. Our study demonstrates that adaptations to stress by slow-growing microorganisms may require long-term observation and is thereby instrumental for a better understanding of methane cycling in acidic ecosystems.

Salmonella enterica encounters acid stress during gastrointestinal transit and within the phagosomal environment of macrophages. Acid stress resistance has been well characterized in Salmonella enterica serovar Typhimurium, but comparative studies in the human-adapted Salmonella enterica serovar Typhi are limited. We compared the growth of S. Typhimurium 14028s and S. Typhi Ty2 at pH values ranging from 3-8 and observed that Salmonella enterica serovar Typhimurium exhibits enhanced growth at pH 4.5 compared to S. Typhi. Comparative transcriptomic profiling of S. Typhimurium and S. Typhi at pH 4.5 and 7.5 identified numerous differentially expressed acid-induced genes (DEGs), including genes encoding membrane proteins (OmpC, PhoE, HydB), a transcriptional regulator (RpoS), and stress response proteins (YciG, STM14_1829, YmdF). Targeted deletion of selected genes in S. Typhimurium significantly suppressed growth at acidic pH, confirming their role in acid stress resistance. These resistance mechanisms are compromised in S. Typhi due to pseudogenization. Heterologous expression of pseudogenized genes in S. Typhi restored acid tolerance. Collectively, these findings suggest that S. Typhi has lost the ability to withstand acid stress due to genomic decay and the loss of multiple genes essential for acid survival in S. Typhimurium, reflecting divergent evolutionary paths in these two serovars. ImportanceSalmonella Typhimurium must adapt to acidic pH conditions in the intestinal tract and the intracellular environment to cause infection. In this study, we show that the enteric fever serovar Salmonella Typhi exhibits impaired growth at pH 4.5, in comparison to Salmonella Typhimurium. We further show that the loss of specific membrane proteins, a transcriptional regulator, and a family of stress response proteins in Salmonella Typhi are responsible for this difference. Collectively, these observations suggest that Salmonella Typhi has evolutionarily lost the ability to withstand acid stress due to differences in its interaction with the human host. This has important implications for the pathogenesis of typhoid fever.

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.

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.