Biology subjects

Doucet, N.

Publications and source records attributed to Doucet, N..

3 recordsLinked to original sources

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↗

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↗

bioRxiv2019

Genome sequencing and functional characterization of a Dictyopanus pusillus fungal extract offers a promising alternative for lignocellulose pretreatment of oil palm residues

The pretreatment of biomass is a critical requirement of bio-renewable fuel production from lignocellulose. Although current processes primarily involve chemical and physical approaches, the biological breakdown of lignin using enzymes and microorganisms is quickly becoming an interesting eco-friendly alternative to classical processes. As a result, bioprospection of wild fungi from naturally occurring lignin-rich sources remains a suitable method to uncover and isolate new species exhibiting ligninolytic activity. In this study, wild species of white rot fungi were collected from Colombian forests based on their natural wood decay ability and high capacity to secrete oxidoreductases with high affinity for phenolic polymers such as lignin. Based on high activity obtained from solid-state fermentation using a lignocellulose source from oil palm as matrix, we describe the isolation and whole-genome sequencing of Dictyopanus pusillus, a wild basidiomycete fungus exhibiting ABTS oxidation as an indication of laccase activity. Functional characterization of a crude enzymatic extract identified laccase activity as the main enzymatic contributor to fungal extracts, an observation supported by the identification of 13 putative genes encoding for homologous laccases in the genome. To the best of our knowledge, this represents the first report of an enzymatic extract exhibiting laccase activity in the Dictyopanus genera, offering means to exploit this species and its enzymes for the delignification process of lignocellulosic by-products from oil palm.

bioengineering↗