Biology subjects

Smalley, N. E.

Publications and source records attributed to Smalley, N. E..

8 recordsLinked to original sources

bioRxiv2025

Quorum sensing antiactivators constrain Pseudomonas aeruginosa RhlR activity

Pseudomonas aeruginosa, an opportunistic pathogen, uses a cell-cell communication system called quorum sensing (QS) to regulate gene expression in response to population density. P. aeruginosa QS involves, in part, two transcription factors, LasR and RhlR, that respond to N-acyl homoserine lactone (AHL) signals. Two proteins known as "antiactivators," QteE and QslA, attenuate QS by inhibiting LasR, RhlR, or both. While initial characterization of antiactivation has revealed the considerable influence these factors may have on dampening QS, details regarding the individual impacts of these antiactivators on P. aeruginosa QS activity remain scant. Additionally, the effects of antiactivators on RhlR QS activity and in QS systems in isolates or strain lacking LasR have yet to be explored. To investigate how QteE and QslA each modulate LasR or RhlR independently, we combined gene deletion and over-expression analysis of each antiactivator in wild-type P. aeruginosa (PAO1) and two strains with rhl-dominated QS: clinical isolate E90 and PAO {Delta}lasR {Delta}mexT. As measured with a transcriptional reporter, over-expression of qteE or qslA notably reduced RhlR activity in PAO1 and PAO {Delta}lasR {Delta}mexT, but only expression of qteE had a marked effect on RhlR activity in E90. Expression analysis indicates LasR and RhlR repress QteE transcription, but not QslA. By over-expressing qslA in the absence of QteE and vice versa, we demonstrate that QslA activity and corresponding effects on QS phenotypes can be QteE-dependent in some scenarios. Our results reveal a nuanced role for individual antiactivator proteins in affecting the layered P. aeruginosa QS circuitry. ImportanceQuorum sensing (QS) is a cell signaling mechanism that enables populations of Pseudomonas aeruginosa to coordinate group behaviors such as biofilm formation, virulence factor production, and antibiotic tolerance once a critical cell-density threshold is reached. P. aeruginosa employs two "antiactivator" proteins that attenuate QS at low cell densities, dampening QS activation. The specific effects of individual antiactivators on the complex and hierarchically-arranged P. aeruginosa QS systems remain undefined. Here, we use two strains with rewired QS circuits to independently assess the effects of QS antiactivators on each QS circuit. We find that while one antiactivator selectively targets one QS circuit, the other can broadly target both with strong effects on QS activity. This work reveals an additional layer of complexity to counter-regulation of QS signalling and further defines antiactivation as a mechanism P. aeruginosa uses to finely tune QS responses.

microbiology↗

bioRxiv2025

Inactivation of MexT in Pseudomonas aeruginosa destabilizes cooperation and favors the emergence of a unique quorum sensing variant

Pseudomonas aeruginosa, an opportunistic gram-negative pathogen, uses a cell-cell signaling system called quorum sensing to coordinate group behaviors. Quorum sensing in P. aeruginosa is a model to study cooperative behaviors in populations. Several studies of cooperation have been conducted using strain PAO1. Wildtype PAO1 harbors a mutation in mexS, which encodes a negative regulator of the transcription factor MexT, which in turn activates many genes, including the efflux pump MexEF-OprN. We hypothesized that the PAO1 mexS mutation might affect cooperative behaviors. When P. aeruginosa is passaged daily on casein as a sole carbon source, quorum sensing is required to induce synthesis of the extracellular proteases needed to acquire carbon and energy. During growth on casein, individuals with inactivating mutations in the quorum sensing regulator LasR are reproducibly enriched in the population. These LasR mutants are cheaters that benefit from cooperatively-produced proteases and have a fitness advantage over cooperators. We passaged wildtype PAO1, PAO1 with a gene-corrected version of mexS, or PAO1 with a null mutation of mexT on casein as the sole carbon and energy source. We found that correcting the mexS mutation resulted in unstable cooperation: bacterial cultures failed to propagate after about 15 days, whereas the wildtype propagated for the duration of our 30-day experiment. The MexS-corrected and MexT-deficient populations also reproducibly exhibited emergence of a particular quorum sensing variant, LasR-V226I. This variant activated a subset of quorum-sensing regulated genes, suggesting an evolutionary pathway to alter P. aeruginosa quorum sensing regulons. ImportanceQuorum sensing governs cooperative activities and has been used as a model for studying cooperative behaviors in bacterial populations. Pseudomonas aeruginosa has two interlinked acyl-homoserine lactone quorum sensing circuits, LasR-I and RhlR-I; the relationship between these circuits is influenced by a regulator called MexT. Inactivation of MexT results in enhanced activity of the quorum sensing transcription factor, RhlR, and we found that it also destabilized cooperative behaviors in this bacterium. MexT-inactive strains also reproducibly support the emergence of a specific LasR variant, V226I, of P. aeruginosa, offering insight into evolutionary pressures that select for mutations in quorum sensing transcription factors and, by extension, a method for bacteria to alter the cohort of genes that are quorum-controlled.

microbiology↗

bioRxiv2025

Modulation of the Pseudomonas aeruginosa quorum sensing cascade by MexT-regulated factors

Pseudomonas aeruginosa (Pa) uses quorum sensing (QS), a cell-cell communication system that enables it to sense cell density and to alter gene expression. Pa has three complete QS circuits controlled by the regulators LasR, RhlR, and PqsR, that together activate hundreds of genes. In the well-studied strain PAO1, QS is organized hierarchically, with PqsR and RhlR activity dependent on LasR. This hierarchy depends on the non-QS transcription factor MexT; deletion of mexT allows for RhlR activity in the absence of LasR. We aimed to identify how MexT modulates the Pa QS architecture. We compared the transcriptome of PAO1 to that of PAO1{Delta}mexT and determined a MexT regulon. We identified two MexT-regulated operons that may affect the QS hierarchy: the efflux pump genes mexEF-oprN and the Pseudomonas quinolone signal (PQS) synthesis genes pqsABCDE. We tested whether the products of these genes affected the QS hierarchy. A mexEF knockout mutant, like a mexT deletion mutant, exhibited RhlR activity earlier, and to a higher magnitude, than wild-type PAO1. MexEF-OprN is known to export quinolones, and we found that exogenous addition of PQS, through PqsE, also resulted in earlier and higher magnitude of RhlR activity compared to wild-type PAO1. We also discovered alternate QS architectures in clinical isolates, where RhlR activity is not fully dependent on LasR. In these isolates, surprisingly, MexT does not influence the relationship between LasR and RhlR. Our work reveals a new suite of factors that regulate QS in Pa, with implications for bacterial behaviors in environmental and clinical settings. ImportanceBacteria interact with both abiotic and biotic factors in their environment. Quorum sensing (QS) is one mechanism that bacteria use to communicate with other bacteria and coordinate behaviors in the population. QS regulates a wide variety of processes ranging from the production of light to modulation of virulence factors; some bacteria use single QS circuits, while others have several. The opportunistic pathogen Pseudomonas aeruginosa uses QS to control some virulence functions and has three complete QS circuits. Our study explores why bacteria might have multiple QS circuits. We show how a non-QS transcription factor, MexT, influences QS regulators in P. aeruginosa and we uncover the diversity of QS architectures in clinical isolates. This study begins to reveal the benefits (or disadvantages) of multiple QS circuits, allowing us to understand behaviors of bacteria that have a range of implications including in health, agriculture, and bioremediation.

microbiology↗

bioRxiv2025

Quorum sensing regulation by the nitrogen phosphotransferase system in Pseudomonas aeruginosa

In the opportunistic pathogen Pseudomonas aeruginosa, the nitrogen-related phosphotransferase system (PTSNtr) influences multiple virulence behaviors. The PTSNtr is comprised of three enzymes: first PtsP, then the PtsO phosphocarrier, and the final PtsN phosphoacceptor. We previously showed that ptsP inactivation increases LasI-LasR quorum sensing, a system by which P. aeruginosa regulates genes in response to population density. LasI synthesizes a diffusible autoinducer that binds and activates the LasR receptor, which activates a feedback loop by increasing lasI expression. In this study, we examined the impact of the PTSNtr on quorum sensing. Disruption of ptsP increased the expression of some, but not all, tested quorum-controlled genes, including lasI, phzM (pyocyanin biosynthesis), hcnA (hydrogen cyanide biosynthesis), and, to a lesser extent, rsaL (quorum sensing regulator). Expression of these genes remained dependent on LasR and the autoinducer, whether provided endogenously or exogenously. Increased lasI expression in {Delta}ptsP (or {Delta}ptsO) cells was partly due to the presence of unphosphorylated PtsN, which alone was sufficient to elevate lasI expression. However, we observed residual increases in {Delta}ptsP or {Delta}ptsO cells even in the absence of PtsN, suggesting that PtsP and PtsO can regulate gene expression independent of PtsN. Indeed, genetically disrupting the PtsO phosphorylation site impacted gene expression in the absence of PtsN, and transcriptomic evidence suggested that PtsO and PtsN have distinct regulons. Our results expand our view of how the PTSNtr components function both within and apart from the classic phosphorylation cascade to regulate key virulence behaviors in P. aeruginosa. IMPORTANCEPseudomonas aeruginosa often causes severe and difficult-to-treat infections. P. aeruginosa virulence requires the nitrogen-related phosphotransferase system (PTSNtr), which comprises the phosphocarrier proteins PtsP and PtsO and the final phosphoacceptor, PtsN. The PTSNtr is known to modulate quorum sensing, but little is known about the mechanism of regulation. Here, we examined quorum sensing regulation by the PTSNtr. We showed that the PTSNtr increases quorum sensing-mediated activation of certain genes through the additive effects of both PtsO and PtsN. We also used transcriptomics to determine the regulons of PtsO and PtsN and found that they are largely nonoverlapping. The results position PtsO and PtsN as independent effectors in the Nitro-PTS and shed new light on virulence regulation in this important pathogen.

microbiology↗

bioRxiv2024

The Relationship of pqs Gene Expression to Acylhomoserine Lactone Signaling in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa has complex quorum sensing (QS) circuitry, which involves two acylhomoserine lactone (AHL) systems, the LasI AHL synthase and LasR AHL-dependent transcriptional activator system and the RhlI AHL synthase-RhlR AHL-responsive transcriptional activator. There is also a quinoline signaling system (the Pseudomonas quinolone signal, PQS, system). Although there is a core set of genes regulated by the AHL circuits, there is substantial strain-to-strain variation in the non-core QS regulated genes. Reductive evolution of the QS regulon, and variation in specific genes activated by QS, occurs in laboratory evolution experiments with the model strain PAO1. We used a transcriptomics approach to test the hypothesis that reductive evolution in the PAO1 QS regulon can in large part be explained by a simple null mutation in pqsR, the gene encoding the transcriptional activator of the pqs operon. We found that PqsR had very little influence on the AHL QS regulon. This was a surprising finding because the last gene in the PqsR-dependent pqs operon, pqsE, codes for a protein, which physically interacts with RhlR and this interaction is required for RhlR-dependent activation of some genes. We used comparative transcriptomics to examine the influence of a pqsE mutation on the QS regulon and identified only three transcripts, which were strictly dependent on PqsE. By using reporter constructs we showed that the PqsE influence on other genes was dependent on experimental conditions and we have gained some insight about those conditions. This work adds to our understanding of the plasticity of the P. aeruginosa QS regulon and to the role PqsE plays in RhlR-dependent gene activation.

microbiology↗

bioRxiv2021

Genetic and transcriptomic characteristics of RhlR-dependent quorum sensing in cystic fibrosis isolates of Pseudomonas aeruginosa

In people with the genetic disease cystic fibrosis (CF), bacterial infections involving the opportunistic pathogen Pseudomonas aeruginosa are a significant cause of morbidity and mortality. P. aeruginosa uses a cell-cell signaling mechanism called quorum sensing (QS) to regulate many virulence functions. One type of QS consists of acyl-homoserine lactone (AHL) signals produced by LuxI-type signal synthases, which bind a cognate LuxR-type transcription factor. In laboratory strains and conditions, P. aeruginosa employs two AHL synthase/receptor pairs arranged in a hierarchy, with the LasI/R system controlling the RhlI/R system and many downstream virulence factors. However, P. aeruginosa isolates with inactivating mutations in lasR are frequently isolated from chronic CF infections. We and others have shown that these isolates frequently use RhlR as the primary QS regulator. RhlR is rarely mutated in CF and environmental settings. We were interested if there were reproducible genetic characteristics of these isolates and if there was a central group of genes regulated by RhlR in all isolates. We examined five isolates and found signatures of adaptation common to CF isolates. We did not identify a common genetic mechanism to explain the switch from Las-to Rhl-dominated QS. We describe a core RhlR regulon encompassing 20 genes encoding 7 products. These results suggest a key group of QS-regulated factors important for pathogenesis of chronic infection, and position RhlR as a target for anti-QS therapeutics. Our work underscores the need to sample a diversity of isolates to understanding QS beyond what has been described in laboratory strains.

microbiology↗

bioRxiv2021

Evolution of the quorum sensing regulon in cooperating populations of Pseudomonas aeruginosa

The bacterium Pseudomonas aeruginosa is an opportunistic pathogen and it thrives in many different saprophytic habitats. In this bacterium acyl-homoserine lactone quorum sensing (QS) can activate expression of over 100 genes, many of which code for extracellular products. P. aeruginosa has become a model for studies of cell-cell communication and coordination of cooperative activities. We hypothesized that long-term growth of bacteria under conditions where only limited QS-controlled functions were required would result in a reduction in the size of the QS-controlled regulon. To test this hypothesis, we grew P. aeruginosa for about 1000 generations in a condition in which expression of QS-activated genes is required for growth. We compared the QS regulons of populations after about 35 generations to those after about 1000 generations in two independent lineages by using quorum quenching and RNA-seq technology. In one evolved lineage the number of QS-activated genes identified was reduced by about 70% and in the other by about 45%. Our results lend important insights about the variations in the number of QS-activated genes reported for different bacterial strains and, more broadly, about the environmental histories of P. aeruginosa.

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↗