Biology subjects

Groleau, M.-C.

Publications and source records attributed to Groleau, M.-C..

At least 19 recordsLinked to original sources

bioRxiv2026

From Exception to Rule: Widespread Production of Methylated Rhamnolipids in the Burkholderia cepacia Complex

Although Burkholderia cenocepacia exhibits biosurfactant-dependent phenotypes and possesses an rhl operon, the chemical identity of its surface-active product has long remained unresolved. The rhl operon of B. cenocepacia harbors an additional gene, rhlM, predicted to encode a methyltransferase, and recently linked to methylated rhamnolipid synthesis in a few Burkholderiaceae. Here, we hypothesized that this biosurfactant remained cryptic due to low expression under standard laboratory conditions. Using a chromosomal Prhl-luxCDABE fusion reporter, we screened diverse nutritional conditions and identified a defined medium containing galactose and urea that strongly induced rhl transcription. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of B. cenocepacia K56-2 extracts identified the biosurfactants as methylated dirhamnolipid congeners, predominantly L-rhamnosyl-L-rhamnosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoate methyl ester (RR-C14-C14Me). Extending our analysis across the Burkholderia cepacia complex (Bcc) revealed that methylated rhamnolipid production is widespread and restricted to rhl-positive species. Comparative genomic and protein structural analyses further demonstrated high conservation of both the rhlM-containing operon architecture and the RhlM protein across the Bcc. Together, our findings identify the long-standing cryptic biosurfactant of B. cenocepacia as methylated rhamnolipids and establish rhamnolipid methylation as a conserved hallmark across the Bcc, shifting the paradigm of rhamnolipid structural diversity from an isolated exception to a widespread rule.

microbiology↗

bioRxiv2026

Identification of Beneficial Bacterial Strains for Tomato Growth Promotion and Biocontrol of Bacterial Canker Caused by Clavibacter michiganensis

Bacterial canker of tomato, caused by Clavibacter michiganensis (Cm), remains difficult to control due to lack of effective management options. In this study, a collection of over 500 bacterial isolates was screened in vitro for antagonistic activity against Cm and plant growth-promoting (PGP) traits. Based on these results, 32 candidates were evaluated in planta, leading to the identification of three highly effective strains: Pantoea agglomerans SO16PY and two Pseudomonas marginalis sensu lato strains, IRDA16 and SO16PC, which consistently enhanced tomato vegetative growth. Notably, P. agglomerans SO16PY delayed disease onset in Cm-inoculated plants by up to 7 days and significantly reduced wilting severity, lowering the disease severity score from 85% to 45%. Strains IRDA16 and SO16PC also restricted disease development, reducing severity scores to 67.5% and 57.5%, respectively. Whole-genome sequencing and comparative genomics revealed that strains IRDA16 and SO16PC form a distinct, specialized rhizosphere lineage within the Pseudomonas marginalis group, exhibiting average nucleotide identity (ANI {approx} 96%) and digital DNA-DNA hybridization (dDDH {approx} 69.5%) values near species delineation thresholds. Genome mining identified diverse biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), the lipopeptide viscosin, and terpenes, which likely drive the biostimulant and antagonistic traits of this novel Pseudomonas lineage. Together, these findings characterize promising bacterial candidates with dual biostimulant and biocontrol capacities while uncovering a genomically distinct Pseudomonas lineage optimized for beneficial plant-microbe interactions in sustainable agriculture. IMPORTANCEClavibacter michiganensis (Cm) is a major bacterial pathogen of tomato and poses a significant economic threat to global production. It is classified as an A2 quarantine pathogen by the European and Mediterranean Plant Protection Organization (EPPO). Current management strategies rely largely on chemical control, including copper-based compounds (e.g., Bordeaux mixture, copper oxychloride), mancozeb, and antibiotics like streptomycin. However, these approaches raise increasing concerns related to environmental contamination, phytotoxicity, and the development of resistant pathogen populations. As a sustainable alternative, plant growth-promoting bacteria (PGPR) have emerged as promising biocontrol agents. In this study, we identified bacterial strains exhibiting antagonistic activity against Cm both in vitro and in planta. Notably, these strains also enhanced tomato growth parameters, demonstrating their dual functionality. Given the environmental drawbacks associated with chemical inputs, the use of such beneficial microorganisms represents a promising strategy for advancing sustainable and ecofriendly tomato production systems.

microbiology↗

bioRxiv2025

Pantoea ananatis-triggered systemic resistance requires root sensing through the LORE receptor kinase in Arabidopsis

Systemic immunity induced by beneficial bacteria helps plants to cope with subsequent pathogen attacks. However, how these bacteria are sensed by plants and how this sensing results in a systemic response is not well understood. Recently, the Brassicaceae LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) receptor kinase was identified as the direct sensor of bacterial medium-chain-length 3-hydroxy fatty acids (mc-3-OH-FAs) and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), which activate systemic Brassicaceae immunity. Here, we show that the bacterium Pantoea ananatis BRT175, which produces HAAs, can colonize Arabidopsis thaliana roots independently of the production of these compounds. P. ananatis triggers induced systemic resistance (ISR) to the necrotrophic pathogen Botrytis cinerea, but not to the hemibiotrophic pathogen Pseudomonas syringae pv. tomato. Importantly, this ISR can be mimicked by HAAs or 3-hydroxy-decanoic acid (3-OH-C10). Both P. ananatis-triggered and 3-OH-C10-triggered ISR against B. cinerea are mediated by LORE sensing, involve salicylic acid, jasmonic acid and ethylene signaling pathways, and activate the expression of similar defense genes in infected leaves. Thus, 3-OH-C10 and HAAs are perceived by a pattern recognition receptor (PRR) in the root and activate a local immune response in roots as well as systemic immunity in leaves. Our study demonstrates that these lipidic microbe-associated molecular patterns (MAMPs), produced by beneficial microorganisms, are necessary and sufficient to trigger rhizobacteria-driven ISR. It further highlights the role of these MAMPs in differentially activating systemic resistance against biotrophic and necrotrophic pathogens.

plant biology↗

bioRxiv2025

PqsE is conserved and functionally relevant in Pseudomonas aeruginosa

The bacterium Pseudomonas aeruginosa is an opportunistic pathogen responsible for several acute and chronic infections. It produces a diverse array of virulence and survival determinants, many of which are tightly regulated by three interlinked quorum sensing (QS) systems named las, rhl and pqs. RhlR, the transcriptional regulator of the rhl system, activates multiple virulence genes upon binding to its cognate autoinducer signal. Meanwhile, the pqs system relies on 4-hydroxy-2-alkylquinolines (HAQs) as signaling molecules to induce the transcriptional regulator MvfR (PqsR). MvfR then activates the transcription of the pqsABCDE operon, encoding enzymes for HAQ biosynthesis. The final gene in this operon encodes PqsE, a multifunctional protein unique to P. aeruginosa. Beyond its thioesterase activity in HAQ biosynthesis, PqsE stabilizes RhlR, facilitating its regulation of target genes, some of which are implicated in virulence. Because of its role in pathogenicity, PqsE is regarded as a promising therapeutic target for combating P. aeruginosa infections. While the role of PqsE towards the RhlR regulon is increasingly understood in prototypical P. aeruginosa strains such as PA14 and PAO1, its broader relevance as an anti-virulence target remains underexplored. Here, we confirm that PqsE is functionally relevant across a panel of twelve genetically diverse P. aeruginosa strains using metabolite quantification and phenotypic assays. Significant strain-to-strain variations further highlight the importance of studying QS regulation among diverse isolates. Moreover, this study underscores PqsE as a key QS regulator with a conserved role in coordinating virulence determinant production and social behaviors across diverse P. aeruginosa populations. IMPORTANCEPseudomonas aeruginosa is a versatile opportunistic pathogen, naturally tolerant and readily acquiring resistance to multiple antibiotics. Consequently, the World Health Organization identified this bacterium as a high priority pathogen for researching and developing new antimicrobial strategies. P. aeruginosa utilizes quorum sensing, a cell-to-cell communication system, to regulate the expression of several of its virulence factors. Here, we confirm that the PqsE protein is conserved and that its function in quorum sensing, especially towards the RhlR regulator, is maintained across a panel of twelve genetically diverse P. aeruginosa strains. Since PqsE is conserved and unique to this bacterium, it could serve as an ideal target for anti-virulence therapies, offering new alternatives to combat antimicrobial resistance.

microbiology↗

bioRxiv2024

Quorum sensing and DNA methylation play active roles in clinical Burkholderia phase variation

Phenotypic diversity in bacteria often results from adaptation to changing environmental conditions and is exemplified by variable colony morphotypes. Discrete genomic changes and modulation in gene expression occur in Burkholderia pseudomallei undergoing adaptation. Alternatively, adapted colony morphotype variants of species belonging to the Burkholderia cepacia complex (Bcc) lose a complete replicon (the pC3 virulence megaplasmid), which affects their production of virulence factors. We report that variants arising in Burkholderia ambifaria clinical isolates - with affected phenotypes - have retained their pC3, suggesting that another phase variation mechanism can take place in this Bcc species. Proteomic and phenotypic characterisation showed that morphotype variants of B. ambifaria strains CEP0996 (pC3-null) and HSJ1 (pC3-positive) share similarities in phenotypes controlled by the Cep quorum sensing system. Thus, we determined the role of quorum sensing in B. ambifaria HSJ1 phase variation and confirmed that the main quorum sensing system Cep is important for the emergence of variants. As DNA methylation is one of the main epigenetic factors involved in bacterial phase variation that regulates some virulence factors of the Bcc species Burkholderia cenocepacia, we hypothesized that B. ambifaria HSJ1 phase variation could also be regulated by adenosine DNA methylation. By deleting the three putative adenosine DNA methyltransferases, we found that an orphan type II DNA methyltransferase prevents the emergence of phase variants. This is the first study to report quorum sensing and adenosine DNA methylation as two antagonistic systems independently controlling phase variation. ImportanceSome Burkholderia species are pathogenic to plants, animals, or humans. In immunocompromised individuals, such as those with cystic fibrosis, infection with Burkholderia cepacia complex (Bcc) bacteria can lead to "cepacia syndrome." In the Australian Aboriginal population, melioidosis caused by B. pseudomallei is prevalent, particularly among those with diabetes or alcoholism. Burkholderias phenotypic plasticity, including colony morphotype variation (CMV), enables rapid adaptation to diverse environments, enhancing survival and pathogenicity. This study reveals phase variation as a new CMV mechanism within the Bcc group. We found that quorum sensing and DNA methylation are involved in phase variation. Understanding the underlying CMV mechanisms could lead to the development of targeted therapies against these highly antibiotic-resistant bacteria.

microbiology↗

bioRxiv2024

Characterization of immunity-inducing rhizobacteria highlights diversity in plant-microbe interactions

The narrow region of soil surrounding roots (rhizosphere) contains an astonishing diversity of microorganisms. Some rhizosphere bacteria can improve plant health and immunity, via direct competition with pathogens or by establishing heightened immunity in aboveground tissues, a phenomenon known as Induced Systemic Resistance (ISR). We screened a bacterial library from agricultural soils to identify strains that, after root treatment, induce immunity in Solanum lycopersicum (tomato) against the fungal pathogen Botrytis cinerea. Here, we report the establishment of a screening method and characterization of a subset of five strains, belonging to the species Bacillus velezensis, Paenibacillus peoriae and Pseudomonas parafulva, that induced systemic resistance in tomato. However interestingly, only two of them triggered canonical ISR in Arabidopsis, indicating plant host specificity and/or alternative modes of actions. Furthermore, some of the strains displayed direct anti-microbial activity. We also found the requirement of the lipid-binding protein DIR1 in ISR establishment, indicating a possible convergence of SAR and ISR signaling. Finally, we found that P. parafulva TP18m, also displayed strong effects on root development. Taken together, we have identified taxonomically diverse immunity-inducing bacteria. Our characterization revealed diverse features, highlighting the complexity of bacteria- host interaction in the rhizosphere. HighlightWe identified taxonomically diverse rhizobacteria that induce systemic resistance in tomato plants to Botrytis after application to the root. These bacteria display diverse modes of action to improve plant health.

plant biology↗

bioRxiv2024

Temperature-responsive control of Pseudomonas aeruginosa virulence determinants through the stabilization of quorum sensing transcriptional regulator RhlR

The versatile bacterium Pseudomonas aeruginosa thrives in diverse environments and is notably recognized for its role as an opportunistic pathogen. In line with its adaptability, P. aeruginosa produces various exoproducts crucial for survival and virulence, several of which regulated through quorum sensing (QS). These factors are also regulated in response to environmental cues, such as temperature changes. As a pathogen, P. aeruginosa is generally thought to activate its virulence factors at temperatures akin to warm-blooded hosts rather than environmental temperatures. Recent studies elucidated the functional structure of the QS transcriptional regulator RhlR, which depends on the stabilizing effects of its cognate autoinducing ligand, N-butanoyl-L-homoserine lactone (C4-HSL), and of the moonlighting chaperone PqsE. Given the influence of temperature on biomolecular dynamics, we investigated how it affects RhlR activity using the RhlR-regulated phzA1 promoter as a proxy. Unexpectedly, we found that RhlR activity is higher at 25{degrees}C than at 37{degrees}C. This temperature-dependent regulation likely stems from altered RhlR turnover, with the presence of PqsE extending RhlR activity tenfold from its basal level at 37{degrees}C to that observed at 25{degrees}C. This lower, environmental-like temperature promotes increased affinity between RhlR and C4-HSL, a trait significantly compromised in the absence of PqsE. These results suggest that this response depends on the structural integrity of the complex, indicating that temperature functions as an additional regulating and stabilizing factor of RhlR function. Accordingly, lower growth temperature fails to increase the activity of a structurally stabilized version of RhlR. The thermoregulation aspect of RhlR activity and signalling impacts the virulence profile of a mutant unable to produce C4-HSL, underscoring its significance in bacterial behaviours and potentially conferring an evolutionary advantage. Author SummaryPseudomonas aeruginosa is recognized for its capacity to colonize vastly different environments, thereby encountering a range of temperatures. The bacteriums ability to adapt to these settings necessitates finely regulated gene expression. Within this regulatory framework lies quorum sensing (QS), the intercellular communication system used by P. aeruginosa to orchestrate the expression of genes responsible for producing diverse exoproducts, including the blue phenazine pyocyanin. RhlR primarily governs the expression of genes required for pyocyanin production, including the phz1 operon. Unlike other QS regulators, RhlR possesses a distinctive characteristic - in addition to its cognate signalling ligand C4-HSL, it depends on the presence of the chaperone-like protein PqsE for stability and activity. This intrinsic instability implies that RhlR may be susceptible to external influences that can modulate its function. Indeed, a lower culture temperature, akin to an environmental-like condition, induces the transcription of the phz1 operon, used as a proxy for RhlR activity. Using a combination of genetic approaches, we present evidence that this thermoregulation is due to an impact on the stability of the RhlR/C4-HSL/PqsE complex. We further show the biological effect of this regulation mechanism in an infection setting, which could underscore a relevant role for other bacterial behaviours.

microbiology↗

bioRxiv2023

Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages. Abbreviated summaryQuorum sensing regulates phage defense in Pseudomonas aeruginosa. The Pf phage protein PfsE inhibits PQS-mediated quorum sensing by binding to the host enzyme PqsA, while also protecting against type IV pili-dependent phage infection. This dual inhibition strategy promotes Pf replication and safeguards the host from competing phages.

microbiology↗

bioRxiv2023

Surface growth of Pseudomonas aeruginosa reveals a regulatory effect of 3-oxo-C12-homoserine lactone in absence of its cognate receptor, LasR

Successful colonization of a multitude of ecological niches by the bacterium Pseudomonas aeruginosa relies on its ability to respond to concentrations of self-produced signal molecules. This intercellular communication system known as quorum sensing (QS) tightly regulates the expression of virulence determinants and a diversity of survival functions, including those required for social behaviours. In planktonic cultures of P. aeruginosa, the transcriptional regulator LasR is generally considered on top of the QS circuitry hierarchy; its activation relies on binding to 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), a product of the LasI synthase. Transcription of lasI is activated by LasR, resulting in a positive feedback loop. Few studies have looked at the function of QS during surface growth even though P. aeruginosa typically lives in biofilm-like communities under natural conditions. Here, we show that surface-grown P. aeruginosa readily produces 3-oxo-C12-HSL in absence of LasR, and that this phenotype is frequent upon surface association in naturally occurring environmental and clinical LasR-defective isolates, suggesting a conserved alternative function for the signal. Indeed, even in the absence of the cognate regulator LasR, 3-oxo-C12-HSL upregulates the autologous expression of pyocyanin and of LasR-controlled virulence determinants in neighboring cells. This highlights a possible role for 3-oxo-C12-HSL in shaping community responses and provides a possible evolutive benefit for mixed populations to carry LasR-defective cells, a common feature of natural of P. aeruginosa. IMPORTANCEThe bacterium Pseudomonas aeruginosa colonizes and thrives in many environments, in which it is typically found in surface-associated polymicrobial communities known as biofilms. Adaptation to this social behavior is aided by quorum sensing (QS), an intercellular communication system pivotal in the expression of social traits. Regardless of its importance in QS regulation, the loss of function of the master regulator LasR is now considered a conserved adaptation of P. aeruginosa, irrespective of the origin of strains. By investigating the QS circuitry in surface-grown cells, we found accumulation of QS signal 3-oxo-C12-HSL in absence of its cognate receptor and activator, LasR. The current understanding of the QS circuit, mostly based on planktonic growing cells, is challenged by investigating the QS circuitry of surface-grown cells. This provides a new perspective on the beneficial aspects that underline the frequency of LasR-deficient isolates.

microbiology↗

bioRxiv2022

Emergence of Small Colony Variants (SCVs) is an adaptive strategy used by Pseudomonas aeruginosa to palliate O2 limitations

The ability to generate a subpopulation of Small Colony Variants (SCVs) is a conserved feature of Pseudomonas aeruginosa and could represent a key adaptive strategy to colonize and persist in multiple niches. However, very little is known about the role of the SCV phenotype, the conditions that promote its emergence and its possible involvement in an adaptive strategy. In the present work, we investigated the in vitro selective conditions promoting the emergence of SCVs from the prototypical strain PA14, which readily forms SCVs in non-agitated standing cultures. We found that O2 limitations, which cause a redox imbalance, is the main factor selecting for the SCV phenotype, which promotes survival of the population via formation of a biofilm at the air-liquid interface to access the electron acceptor. When this selective pressure is relieved by aeration or supplementation of an alternative electron acceptor, SCVs are barely detectable. We also observed that SCV emergence contributes to redox rebalancing, suggesting that it is involved in an adaptive strategy. We conclude that selection for the SCV phenotype is an adaptive solution adopted by P. aeruginosa to access poorly available O2. IMPORTANCEThe bacterium P. aeruginosa is an opportunistic pathogen that thrives in many environments. It poses a significant health concern, notably because it is a causative agent of nosocomial infections and the most prevalent pathogen found in the lungs of people with cystic fibrosis. In infected hosts, its persistence is often related to the emergence of an alternative phenotype known as Small Colony Variant (SCV). Identification of conditions selecting for the SCV phenotype contributes to knowledge regarding adaptive mechanisms exploited by P. aeruginosa to survive in multiple niches and persist during infections. Hindering this adaptation strategy could help control P. aeruginosa persistent infections.

microbiology↗

bioRxiv2022

Community composition shapes microbial-specific phenotypes in a cystic fibrosis polymicrobial model system

Interspecies interactions can drive the emergence of unexpected microbial phenotypes that are not observed when studying monocultures. The cystic fibrosis (CF) lung consists of a complex environment where particular microbes, living as polymicrobial biofilm-like communities, are associated with negative clinical outcomes for persons with CF (pwCF). However, the current lack of in vitro models integrating the microbial diversity observed in the CF airway hampers our understanding of why polymicrobial communities are recalcitrant to therapy in this disease. Here, integrating computational approaches informed by clinical data, we built a mixed community of clinical relevance to the CF lung composed of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis and Prevotella melaninogenica. We developed and validated this model biofilm community with multiple isolates of these four genera. When challenged with tobramycin, a front-line antimicrobial used to treat pwCF, the microorganisms in the polymicrobial community show altered sensitivity to this antibiotic compared to monospecies biofilms. We observed that wild-type P. aeruginosa is sensitized to tobramycin in a mixed community versus monoculture, and this observation holds across a range of community relative abundances. We also report that LasR loss-of-function, a variant frequently detected in the CF airway, induces tolerance of P. aeruginosa to tobramycin specifically in the mixed community. The molecular basis of this community-specific recalcitrance to tobramycin for the LasR mutant variant is the increased production of redox-active phenazines. Our data support the importance of studying clinically-relevant model polymicrobial biofilms to understand community-specific traits relevant to infections. SIGNIFICANCE STATEMENTThe CF lung is colonized by biofilm-like microbial communities that exhibit both resistance and tolerance (collectively called "recalcitrance") to antimicrobials used in the clinic. Here, we leveraged clinical data from pwCF to inform our understanding of communities exhibiting recalcitrance. We developed and validated an in vitro model that revealed novel, community-specific phenotypes relevant to the clinic. We used this model to explore the underlying mechanism associated with a community-specific emergent behavior. We posit that in vitro models of polymicrobial communities may help in developing new antimicrobial strategies to improve patient outcomes, and that the approach used here can be applied to other polymicrobial models.

microbiology↗

bioRxiv2022

Pseudomonas aeruginosa strains from both clinical and environmental origins readily adopt a stable small colony variant (SCV) phenotype resulting from single mutations in c-di-GMP pathways

A subpopulation of Small Colony Variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolated from colonized cystic fibrosis lungs. Since most SCVs have until now been isolated from clinical samples, it remains unclear how widespread is the ability of P. aeruginosa to develop this phenotype and the genetic mechanism(s) behind SCVs emergence according to the origin of the isolate. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to, under laboratory culture conditions, spontaneously adopt a SCV-like smaller alternative morphotype distinguishable from the ancestral parent strain. We found that all the P. aeruginosa strains tested could adopt a SCV phenotype, regardless of their origin. Whole genome sequencing of SCVs obtained from clinical and environmental sources revealed single mutations exclusively in two distinct c-di-GMP signaling pathways, Wsp and YfiBNR. We conclude that the ability to switch to a SCV phenotype is a conserved feature of P. aeruginosa and results from the acquisition of a stable genetic mutation, regardless of the origin of the strain. IMPORTANCEP. aeruginosa is an opportunistic pathogen that thrives in many environments. It poses a significant health concern, notably because this bacterium is the most prevalent pathogen found in the lungs of people with cystic fibrosis. In infected hosts, its persistence is considered related to the emergence of an alternative small colony variant (SCV) phenotype. By reporting the distribution of P. aeruginosa SCVs in various non-clinical environments and the involvement of c-di-GMP in SCV emergence from both clinical and environmental strains, this work contributes to understanding a conserved adaptation mechanism used by P. aeruginosa to adapt readily in all environments. Hindering this adaptation strategy could help control P. aeruginosa persistent infection.

microbiology↗

bioRxiv2022

Surface motility favors co-dependent interaction between Pseudomonas aeruginosa and Burkholderia cenocepacia

Interactions between different bacterial species shape bacterial communities and their environments. The opportunistic pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia both can colonize the lungs of individuals affected by cystic fibrosis. Using the social surface behavior called swarming motility as a study model of interactions, we noticed intricate interactions between B. cenocepacia K56-2 and P. aeruginosa PA14. While strain K56-2 does not swarm under P. aeruginosa favorable swarming conditions, co-inoculation with a non-motile PA14 flagellum-less {Delta}fliC mutant restored spreading for both strains. We show that P. aeruginosa provides the wetting agent rhamnolipids allowing K56-2 to perform swarming motility, while aflagellated PA14 seems able to < > along with K56-2 cells in the swarming colony. ImportancePseudomonas aeruginosa and Burkholderia cenocepacia are important opportunistic pathogens often found together in the airways of persons with cystic fibrosis. Laboratory co-culture of both species often ends with one taking over the other. We used a surface motility assay to study the social interactions between population of these bacterial species. Under our conditions, B. cenocepacia cannot swarm without supplementation of the wetting agent produced by P. aeruginosa. In a mixed colony of both species, an aflagellated mutant of P. aeruginosa provides the necessary wetting agent to B. cenocepacia, allowing both bacteria to swarm and colonize a surface. We highlight this peculiar interaction where both bacteria set aside their antagonistic tendencies to cooperate.

microbiology↗

bioRxiv2021

Pseudomonas aeruginosa isolates defective in function of the LasR quorum sensing regulator are frequent in diverse environmental niches

The saprophyte Pseudomonas aeruginosa is a versatile opportunistic pathogen causing infections in immunocompromised individuals. To facilitate its adaptation to a large variety of niches, this bacterium exploits population density-dependant gene regulation systems called quorum sensing. In P. aeruginosa, three distinct but interrelated quorum sensing systems (las, rhl and pqs) regulate the production of many survival and virulence functions. In prototypical strains, the las system, through its transcriptional regulator LasR, is important for the full activation of the rhl and pqs systems. Still, LasR-deficient isolates have been reported, mostly sampled from the lungs of people with cystic fibrosis, where they are considered selected by the chronic infection environment. In this study, we show that a defect in LasR activity appears to be an actually widespread mechanism of adaptation in this bacterium. Indeed, we found abundant LasR-defective isolates sampled from hydrocarbon-contaminated soils, hospital sink drains, and meat/fish market environments, using an approach based on phenotypic profiling, supported by gene sequencing. Interestingly, several LasR-defective isolates maintain an active rhl system or are deficient in pqs system signaling. The high prevalence of a LasR-defective phenotype among environmental P. aeruginosa isolates questions the role of quorum sensing in niche adaptation.

microbiology↗

bioRxiv2021

The ability of Pseudomonas aeruginosa to adopt a Small Colony Variant (SCV) phenotype is conserved, and not restricted to clinical isolates

A subpopulation of Small Colony Variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolated from cystic fibrosis (CF) lungs biofilms. SCVs have almost exclusively been reported from infected hosts, essentially CF individuals or, by extension, from laboratory cultivation of strains originated from infected hosts. We previously reported the identification of P. aeruginosa SCVs emerging from a non-clinical strain and displaying features shared with clinical SCVs. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to, under laboratory culture conditions, spontaneously adopt a SCV-like smaller alternative morphotype distinguishable from the ancestral parent strain. Unexpectedly, we found that all the P. aeruginosa strains tested have the ability to adopt a SCV morphotype, regardless of their origin. Based on the phenotypes already described for SCVs, the SCV-like morphotypes obtained were clustered in two groups displaying various phenotypic profiles, including one characteristic of already described SCVs. We conclude that the ability to switch to a SCV phenotype is a conserved feature in Pseudomonas aeruginosa. IMPORTANCEP. aeruginosa is an opportunistic pathogen that thrives in many environments. It is significant public health concern, notably because it is the most prevalent pathogen found in the lungs of people with cystic fibrosis (CF). In infected hosts, its persistence is believed to be related to the emergence of an alternative small colony variant (SCV) phenotype. By reporting the distribution of P. aeruginosa SCVs in various non-clinical environments, this work contributes to understanding a conserved adaptation mechanism used by P. aeruginosa to rapidly adapt in all environments. Counteraction of this strategy could prevent P. aeruginosa persistent infection in the future.

microbiology↗

bioRxiv2020

An experimentally evolved variant of RsmA confirms its central role in the control of Pseudomonas aeruginosa social motility

Bacteria can colonize a variety of different environments by modulating their gene regulation using two-component systems. The versatile opportunistic pathogen Pseudomonas aeruginosa has been studied for its capacity to adapt to a broad range of environmental conditions. The Gac/Rsm pathway is composed of the sensor kinase GacS, that detects environmental cues, and the response regulator GacA, that modulates the expression of a specific genes. This system, through the sRNA repressors RsmY and RsmZ, negatively controls the activity of the protein RsmA, which is centrally involved in the transition from chronic to acute infections by post-transcriptionally regulating several virulence functions. RsmA positively regulates swarming motility, a social surface behaviour. Through a poorly defined mechanism, RsmA is also indirectly regulated by HptB, and a {Delta}hptB mutant exhibits a severe swarming defect. Since a {Delta}hptB mutant retains all the known functions required for that type of motility, we used an experimental evolution approach to identify elements responsible for its swarming defect. After a few passages under swarming conditions, the defect of the {Delta}hptB mutant was rescued by the emergence of spontaneous single nucleotide substitutions in the gacA and rsmA genes. Since GacA indirectly represses RsmA activity, it was coherent that an inactivating mutation in gacA would compensate the {Delta}hptB swarming defect. However, the effect of the mutation in rsmA was unexpected since RsmA promotes swarming; indeed, using expression reporters, we found that the mutation that does not abolish its activity. Instead, using electrophoretic mobility shift assays and molecular simulations, we show that this variant of RsmA is actually less amenable to titration by its cognate repressor RsmY, supporting the other phenotypes observed for this mutant. These results confirm the central role of RsmA as a regulator of swarming motility in P. aeruginosa and identify residues crucial for RsmA function in social motility. Author summaryBacteria need to readily adapt to their environment. Two-component systems (TCS) allow such adaption by triggering bacterial regulation changes through the detection of environmental cues. The opportunistic pathogen Pseudomonas aeruginosa possesses more than 60 TCS in its genome. The Gac/Rsm is a TCS extensively studied for its implication in virulence regulation. This system regulates the transition between chronic and acute bacterial infection behaviours. To acquire a better understanding of this regulation, we performed a directed experimental evolution on a swarming-deficient mutant in a poorly understood regulatory component of the Gac/Rsm pathway. We observed single nucleotide substitutions that allowed restoration of a swarming phenotype similar to the wild-type behaviour. More specifically, mutations were found in the gacA and rsmA genes. Interestingly, the observed mutation in rsmA does not result in loss of function of the protein but rather alters its susceptibility to repression by its cognate interfering sRNA. Since modification in the RNA sequence of RsmA results in the rescue of swarming motility, we confirm the central role of this posttranscriptional repressor in this social lifestyle.

microbiology↗

bioRxiv2020

Burkholderia thailandensis methylated hydroxy-alkylquinolines: biosynthesis and antimicrobial activity in co-culture experiments

The bacterium Burkholderia thailandensis produces an arsenal of secondary metabolites that have diverse structures and roles in the ecology of this soil-dwelling bacterium. In liquid co-culture experiments, B. thailandensis secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis. To identify the antimicrobial, we used a transposon mutagenesis approach. This screen identified antimicrobial-defective mutants with insertions in the hmqA, hmqC and hmqF genes involved in biosynthesis of a family of 2-alkyl-4(1H)-quinolones called 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs), which are closely related to the Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs). Insertions also occurred in the previously uncharacterized gene BTH_II1576. Results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NO) in B. thailandensis and that HMAQ-NOs are sufficient to eliminate B. subtilis in co-cultures. Moreover, synthetic HMAQ-NO is [~]50-fold more active than HMAQ. Both the methyl group and the length of the carbon side chain account for high activity of HMAQ-NO against B. subtilis. The results provide new information on the biosynthesis and activities of HMAQs and reveal new insight into how these molecules might be important for the ecology of B. thailandensis. IMPORTANCEThe soil bacterium Burkholderia thailandensis produces 2-alkyl-4(1H)-quinolones, mostly methylated 4-hydroxy-alkenylquinolines, a family of relatively unstudied metabolites similar to molecules also synthesized by Pseudomonas aeruginosa. Several of the methylated 4-hydroxy-alkenylquinolines have antimicrobial activity against other species. We show that N-oxidated methyl-alkenylquinolines are particularly antimicrobial and sufficient to kill Bacillus subtilis in co-cultures. We confirmed their biosynthesis requires the previously unstudied protein HmqL. These results provide new information about the biology of 2-alkyl-4(1H)-quinolones, particularly the methylated 4-hydroxy-alkenylquinolines, which are unique to B. thailandensis. This study also has importance for understanding B. thailandensis secondary metabolites and has implications for potential therapeutic development.

microbiology↗

bioRxiv2019

ScmR, a global regulator of gene expression, quorum sensing, pH homeostasis, and virulence in Burkholderia thailandensis

The nonpathogenic soil saprophyte Burkholderia thailandensis is a member of the Burkholderia pseudomallei-thailandensis-mallei (Bptm) group, which also comprises the closely related human pathogens Burkholderia pseudomallei and Burkholderia mallei responsible for the diseases melioidosis and glanders, respectively. ScmR, a recently identified LysR-type transcriptional regulator (LTTR) in B. thailandensis acts as a global transcriptional regulator throughout the stationary phase, and modulates the production of a wide range of secondary metabolites, including N-acyl-L-homoserine lactones (AHLs) and 4-hydroxy-3-methyl-2-alkylquinoline (HMAQ), virulence in the model host Caenorhabditis elegans, as well as several quorum sensing (QS)-dependent phenotypes. We have investigated the role of ScmR in B. thailandensis strain E264 during the exponential phase. We used RNA-Sequencing (RNA-Seq) transcriptomic analyses to identify the ScmR regulon, which was compared to the QS-controlled regulon, showing a considerable overlap between the ScmR-regulated genes and those controlled by QS. We characterized several genes modulated by ScmR, using quantitative reverse transcription-PCR (qRT-PCR) or mini-CTX-lux transcriptional reporters, including the oxalate biosynthetic gene obc1 required for pH homeostasis, the orphan LuxR-type transcriptional regulator BtaR5-encoding gene, the bsa (Burkholderia secretion apparatus) type III secretion system (T3SS) genes essential for both B. pseudomallei and B. mallei pathogenicity, as well as the scmR gene itself. We confirmed that the transcription of scmR is under QS control, presumably ensuring fine-tuned modulation of gene expression. Finally, we demonstrate that ScmR influences virulence using the fruit fly model host Drosophila melanogaster. We conclude that ScmR represents a central component of the B. thailandensis QS regulatory network. ImportanceCoordination of the expression of genes associated with bacterial virulence and environmental adaptation is often dependent on quorum sensing (QS). The QS circuitry of the nonpathogenic bacterium Burkholderia thailandensis, which is widely used as a model system for the study of the human pathogen Burkholderia pseudomallei, is complex. We found that the recently identified LysR-type transcriptional regulator (LTTR), ScmR, which is highly conserved and involved in the control of virulence/survival factors in the Burkholderia genus, is a global regulator mediating gene expression through the multiple QS systems coexisting in B. thailandensis, as well as independently of QS. We conclude that ScmR represents a key QS modulatory network element, ensuring tight regulation of the transcription of QS-controlled genes, particularly those required for acclimatization to the environment.

microbiology↗