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PubMed2026

EprX associates with concurrent shifts in antimicrobial resistance and virulence in clinical bloodstream E. coli: a putative adaptive node for bacterial fitness.

Bloodstream infections (BSIs) caused by E. coli represent a growing global threat, driven by escalating antimicrobial resistance (AMR) and sustained virulence. However, the regulatory mechanisms linking these two phenotypes remain poorly understood. Here, we identify EprX, a previously uncharacterized YjbI-type pentapeptide repeat protein (PRP), a locus that our data suggest may influence metabolic and transcriptional profiles in clinical BSI E. coli isolates. Genomic screening of 85 clinical BSI strains reveals that eprX is present in 21.2% of isolates, often within distinct genomic contexts suggestive of mobile acquisition. Using λ-Red recombineering, we constructed eprX knockout mutants. Loss of eprX is associated with altered antimicrobial resistance profiles, increasing susceptibility to gentamicin, ciprofloxacin, and levofloxacin. This phenotype is consistent with upregulation of outer membrane porin genes (ompC, ompF) and downregulation of multidrug efflux pump genes (macB, mdtC, emrB) and two-component regulatory system genes. eprX deficiency also appears to correlate with attenuated virulence in our assays, as evidenced by improved survival of Galleria mellonella larvae (65-95% at 72 h post-infection vs. 40-60% for wild-type strains) and reduced adhesion to and invasion of human HeLa cells. Transcriptomic profiling reveals that eprX carriage is associated with broad, coordinated shifts in the expression of genes involved in LPS transport (lptG/lptF), type ;II secretion system components (gspD/gspE/gspF), autotransporter adhesins (ag43), and flagellar assembly, suggesting potential disruptions in outer-membrane integrity, biofilm formation, and virulence programs. Our data suggests that eprX is a genetic locus whose presence correlates with concurrent shifts in resistance maintenance and virulence traits, representing a putative adaptive node within the E. coli fitness landscape.

Animals↗

PubMed2026

Experimental insights in taxon-specific functional responses to droughts in glacier-fed stream biofilms.

BACKGROUND: Glacier-fed streams are predicted to face increasingly frequent and intense droughts. However, the impacts of drought events on benthic biofilm, including bacteria, eukaryotes, and viruses, the dominating life form in glacier-fed streams, remain poorly understood. RESULTS: Using streamside flume mesocosms in the Swiss Alps, we grew glacier-fed stream biofilms over 103 days and exposed them to three droughts. Using a multi-omics approach (metagenomics, metatranscriptomics, and metaproteomics), we assessed the effects of a series of droughts on the taxonomy and metabolic activity of bacterial, eukaryotic, and viral metagenome-assembled genomes (MAGs). We found that the first drought (6 h) caused only minor changes, including mild upregulation of heterotrophic metabolism and signs of stress in diatoms. In contrast, the second drought (24 h) significantly altered both the composition and functionality of the microbiome, shifting phototrophic dominance from diatoms to Cyanobacteriota, while maintaining overall phototropic biomass and further upregulating the heterotrophic metabolism. Interestingly, a third 24 h drought had no detectable transcriptomic effect between pre- and post-drought conditions, suggesting a certain level of adaptive responses to droughts, but with the low diatom abundance being maintained. CONCLUSIONS: These findings indicate that glacier-fed biofilm microorganisms initially resisted short-term drought, but a second longer drought caused important shifts in their community structure, activity, and function. Climate-induced increases in drought frequency or duration may therefore have a lasting impact on microbial ecosystem functioning in glacier-fed streams. Video Abstract.

Biofilms↗

PubMed2025

Variability in intrinsic promoter strength underlies the temporal hierarchy of the Caulobacter SOS response induction.

Bacteria encode for gene regulatory networks crucial for sensing and repairing DNA damage. Upon exposure to genotoxic stress, these transcriptional networks are induced in a temporally structured manner. A case in point is of the highly conserved SOS response that is regulated by the LexA repressor. Studies have proposed that affinity of LexA towards promoters of SOS response genes is the primary determinant of its expression dynamics. Here, we describe an additional level of regulation beyond LexA box properties that modulates the SOS response gene expression pattern. Using transcriptomic analyses, we reveal a distinct temporal hierarchy in the induction of SOS-regulated genes in Caulobacter crescentus. We observe that LexA box properties are insufficient in predicting the temporal hierarchy of these genes. Instead, we find that intrinsic promoter strength underlies the order of gene activation, with differential sigma factor association as one of the factors modulating gene expression timing. Our findings highlight a novel regulatory layer in SOS dynamics and underscore the importance of promoter properties in shaping bacterial stress responses.

Promoter Regions, Genetic↗

PubMed2026

Endozoicomonas acroporae enhances coral thermal resilience through host-microbe coordination.

Probiotics hold promise for enhancing coral resilience under climate-driven thermal stress, yet their mechanisms remain poorly understood. Although the bacterial genus Endozoicomonas has been proposed to benefit corals, in vivo evidence of beneficial effects on the host remains limited. Here, we establish Endozoicomonas acroporae Acr-14T as a coral probiotic and characterize its effects on the reef-building coral Stylophora pistillata. We show that E. acroporae Acr-14T enhances host thermal tolerance, colonizes coral tissues, and forms coral-associated microbial aggregates. Microbial profiling indicates that probiotic treatment is associated with reduced relative abundances of opportunistic microbes and enrichment of putatively beneficial taxa. To support transcriptomic analyses, we assembled a chromosome-level genome of S. pistillata clade 1 (Pacific lineage) and found that E. acroporae Acr-14T treatment mitigates heat-induced protein-folding stress and apoptotic signaling. Single-cell transcriptomics further revealed altered expression of genes involved in S-adenosylmethionine (SAMe) metabolism and pro-survival signaling in gastrodermal cells of probiotic-treated corals. Together, our results provide a cell-type-resolved view of host responses linked to Endozoicomonas-mediated coral thermal resilience and offer insight into molecular mechanisms implicated in host-microbe interactions under environmental stress.

Animals↗

PubMed2024

Adaptation for Staphylococcus aureus to hosts via insertion mutation in the accessory gene regulator agrC gene: decreased virulence and enhanced persistence capacity.

UNLABELLED: Staphylococcus aureus is an important human pathogen due to its vast array of virulence factors regulated by multiple regulatory mechanisms, including the accessory gene regulator. In this study, two S. aureus strains were simultaneously isolated from the blood of a febrile patient, belonging to the same clone, designated as 23H with a complete hemolytic phenotype, and 23B, exhibiting an incomplete hemolytic phenotype. The genomic comparison between strains 23B and 23H revealed that 23B had a single adenine base insertion at position 923 in the agrC gene, leading to a functional loss of the encoded AgrC. Experimental findings showed that strain 23B had decreased hemolytic activity, lower cytotoxicity against human alveolar epithelial A549 cells and in the Galleria mellonella model, and a reduced ability to survive intracellularly after infecting macrophages, in comparison to 23H. Conversely, 23B exhibited enhanced biofilm formation, greater adherence to A549 cells, and increased persistence in the face of vancomycin and daptomycin treatment. Transcriptomic analysis revealed that 23B upregulated surface protein-encoding genes while simultaneously reducing the expression levels of virulence factors, highlighting the intricate regulatory adjustments facilitating its persistence and reducing pathogenic potential. ATP assay results indicated that 23B maintained elevated ATP levels during the exponential phase yet exhibited reduced levels in the stationary phase when compared with 23H. Our findings suggested that the mutation in the agrC gene of S. aureus results in diminished virulence but markedly enhances persistence. This mutated strain warrants clinical attention because it may lead to treatment failures and persist in patients. IMPORTANCE: In clinical antimicrobial therapy, bacterial strains often develop resistance to antimicrobial agents. Additionally, mutations in their gene regulatory networks can increase their persistence, especially in immunocompromised patients. This study identified an insertion mutation in the accessory gene regulator, agrC gene, carried by a Staphylococcus aureus strain isolated from the blood of a febrile patient, leading to the functional loss of AgrC. Further research revealed that despite the reduced virulence of the mutated strain, it significantly bolstered the capacity to adapt and endure within the host during prolonged infections. This was evidenced by increased adhesion and biofilm formation capabilities, development of antimicrobial tolerance, and decreased ATP levels linked to persistence. Therefore, monitoring these mutations in S. aureus is crucial clinically, as they can complicate treatment strategies.

Staphylococcus aureus↗

PubMed2025

Live-Cell Monitoring and Omics Analysis of Liquid-Solid Transitions of Biomolecular Condensates.

Biomolecular condensates, or so-called membraneless organelles, transition from liquid into more solid-like states over time, contributing to the development of pathological conditions. The present study proposes a simple method using photoactive yellow protein (PYP) and its specific fluorescent covalent ligands to distinguish between the liquid and solid states of protein condensates in live cells. The method, compatible with fluorescence-activated cell sorting (FACS), correlates the stiffness of specific protein condensates with their accessibility to PYP ligands, enabling quantitative multicolor monitoring of condensate solidification. We applied this technique to 12 phase-separating proteins and their mutants, finding that TDP-43, particularly its A315T mutant linked to familial amyotrophic lateral sclerosis, most readily forms solid aggregates. Furthermore, this FACS-compatible strategy enabled the isolation of distinct cell populations based on condensate states, allowing for subsequent proteomic and transcriptomic analyses. Our findings demonstrate that condensate solidification is accompanied by the upregulated expression of extracellular matrix proteins, suggesting a previously unrecognized link between solid aggregate formation and extracellular matrix hardening.

Humans↗

PubMed2026

An XRE-type regulator in Streptococcus mutans plays an important role in brpA expression and oxidative stress tolerance response.

This study used a functional genomics approach to explore the role of a xenobiotic response element (XRE)-type regulator (SMU.405c) in Streptococcus mutans physiology, including the expression of biofilm regulatory protein BrpA. Results showed that deletional mutation of xre significantly reduced the ability of the deficient mutant to grow in the presence of methyl viologen, a commonly used oxidative stressor (P < 0.001). When challenged in a hydrogen peroxide killing assay, the survival rate of the ∆xre mutant was >2-log less than the parent strain after 60 min (P < 0.001). Luciferase reporter fusion assays showed that xre deficiency had no significant effect on luciferase expression when it was under the control of the intact brpA promoter, but the reporter activity increased by >6-fold (P < 0.001) when the reporter gene was fused to a brpA promoter derivative with deletion of a putative XRE-binding box. Electrophoretic mobility shift assay (EMSA) showed that recombinant XRE interacted with the brpA promoter, resulting in an electrophoretic shift of the promoter probes. In vitro transcription assay also showed that inclusion of XRE caused transcription to fall off, significantly reducing full-length brpA transcripts. RNA-seq analysis revealed that deficiency of XRE led to altered expression of >102 genes by >2-fold (P < 0.05), including 28 with increased expression, and 74 with decreased expression. Among the down-regulated were genes for DNA repair and oxidative stress tolerance response. These results suggest that XRE (SMU.405c) in S. mutans plays an important role in brpA expression and oxidative stress tolerance response.IMPORTANCEStreptococcus mutans, a keystone pathogen in human dental caries, primarily lives in the highly diverse microbiota on tooth surfaces, where the conditions are often harsh and fluctuate frequently. Locus SMU.405c was annotated to encode a xenobiotic response element (XRE)-like transcriptional regulator, but no information is available concerning the role of this protein in S. mutans pathophysiology. This study used a functional genomics approach along with molecular and transcriptomic analysis to characterize a deletional xre mutant, and the results showed that xre deficiency in S. mutans resulted in weakened oxidative stress tolerance response and alterations in transcription of >102 genes, including those known to play an important role in cell envelope biogenesis and stress tolerance response. Reporter fusion assay, electrophoretic mobility shift assay (EMSA), and in vitro transcription further demonstrated that the XRE-like regulator encoded by SMU.405c is a repressor of brpA expression and plays an important role in oxidative stress tolerance response.

Streptococcus mutans↗

PubMed2022

Genome-scale metabolic modeling reveals increased reliance on valine catabolism in clinical isolates of Klebsiella pneumoniae.

Infections due to carbapenem-resistant Enterobacteriaceae have recently emerged as one of the most urgent threats to hospitalized patients within the United States and Europe. By far the most common etiological agent of these infections is Klebsiella pneumoniae, frequently manifesting in hospital-acquired pneumonia with a mortality rate of ~50% even with antimicrobial intervention. We performed transcriptomic analysis of data collected previously from in vitro characterization of both laboratory and clinical isolates which revealed shifts in expression of multiple master metabolic regulators across isolate types. Metabolism has been previously shown to be an effective target for antibacterial therapy, and genome-scale metabolic network reconstructions (GENREs) have provided a powerful means to accelerate identification of potential targets in silico. Combining these techniques with the transcriptome meta-analysis, we generated context-specific models of metabolism utilizing a well-curated GENRE of K. pneumoniae (iYL1228) to identify novel therapeutic targets. Functional metabolic analyses revealed that both composition and metabolic activity of clinical isolate-associated context-specific models significantly differs from laboratory isolate-associated models of the bacterium. Additionally, we identified increased catabolism of L-valine in clinical isolate-specific growth simulations. These findings warrant future studies for potential efficacy of valine transaminase inhibition as a target against K. pneumoniae infection.

Humans↗

PubMed2025

Staphylococcus aureus COL: An Atypical Model Strain of MRSA That Exhibits Slow Growth and Antibiotic Tolerance due to a Mutation in PRPP Synthetase.

Methicillin-resistant Staphylococcus aureus (MRSA) has been a pathogen of global concern since its emergence in the 1960s. As one of the first MRSA strains isolated, COL has become a common model strain of S. aureus. Here we report that COL is, in fact, an atypical strain of MRSA that exhibits slow growth and multidrug tolerance. Genomic analysis identified three mutated genes in COL (rpoB, gltX and prs) with links to tolerance. Allele swapping experiments between COL and the closely-related, nontolerant Newman strain uncovered a complex interplay between these genes. However, Prs (phosphoribosyl pyrophosphate [PRPP] synthetase) accounted for most of the growth and tolerance phenotype of COL. Biochemical and transcriptomic analysis revealed that COL does not exhibit slow growth as a result of partial stringent response activation, as previously proposed. Instead, the COL Prs mutation greatly reduces the PRPP synthetase activity of the enzyme and leads to downregulation of pyrimidine, histidine, and tryptophan synthesis, three pathways that rely on PRPP. Overall, our findings indicate that COL is an atypical, antibiotic-tolerant strain of MRSA whose isolation predates the previous first report of tolerance among clinical isolates. Characterization of clinical Prs mutations and their relationship with tolerance requires further investigation.

Methicillin-Resistant Staphylococcus aureus↗

PubMed2026

Translating single-cell RNA sequencing into monocyte direct leukocyte subpopulation-transcript abundance assay ratio-based biomarkers (IFI27/PSAP or IFI27/CTSS) for clinical detection of viral infection.

A rapid method for triaging febrile patients by aetiology (e.g., viral or bacterial infection) using gene expression in peripheral blood (PB) is an intensively researched area. However, gene expression in blood represents a composite sum of gene expression of all the component cell types present in the sample. As a result, numerous genes are measured in most proposed signatures. Herein, we propose a simple ratio-based biomarker (RBB) called direct leukocyte subpopulation-transcript abundance assay (DIRECT LS-TA) that recapitulates gene expressions of a single cell type in PB (i.e., monocytes). Based on single-cell RNA sequencing (scRNAseq) data and bulk expression data, IFI27 and SIGLEC1 are found as interferon-stimulated genes (ISGs) predominantly expressed by monocytes. The DIRECT LS-TA method can use a simple ratio of two genes measured in PB as an RBB to represent the target gene expression in monocytes without the need for monocyte purification. Both scRNAseq and bulk RNA sequencing datasets were used to evaluate the correlation between ISG expression in monocytes and PB, with a particular focus on monocyte expression of IFI27. An iceberg plot of bulk transcriptome data was used to identify genes that were predominantly expressed by monocytes in PB. DIRECT LS-TA RBBs of the three genes (IFI27, IFI44L and SIGLEC1) were evaluated by group-wise comparison, receiver operating characteristic and meta-analysis. In addition, the conventional interferon (IFN) score was evaluated for comparison of diagnostic performance. In viral infection datasets, DIRECT LS-TA of IFI27 (IFI27/PSAP or IFI27/CTSS) was most intensely activated (p value by t test <1e-9) and had the best area under the curve (0.94) among the three potential monocyte ISGs analysed. DIRECT LS-TA SIGLEC1 was also another monocyte biomarker but showed a lower activation (p<9e-5). IFI27/PSAP showed better diagnostic performance than the conventional IFN score. On the other hand, IFI44L was not a predominant monocyte expression gene. DIRECT LS-TA of IFI27 (IFI27/PSAP or IFI27/CTSS) measured in PB was the best biomarker of viral infection and IFN activation among ISGs predominantly expressed by monocytes. It performed even better than the conventional IFN score which required quantification of eight genes. The results suggest that DIRECT LS-TA of IFI27 is a monocyte-informative biomarker which is easy to determine in PB without the need for cell sorting.

Humans↗

PubMed2026

Enhancing the fiber degradation efficiency in dairy cattle rumen through engineered bacterial communities.

BACKGROUND: The rumen functions as an anaerobic fermentation chamber, housing microorganisms with cellulolytic and proteolytic capabilities that facilitate feed utilization. Fiber-degrading bacteria possess the capability to enhance the productivity of cellulolytic feed. The application of omics technologies has greatly improved our understanding of the rumen microbiome. Determining microbial composition and functional patterns in the rumen does not equate to a comprehensive exploration of rumen microbial resources and their mechanisms of action. This study seeks to integrate high throughput 16S rRNA data with information on culturomics, cellulolytic activities, nutrition, and synthetic microbial communities (SynCom) engineering. The objective is to evaluate the relationship between rumen microbial activity and fiber utilization efficiency in cattle, ultimately aiming to develop a more powerful intervention strategy for the ruminant industry. RESULTS: The enrichment culture with various carbon sources led to significant alterations in the composition and structure of rumen microbiota, particularly enhancing those associated with carbohydrate metabolism. Employing the culturomics methodology, 896 strains from 78 species (including 8 novel species) were isolated, resulting in a 10.1% isolation rate relative to the rumen bacterial community. Among them, 35 strains demonstrated boosted cellulose-degrading capability on plates, while 25 exhibited the ability to degrade hemicellulose as well. SynComs of these candidates were prepared based on the ratio observed in rumen microbiota exhibiting high cellulolytic performance. SynCom 3 improved the neutral detergent fiber degradation (NDFD) by 20.39% averagely. Additionally, both in vitro and in situ assessments indicated that the optimization of dose/strain in SynCom 3 significantly improved the in vitro NDFD by 20.56% and increased the in situ NDFD by 7.81%, along with the acidic detergent fiber (ADF, + 11.47%). Genomic analysis revealed that the SynCom 3 functioned well in fiber degradation through the synergistic action of key carbohydrate-active enzymes. CONCLUSIONS: This study strengthens rumen microbiome research by integrating omics and SynCom engineering within a microbiota-bacteria-enzymes-genes framework, revealing the significance of enzymatic synergy in carbohydrate metabolism. The findings establish a framework for utilizing low-abundance microbes and engineering functional consortia, which are crucial for improving ruminant feed utilization and biomass conversion. Future research should investigate the transcriptomic profiles and the metabolic cross-feeding mechanisms of fiber-degrading strains in the rumen. Video Abstract.

Animals↗

PubMed2026

Influenza A Virus Coinfection Alters Streptococcus pneumoniae Gene Expression during Upper Respiratory Tract Colonization.

Streptococcus pneumoniae (Spn) asymptomatically colonizes the upper respiratory tract (URT), a niche from which it can transmit to another host or cause invasive disease in the same host. The in vivo transcriptional adaptations that Spn undergoes during nasopharyngeal colonization, particularly during influenza A virus (IAV) coinfection, are poorly understood. Here, we leveraged an established infant mouse model of colonization, shedding, and transmission to perform genome-wide transcriptomic profiling of Spn during mono- and during IAV co-infection. Compared with broth-grown controls, pneumococci isolated from the URT exhibited distinct transcriptional programs, with over 200 genes differentially expressed across time points. Genes involved in carbohydrate uptake and metabolism, glycan degradation, amino sugar and nucleotide sugar metabolism, and amino acid biosynthesis were consistently enriched during colonization, highlighting metabolic adaptation to the nasopharyngeal niche. In contrast, IAV coinfection induced a markedly distinct transcriptional signature, including upregulation of branched-chain amino acid biosynthesis, bacteriocin production, and phosphate acquisition systems. Notably, the pilus islet-1 locus was upregulated during Spn-IAV coinfection. Functional studies demonstrated that while the pilus was dispensable for colonization under mono- and coinfection conditions, it promoted high-shedding events and enhanced inflammatory responses during IAV coinfection. However, reduced inflammation and reduced high shedding events from pups inoculated with a pilus-deficient mutant did not alter transmission frequency in the infant mouse model. Collectively, our findings define the in vivo transcriptional landscape of Spn during URT colonization and reveal distinct bacterial adaptations during viral coinfection, providing insight into mechanisms that influence pneumococcal persistence, inflammation, and transmission.

Journal Article↗

PubMed2026

Influenza A virus co-infection alters Streptococcus pneumoniae gene expression during upper respiratory tract colonization.

Streptococcus pneumoniae (Spn) asymptomatically colonizes the upper respiratory tract (URT), a niche from which it can transmit to another host or cause invasive disease in the same host. The in vivo transcriptional adaptations that Spn undergoes during nasopharyngeal colonization, particularly during influenza A virus (IAV) co-infection, are poorly understood. Here, we leveraged an established infant mouse model of colonization, shedding, and transmission to perform genome-wide transcriptomic profiling of Spn during mono- and during IAV co-infection. Compared with broth-grown controls, pneumococci isolated from the URT exhibited distinct transcriptional programs, with over 200 genes differentially expressed across time points. Genes involved in carbohydrate uptake and metabolism, glycan degradation, amino sugar and nucleotide sugar metabolism, and amino acid biosynthesis were consistently enriched during colonization, highlighting metabolic adaptation to the nasopharyngeal niche. In contrast, IAV co-infection induced a markedly distinct transcriptional signature, including upregulation of branched-chain amino acid biosynthesis, bacteriocin production, and phosphate acquisition systems. Notably, the pilus islet-1 locus was upregulated during Spn-IAV co-infection. Functional studies demonstrated that while the pilus was dispensable for colonization under mono- and co-infection conditions, it promoted high-shedding events and enhanced inflammatory responses during IAV co-infection. However, reduced inflammation and reduced high-shedding events from pups inoculated with a pilus-deficient mutant did not alter transmission frequency in the infant mouse model. Collectively, our findings define the in vivo transcriptional landscape of Spn during URT colonization and reveal distinct bacterial adaptations during viral co-infection, providing insight into mechanisms that influence pneumococcal persistence, inflammation, and transmission.

RNA-seq↗

PubMed2024

A distinct, high-affinity, alkaline phosphatase facilitates occupation of P-depleted environments by marine picocyanobacteria.

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus, the two most abundant phototrophs on Earth, thrive in oligotrophic oceanic regions. While it is well known that specific lineages are exquisitely adapted to prevailing in situ light and temperature regimes, much less is known of the molecular machinery required to facilitate occupancy of these low-nutrient environments. Here, we describe a hitherto unknown alkaline phosphatase, Psip1, that has a substantially higher affinity for phosphomonoesters than other well-known phosphatases like PhoA, PhoX, or PhoD and is restricted to clade III Synechococcus and a subset of high light I-adapted Prochlorococcus strains, suggesting niche specificity. We demonstrate that Psip1 has undergone convergent evolution with PhoX, requiring both iron and calcium for activity and likely possessing identical key residues around the active site, despite generally very low sequence homology. Interrogation of metagenomes and transcriptomes from TARA oceans and an Atlantic Meridional transect shows that psip1 is abundant and highly expressed in picocyanobacterial populations from the Mediterranean Sea and north Atlantic gyre, regions well recognized to be phosphorus (P)-deplete. Together, this identifies psip1 as an important oligotrophy-specific gene for P recycling in these organisms. Furthermore, psip1 is not restricted to picocyanobacteria and is abundant and highly transcribed in some α-proteobacteria and eukaryotic algae, suggesting that such a high-affinity phosphatase is important across the microbial taxonomic world to occupy low-P environments.

Alkaline Phosphatase↗

PubMed2026

Azotobacter vinelandii AmrZ is a global regulator linking alginate production and c-di-GMP homeostasis.

Azotobacter vinelandii, a member of the Pseudomonadaceae, produces the exopolysaccharide alginate during vegetative growth; however, the circuitry linking alginate biosynthesis to lifestyle transitions remains poorly defined. Here, we show that the Ribbon-Helix-Helix transcription factor AmrZ coordinates alginate production, intracellular c-di-GMP levels and motility. Deletion of amrZ abolished alginate synthesis, whereas chromosomal complementation restored it. A PalgD-gusA fusion and RT-qPCR demonstrated that algD, the first gene in the alginate biosynthetic cluster, depends on AmrZ for expression. Motif analysis identified multiple AmrZ sites upstream of algD, and electrophoretic mobility-shift assays (EMSAs) confirmed specific binding to these regions. AmrZ also positively autoregulates: PamrZ-gusA activity decreased in ΔamrZ, and purified AmrZ bound the amrZ promoter in EMSA. Moreover, PamrZ activity required the sigma factor AlgU, consistent with the presence of an AlgU promoter; this positive, AlgU-dependent feedback may stabilize AmrZ under alginate-inducing conditions. To probe AmrZ control of c-di-GMP, we implemented a riboswitch-based biosensor in A. vinelandii. The ΔamrZ strain showed a markedly reduced signal, similar to a diguanylate cyclase (DGC) mutant, whereas a phosphodiesterase mutant displayed elevated output, validating the assay. RNA-seq and RT-qPCR identified two DGC genes, AVAEIV_RS11610 and AVAEIV_RS18795, as AmrZ-activated targets; EMSA verified direct binding at the RS11610 regulatory region. By contrast, transcription of the principal vegetative DGC AvGReg was not AmrZ-regulated. Lower c-di-GMP in ΔamrZ correlated with larger swimming halos. Collectively, these genetic, biochemical and transcriptomic data support a model in which AmrZ directly activates algD and elevates c-di-GMP via selected DGCs, thereby promoting alginate synthesis while reducing motility. RNA-seq data also indicate that AmrZ influences broader cellular programmes, including metabolism and iron homeostasis, positioning AmrZ as a central regulator that links c-di-GMP homeostasis to coordinated exopolysaccharide production in A. vinelandii. This work contributes to our understanding of the regulatory networks controlled by AmrZ outside the Pseudomonas genus and reveals important differences in its targets and regulatory mechanisms.

Azotobacter vinelandii↗

PubMed2026

Phenylacetic acid mediates Acinetobacter baumannii entry into a viable but non-culturable state.

Desiccation tolerance is central to the pathogenic success of the opportunistic pathogen Acinetobacter baumannii, allowing its survival on hospital surfaces in the absence of water and nutrients for months at a time, compromising surface decontamination and aiding cross-contamination between staff and patients. Despite the importance of desiccation tolerance, the regulation underpinning this behaviour remains largely elusive. In this work, transcriptomic analyses of desiccated cells revealed phenylacetic acid (PAA) catabolism as an essential mediator of desiccation tolerance. We subsequently demonstrate that deletion of the paa operon abolished the clonogenicity of desiccated cells. Strikingly, these A. baumannii cells remained viable by entering the viable but non-culturable (VBNC) state, a means to survive extreme stressors like antibiotic exposure. Furthermore, we uncover that PAA catabolism is necessary to mediate PAA-driven biofilm regulation. These findings highlight PAA catabolism as a modulator of biofilm formation and a key pathway for entry into the VBNC state in response to desiccation. This reveals PAA catabolism as a target for novel infection prevention strategies.

Phenylacetates↗

PubMed2025

Fecal microbiota transplantation promotes type 2 mucosal immune responses with colonic epithelium proliferation in patients with recurrent Clostridioides difficile.

BACKGROUNDFecal microbiota transplantation (FMT) is the most effective therapy for recurrent Clostridioides difficile infection (rCDI), yet its mechanism of action remains poorly understood.METHODSWe report the results of a clinical trial of patients undergoing FMT therapy for rCDI (n = 16), which analyzed colon biopsies, plasma, PBMCs, and stool at the time of FMT and 2-month follow-up. Plasma and colon biopsy samples were also collected from healthy controls for comparison with patients with rCDI. Microbiome composition, colonic gene expression, and immune changes were evaluated through high-throughput sequencing and immunoprofiling via flow cytometry.RESULTSNo patients experienced recurrence at follow-up. FMT significantly altered the intestinal microbiome but had no significant impact on the systemic immune system. In contrast, FMT promoted broad changes in colonic transcriptional profiles compared with both pre-FMT and healthy control biopsies, inhibiting genes associated with proinflammatory signaling and upregulating type 2 immunity and proliferative pathways (Myc and mTORC1). FMT increased expression of IL-33 and the type 2 immune EGFR family ligand amphiregulin, potentially explaining upregulation of Myc and mTORC1 pathways. Spatial transcriptomics demonstrated that these changes were localized to the colonic epithelium. Comparison of transcriptional profiles with available single-cell gene sets determined that post-FMT biopsies were enriched in signatures associated with proliferative cell types while repressing signatures of differentiated colonocytes.CONCLUSIONWe conclude that FMT promotes proliferation of the colonic epithelium in patients with rCDI, which may drive regeneration and protect against subsequent CDI.TRIAL REGISTRATIONClinicaltrials.gov NCT02797288.FUNDINGThis work was funded by grants from the NIH.

Adult↗

PubMed2024

Designer TALEs enable discovery of cell death-inducer genes.

Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.

Cell Death↗