Biology subjects

Krol, E.

Publications and source records attributed to Krol, E..

7 recordsLinked to original sources

bioRxiv2026

Spatial Transcriptomics Reveals Region-Specific Host Responses in Amoebic Gill Disease of Atlantic Salmon

Amoebic gill disease (AGD), caused by the parasite Neoparamoeba perurans, is a major welfare and economic burden for the Atlantic salmon aquaculture industry. However, the spatial organisation of immune functions within the gill, the site of infection, remains poorly resolved. Here, we present the first spatial transcriptomic atlas of the Atlantic salmon gill affected by AGD, generated using 10x Genomics Visium on four AGD-positive fish, confirmed by gill scoring and quantitative PCR for parasite load. As no uninfected gills were included, this study maps the anatomical and immunological organisation of AGD-affected gills rather than defining AGD-specific pathology. Unsupervised clustering of spatial gene expression resolved distinct transcriptional domains that grouped into four principal anatomical compartments of the gill, including (i) epithelial and lamellar regions defined by keratin and osmoregulatory gene expression, (ii) gill arch regions enriched for connective tissue and smooth muscle markers, (iii) an erythroid-associated vascular cluster, and (iv) a skeletal muscle cluster with signatures of glycolysis and oxidative phosphorylation. Across these domains, we identified a spatially organised and diffused immune landscape, with pattern recognition receptors, antimicrobial effectors and chemokines concentrated at the filament surface, and immunoglobulin light chain transcripts consistent with B lymphocyte activity enriched toward the gill arch. Integration of the spatial data with a published single-nucleus reference atlas using reference-based deconvolution resolved the cellular identity underlying these domains and revealed frequent co-localisation of immune and structural cell types, suggesting regulated cell to cell communication within the gill-associated lymphoid tissue. Overall, our results show that the Atlantic salmon gill is not a transcriptionally uniform surface but a regionally specialised organ, with immune functions distributed across, and specialised within, distinct anatomical compartments

immunology↗

bioRxiv2025

Rhizobial motility preference in root colonization of Medicago truncatula

O_LITunnel-like infection thread (IT) structures support root colonization by symbiotic nitrogen-fixing rhizobia bacteria in most legume species. These tip-grown structures are key to direct rhizobia from root hairs to developing nodules, where they are hosted to fix nitrogen. Rhizobia likely progress inside ITs by combining growth and motility, by modes not yet defined. Here, we tackled this question by combining mathematical modeling, live cell imaging, and bacterial mutant phenotyping in Medicago truncatula. C_LIO_LIModeling the motion of fluorescently-labeled Sinorhizobium meliloti inside early root hair IT compartments estimated slow movement (2 to 6 {micro}m/h), compatible with passive rather than active motility. Consistent with this model, flagella-less fliF and fliF-fliRdel S. meliloti mutants were impaired in active swimming motility in vitro, yet could colonize host roots and nodules in planta. In contrast, mutation in the rhizobactin 1021 siderophore rhbE biosynthesis gene affected both surface motility in vitro, and host root and nodule colonisation. This mutation also promoted the formation of branched ITs in root hairs, which ultimately resulted in impaired nodule development and infection. C_LIO_LIOur findings support the model estimation and suggest that S. meliloti prioritises flagella-independent surface translocation, partially by secreting rhizobactin 1021 surfactants to reach developing nodules in M. truncatula. C_LI

plant biology↗

bioRxiv2025

A systems-level insight into PHB-driven metabolic adaptation orchestrated by the PHB-binding transcriptional regulator AniA (PhaR)

Polyhydroxybutyrate (PHB) is a carbon and energy storage polymer, whose accumulation under nutrient imbalances with excess carbon is a widespread phenomenon in bacteria. PhaR is a conserved transcriptional regulator found to be associated to PHB granules in several species. Although its role in modulating PHB storage and metabolism has been extensively studied across the bacterial phylogeny, a comprehensive analysis of the PhaR regulon within the context of its dual role as a metabolic sensor and regulator remains missing. To bridge this gap, we integrated co-expression network analysis with proteome profiling across multiple mutant backgrounds (lack of PhaR (AniA) and/or PHB synthesis) in the free-living state of the PHB-accumulating alphaproteobacterial root nodule symbiont Sinorhizobium meliloti. This analysis was enriched by identifying direct regulatory targets of PhaR through a regulon-centric computational multi-step search for DNA binding site motifs combined with PhaR-DNA binding and promoter-reporter assays. We confirmed that the model of accumulated PHB sequestering PhaR and thereby relieving phasin and PHB depolymerase gene repression to control cellular PHB levels also applies to S. meliloti, and showed that PhaR-mediated regulation also occurs the symbiotic state. A comprehensive picture of the impact of PHB-mediated PhaR titration on cellular functions revealed exopolysaccharide production as well as central carbon metabolism (pdh and bkd), gluconeogenesis (ppdK and pyc), entry into the TCA cycle (gltA), and the initial steps of the Entner-Doudoroff pathway (zwf, pgl and edd) as major regulatory targets, and beyond carbon metabolism several target genes of yet unknown function. Our findings highlight a pivotal role for PhaR in orchestrating carbon metabolism.

microbiology↗

bioRxiv2024

Xenometabolome of Early-Life Stage Salmonids Exposed to 6PPD-Quinone

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a ubiquitous transformation product (TP) derived from the rubber tire antioxidant N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and is acutely toxic to certain species of Salmonidae. Not all salmonids are sensitive to acute lethality caused by 6PPD-Q, with 6PPD-Q potency varying by several orders of magnitude among teleosts. The main driver(s) of species sensitivity differences is (are) a pressing question, with one area of interest examining whether differences in teleosts ability to biotransform and detoxify 6PPD-Q could be a key factor. This study utilized liquid-chromatography high-resolution mass spectrometry (LC-HRMS) to assess biotransformation and metabolome-wide effects of 6PPD-Q on early-life stage salmonids, including two sensitive species, rainbow trout (Oncorhynchus mykiss) and lake trout (Salvelinus namaycush), and one tolerant species, brown trout (Salmo trutta). Three phase I TPs and seven phase II TPs were detected, with differences in peak areas revealing that brown trout had the greatest ability to detoxify 6PPD-Q. Monohydroxylated TPs were verified using co-developed analytical standards that will be of use for future biomonitoring and exposure assessment. Several endogenous metabolites were found to be dysregulated in rainbow and lake trout, indicative of mitochondrial dysfunction, altered metabolism, and disrupted membrane permeability. Results of this study indicate a difference in the biotransformation capability of 6PPD-Q among Salmonidae fish species and subsequent unique metabolome responses. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/598661v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1931a42org.highwire.dtl.DTLVardef@a0f446org.highwire.dtl.DTLVardef@10540d0org.highwire.dtl.DTLVardef@1e21b49_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisInter-species salmonid differences in the biotransformation capability of 6PPD-Q leads to sensitivity variability and subsequent unique metabolome responses.

pharmacology and toxicology↗

bioRxiv2022

Structural basis of bifunctionality of Sinorhizobium meliloti Clr, a cAMP and cGMP receptor protein

In bacteria, Crp-Fnr superfamily transcription factors are the most ubiquitous receptor proteins of 3,5-cyclic adenosine monophosphate (cAMP) and 3,5-cyclic guanosine monophosphate (cGMP). The prototypic Escherichia coli CAP protein represents the main CRP subclass and is known to bind cAMP and cGMP, but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by CRP subclass G protein Clr of Sinorhizobium meliloti. We present crystal structures of apo-Clr, and Clr*cAMP and Clr*cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr*cNMP*CBS-DNA complexes to almost identical active conformations. Unlike the situation known for the E. coli CAP*cNMP complex, in the Clr*cNMP complex, the nucleobases of cGMP and cAMP are in the syn- and anti-conformation, respectively, allowing a shift to the active conformations in both cases. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in presence of CBS core motif DNA (KDcNMP 16 M). However, different affinities were determined in absence of this DNA (KDcGMP 24 M; KDcGMP 6 M). Sequencing of Clr co-immunoprecipitated DNA as well as Electrophoretic Mobility Shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases, which are in agreement with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr*cNMP*CBS-DNA crystal structures. IMPORTANCECyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) are both long known as important nucleotide second messenger in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins. Escherichia coli CAP, the prototypic transcription regulator of the main CRP subclass, binds both cyclic mononucleotides, but only the CAP*cAMP complex promotes transcription activation. In contrast, CRP subclass G proteins studied so far, are activated by cGMP, or both by cAMP and cGMP. Here, we report a structural analysis of the bifunctional cAMP- and cGMP-activatable Clr from Sinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.

microbiology↗

bioRxiv2021

Unipolar peptidoglycan synthesis in the Rhizobiales requires an essential class A penicillin-binding protein

Members of the Rhizobiales are polarly-growing bacteria that lack homologs of the canonical rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant-pathogen Agrobacterium tumefaciens. The development of fluorescent O_SCPLOWDC_SCPLOW-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis convincingly demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an alternative mode of cell wall synthesis that likely requires O_SCPLOWLDC_SCPLOW-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation.

microbiology↗

bioRxiv2020

Tol-Pal system and Rgs proteins interact to promote unipolar growth and cell division in Sinorhizobium meliloti

Sinorhizobium meliloti is an -proteobacterium belonging to the Rhizobiales. Bacteria from this order elongate their cell wall at the new cell pole, generated by cell division. Screening for protein interaction partners of the previously characterized polar growth factors RgsP and RgsM, we identified the inner membrane components of the Tol-Pal system (TolQ and TolR) and novel Rgs (rhizobial growth and septation) proteins with unknown functions. TolQ, Pal and all Rgs proteins, except for RgsE, were indispensable for S. meliloti cell growth. Six of the Rgs proteins, TolQ and Pal localized to the growing cell pole in the cell elongation phase and to the septum in pre-divisional cells, and three Rgs proteins localized to growing cell pole only. The FtsN-like protein RgsS contains a conserved SPOR domain and is indispensable at the early stages of cell division. The components of the Tol-Pal system were required at the late stages of cell division. RgsE, a homolog of the Agrobacterium tumefaciens growth pole ring protein GPR, has an important role in maintaining the normal growth rate and rod cell shape. RgsD is a novel periplasmic protein with the ability to bind peptidoglycan. Analysis of the phylogenetic distribution of novel Rgs proteins showed that they are conserved in Rhizobiales and mostly absent from other -proteobacterial orders, suggesting a conserved role of these proteins in polar growth. IMPORTANCEBacterial cell proliferation involves cell growth and septum formation followed by cell division. For cell growth, bacteria have evolved different complex mechanisms. The most prevalent growth mode of rod shaped bacteria is cell elongation by incorporating new peptidoglycan in a dispersed manner along the sidewall. A small share of rod-shaped bacteria, including the -proteobacterial Rhizobiales, grow unipolarly. Here, we identified and initially characterized a set of Rgs (rhizobial growth and septation) proteins, which are involved in cell division and unipolar growth of Sinorhizobium meliloti and highly conserved in Rhizobiales. Our data expand the knowledge of components of the polarly localized machinery driving cell wall growth and suggest a complex of Rgs proteins with components of the divisome, differing in composition between the polar cell elongation zone and the septum.

microbiology↗