Biology subjects

Eberl, L.

Publications and source records attributed to Eberl, L..

13 recordsLinked to original sources

bioRxiv2026

Tn-Seq-based identification of genes that play a role in antimicrobial tolerance of Pseudomonas aeruginosa aggregates

Microbial biofilms, whether surface-associated or in the form of aggregated microbes, cause a variety of persistent infections due to their inherent tolerance to antibiotic treatment. Current knowledge of the mechanistic basis of biofilm-associated antibiotic tolerance is fragmented, and the relative importance of previously identified factors remains unknown. We developed a Tn-Seq-based workflow that enables simultaneous assessment of the relative antibiotic tolerance of thousands of individual clonal bacterial aggregates, each established by a single transposon mutant. Using this genome-wide Tn-Seq analysis approach, we identified several factors and pathways that play a role in antimicrobial tolerance of Pseudomonas aeruginosa aggregates. The majority of these factors are important for aggregate-associated tolerance to specific antibiotics, whereas some factors play a role in aggregate-associated tolerance regardless of the class of antibiotic. This methodology provides a framework that can be adapted to investigate aggregate-associated tolerance for other bacterial species and in response to different antimicrobial agents and stressors.

microbiology↗

bioRxiv2025

Structural Elucidation and Revised Biosynthetic Pathway of the Membrane Vesicle-Associated Antifungal Compound AFC

Bacterial membrane vesicles (MVs) serve as delivery vehicles for hydrophobic and membrane-associated secondary metabolites, enhancing their solubility, stability, and bioactivity. Here, we show that the antifungal compound AFC-BC11 (AFC), produced by Burkholderia cenocepacia K56-2, is selectively packaged into and released via MVs. Using HR-MS/MS, NMR, and stable isotope feeding experiments, we determined the chemical structure of AFC and analyzed its biosynthesis. Our results confirm that the structure largely matches the recent report by Zhong et al., with a key difference: the double bond in the fatty acid moiety is positioned between C11 and C12. We provide compelling evidence that this constitution reflects the direct incorporation of cis-vaccenic acid, the most abundant fatty acid in B. cenocepacia, rather than a tailoring modification. Comparative analysis of afcU, afcF, and afcS mutants suggests a biosynthetic pathway involving {omega}-modification of cis-vaccenic acid, revising previous proposals of citric acid conjugation to myristic acid and opening avenues for acyl chain engineering. Together, these findings establish AFC as an MV-associated antifungal metabolite, provide a refined structural and biosynthetic model, and highlight the role of MVs in the dispersal of hydrophobic bioactive compounds. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/685075v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f4d736org.highwire.dtl.DTLVardef@15d2683org.highwire.dtl.DTLVardef@8b891corg.highwire.dtl.DTLVardef@d6b693_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Signal Antagonists Suppress Pseudomonas syringae Pathogenicity

The bacterial plant pathogen Pseudomonas syringae causes significant damage to economically important crops worldwide. These bacteria coordinate their behavior and virulence through specific signaling compounds, such as the diazeniumdiolate leudiazen. Conventional antibacterial treatments enable the development of resistant strains. A more attractive treatment strategy would involve antagonists that suppress the expression of virulence factors without killing the pathogen, potentially reducing the risk of resistance development. Herein, we present the design and synthesis of analogs of leudiazen, which positively regulates the production of mangotoxin in P. syringae pv. syringae (Pss). Several compounds display inhibitory activity towards mangotoxin production, and a lead compound abolishes necrosis in infected tomato leaves, without significantly affecting bacterial growth. Thus, this study represents a promising advance towards developing effective and sustainable methods for bacterial disease control.

microbiology↗

bioRxiv2024

Functional Biosynthetic Stereodivergence in a Gene Cluster via a Dihydrosydnone N-oxide

Chirality features a critical role in the biochemistry of life and often only one enantiomeric series is observed (homochirality). Only few natural products have been obtained as racemates, e.g. the quorum-sensing signal valdiazen produced by Burkholderia cenocepacia H111. In this study, we investigated its biosynthetic gene cluster and discovered that both the enantiomerically pure (R)-fragin and the racemic valdiazen are obtained from the same pathway. This stereodivergence is based on the unusual heterocycle dihydrosydnone N-oxide intermediate, as evident from gene knockout, stable isotope feeding experiments, and mass spectrometry experiments. Both non-enzymatic racemisation via keto-enol tautomerisation and enzyme-mediated dynamic kinetic resolution were found to be crucial to this stereodivergent pathway. This novel mechanism underpins the production of configurationally and biologically distinct metabolites from a single gene cluster. Our findings highlight the intricate design of an intertwined biosynthesis pathway, providing a deeper understanding of microbial secondary metabolism related to microbial communication. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/591611v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@11b63corg.highwire.dtl.DTLVardef@d339e6org.highwire.dtl.DTLVardef@17e34b1org.highwire.dtl.DTLVardef@b58692_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

RNAseq reveals that Pseudomonas aeruginosa mounts medium-dependent competitive responses when sensing diffusible cues from Burkholderia cenocepacia

Most habitats host diverse bacterial communities, offering opportunities for inter-species interactions. While competition might often dominate such interactions, little is known about whether bacteria can sense competitors and mount adequate responses. The competition-sensing hypothesis proposes that bacteria can use cues such as nutrient stress and cell damage to prepare for battle. Here, we tested this hypothesis by measuring transcriptome changes in Pseudomonas aeruginosa exposed to the supernatant of its competitor Burkholderia cenocepacia. We found that P. aeruginosa exhibited significant and growth-medium-dependent transcriptome changes in response to competition. In iron-rich medium, P. aeruginosa up-regulated genes encoding the type-VI secretion system and the siderophore pyoverdine, whereas genes encoding phenazine toxins and hydrogen cyanide were upregulated under iron-limited conditions. Moreover, general stress response and quorum-sensing regulators were upregulated upon supernatant exposure. Altogether, our results reveal nuanced competitive responses of P. aeruginosa when confronted with B. cenocepacia supernatant, integrating both environmental and social cues.

microbiology↗

bioRxiv2021

The structural role of bacterial eDNA in the formation of biofilm streamers

Across diverse habitats, bacteria are mainly found as biofilms, surface-attached communities embedded in a self-secreted matrix of extracellular polymeric substances (EPS), which enhances bacterial resistance to antimicrobial treatment and mechanical stresses. In the presence of flow and geometric constraints such as corners or constrictions, biofilms take the form of long, suspended threads known as streamers, which bear important consequences in industrial and clinical settings by causing clogging and fouling. The formation of streamers is thought to be driven by the viscoelastic nature of the biofilm matrix. Yet, little is known about the structural composition of streamers and how it affects their mechanical properties. Here, using a microfluidic platform that allows growing and precisely examining biofilm streamers, we show that extracellular DNA (eDNA) constitutes the backbone and is essential for the mechanical stability of Pseudomonas aeruginosa s streamers. This finding is supported by the observations that DNA-degrading enzymes prevent the formation of streamers and clear already formed ones, and that the antibiotic ciprofloxacin promotes their formation by increasing the release of eDNA. Furthermore, using mutants for production of the exopolysaccharide Pel, an important component of P. aeruginosa s EPS, we reveal a new, although indirect role of Pel, in tuning the mechanical properties of the streamers. Taken together, these results highlight the importance of eDNA and of its interplay with Pel in determining the mechanical properties of P. aeruginosa streamers, and suggest that targeting the composition of streamers can be an effective approach to control the formation of these biofilm structures.

biophysics↗

bioRxiv2020

NirA is an alternative nitrite reductase from Pseudomonas aeruginosa with potential as an anti-virulence target

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, anti-virulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we have identified a Tn5 mutant with an insertion in PA4130 showing reduced levels in a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented similar results as those from the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster, Caenorhabditis elegans as well as decreased toxicity in human cell lines. This mutant also presented an 80% increased survival in murine acute and agar-bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulphite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this protein is a nitrite reductase operating in a siroheme and 4Fe-4S dependant manner. The preference for nitrite and the production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named as NirA.

microbiology↗

bioRxiv2020

Characterization of the antimicrobial activity of the cell-penetrating peptide TAT-RasGAP317-326

Antibiotic resistance is an increasing threat for public health, underscoring the need for new antibacterial agents. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics. TAT-RasGAP317-326 is a recently described AMP effective against a broad range of bacteria, but little is known about the conditions that may influence its activity. Using RNA-sequencing and screening of mutant libraries, we show that Escherichia coli and Pseudomonas aeruginosa respond to TAT-RasGAP317-326 by regulating metabolic and stress response pathways, possibly implicating two-component systems. Our results also indicate that bacterial surface properties, in particular integrity of the lipopolysaccharide layer, influence peptide binding and entry. Finally, we found differences between bacterial species with respect to their rate of resistance emergence against this peptide. Our findings provide the basis for future investigation on the mode of action of this peptide and its potential clinical use as an antibacterial agent.

microbiology↗

bioRxiv2020

A Volatile Signal Controls Virulence in the Plant Pathogen Pseudomonas syringae pv. syringae and a Strategy for Infection Control in Organic Farming

Pseudomonas syringae is an important pathogen of many agriculturally valuable crops. Among the various pathovars described P. syringae pv. syringae (Pss) has a particularly wide host range, infecting primarily woody and herbaceous host plants. The ability of Pss to cause bacterial apical necrosis of mango trees is dependent on the production of the antimetabolite toxin mangotoxin. The production of this toxin was shown to be regulated by a self-produced signaling molecule. In this study, we determined the structure of the Pss signal molecule belonging to the recently described family of diazeniumdiolate communication molecules. Employing a targeted mass spectrometry-based approach, we provide experimental evidence that the major signal produced by Pss is the volatile compound leudiazen, which controls mangotoxin production and virulence in a detached tomato leaflet infection model. Experimental results demonstrate that KMnO4 solution inactivates leudiazen and that treatment of infected leaves with KMnO4 abolishes necrosis. This strategy represents the first example of chemically degrading a signaling molecule to interfere with bacterial communication. The application of KMnO4 solution, which is regulatorily approved in organic farming, may constitute an environmentally friendly strategy to control Pss infections.

microbiology↗

bioRxiv2020

An investigation of Burkholderia cepacia complex methylomes via SMRT sequencing and mutant analysis

The Burkholderia cepacia complex (Bcc) is a group of 22 closely related opportunistic pathogens which produce a wide range of bioactive secondary metabolites with great biotechnological potential, for example in biocontrol and bioremediation. This study aimed to investigate methylation in the Bcc by SMRT sequencing, and to determine the impact of restriction-methylation (RM) systems on genome protection and stability and on phenotypic traits. We constructed and analysed a mutant lacking all RM components in the clinical isolate B. cenocepacia H111. We show that a previously identified essential gene of strain H111, gp51, encoding a methylase within a prophage region, is required for maintaining the bacteriophage in a lysogenic state. We speculate that epigenetic modification of a phage promoter provides a mechanism for a constant, low level of phage production within the bacterial population. We also found that, in addition to bacteriophage induction, methylation was important in biofilm formation, cell shape, motility, siderophore production and membrane vesicle production. Moreover, we found that DNA methylation had a massive effect on the maintenance of the smallest replicon present in this bacterium, which is essential for its virulence. In silico investigation revealed the presence of two core RM systems, present throughout the Bcc and beyond, suggesting that the acquisition of these RM systems occurred prior to the phylogenetic separation of the Bcc. We used SMRT sequencing of single mutants to experimentally assign the B. cenocepacia H111 methylases to their cognate motifs. Analysis of the distribution of methylation patterns suggested roles for m6A methylation in replication, since motifs recognised by the core Type III RM system were more abundant at the replication origins of the three H111 replicons, and in regions encoding functions related to cell motility and iron uptake. Author summaryWhile nucleotide sequence determines an organisms proteins, methylation of the nucleotides themselves can confer additional properties. In bacteria, methyltransferases methylate specific motifs to allow discrimination of self from non-self DNA, e.g. from bacteriophages. Restriction enzymes detect non-self methylation patterns and cut foreign DNA. Furthermore, methylation of promoter regions can influence gene expression and hence affect phenotype. In this study, we determined the methylated motifs of four strains from the Burkholderia cepacia complex of opportunistic pathogens. Three novel motifs were found, and two that were previously identified in a related species. We deleted the genes encoding the restriction and modification components in a representative strain from among the four sequenced. In this study, methylation is shown to affect various phenotypes, among which maintenance of the lysogenic state of a phage and segregational stability of the smallest megareplicon are most remarkable.

microbiology↗

bioRxiv2020

DNA methylation epigenetically regulates gene expression in Burkholderia cenocepacia

Respiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation has shown to be an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2, and their role in regulating gene expression. In silico predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant {Delta}BCAL3494 showed changes in biofilm structure and cell aggregation, {Delta}BCAM0992 was less motile. B. cenocepacia wild type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-Molecule Real-Time sequencing was used to characterize the methylome of B. cenocepacia, including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified and qPCR experiments showed an upregulation of several genes, including biofilm and motility related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of B. cenocepacia genes involved in biofilm formation and motility. ImportanceCF patients diagnosed with B. cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multi-replicon genome and a lot remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of B. cenocepacia genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to lead to new insights in how to tackle B. cenocepacia infections in CF patients.

microbiology↗

bioRxiv2019

The effect of flow on swimming bacteria controls the initial colonization of curved surfaces

The colonization of solid surfaces by bacteria is a widespread phenomenon with major consequences on environmental processes, biotechnology and human health. While much is known about the molecular mechanisms of surface colonization, the influence of the physical environment remains poorly understood. Here we show that the magnitude and location of colonization of non-planar surfaces by motile bacteria is largely controlled by the local flow conditions. Using microfluidic experiments with Pseudomonas aeruginosa and Escherichia coli, we demonstrate that the velocity gradients created by a curved surface drive preferential attachment to specific regions of the collecting surface, namely the leeward side of cylinders and immediately downstream of apexes on corrugated surfaces, locations that are in stark contrast to where non-motile cells attach. The preferential attachment location depends on the local hydrodynamic conditions and, as revealed by a mathematical model benchmarked on the observations, on cell morphology and swimming traits, while it is independent of the physicochemical properties of the surface. The interplay between imposed flow and bacterial motility further affects the overall attachment rate, increasing it by up to two orders of magnitude compared to the non-motile case at moderate flow velocities of up to twenty times the bacterial swimming speed. These results highlight the importance of fluid flow on the magnitude and location of bacterial colonization of surfaces and provide a mechanistic model to predict colonization in flow for a wide range of applications, from infection biology to bioremediation and biofouling.

biophysics↗

bioRxiv2019

Genetic architecture constrains exploitation of siderophore cooperation in Burkholderia cenocepacia

Explaining how cooperation can persist in the presence of cheaters, exploiting the cooperative acts, is a challenge for evolutionary biology. While microbial systems have proved extremely useful to test evolutionary theory and identify mechanisms maintaining cooperation, our knowledge is often limited to insights gained from a few model organisms. Here, we introduce siderophore secretion by the bacterium Burkholderia cenocepacia as a novel study system. Using a combination of phenotypic and competition assays we found that ornibactin, the main siderophore used for iron scavenging in this species, is secreted into the media, can be shared as public good between cells, but cannot be exploited by ornibactin-defective mutants. Molecular analysis revealed that cheating is compromised because the ornibactin receptor gene and genes involved in ornibactin synthesis are co-expressed from the same operon, such that disruptive mutations in the upstream synthesis genes compromise receptor availability. To prove that it is the genetic architecture of the siderophore locus that prevents cheating, we broke the linked traits by expressing the ornibactin receptor from a plasmid, a measure that turned the ornibactin mutant into a functional cheater. A literature survey across Burkholderia species suggests that the genetic linkage independently broke over evolutionary time scales in several lineages, indicating that cheating and countermeasures might be under selection. Altogether, our results highlight that expanding our repertoire of microbial study systems leads to new discoveries and reinforce the view that social interactions shape evolutionary dynamics in microbial communities.

evolutionary biology↗