Two-component System Research Articles

Vapor-liquid equilibria for the CO2 + trimethoxymethylsilane and CO2 + triethoxymethylsilane systems under high-pressure conditions

New binary isotherms are crucial for designing chemical separation processes within supercritical carbon dioxide (CO2) + trialkoxysilane systems. Vapor-liquid equilibria (VLE) were investigated for two-component systems, trimethoxymethylsilane + CO2 and triethoxymethylsilane + CO2, at five temperatures (313.2, 333.2, 353.2, 373.2, and 393.2 K) and pressures up to 14.07 MPa using a synthetic high-pressure phase equilibria apparatus. The pressure-temperature (P-T) plot indicates that the critical mixture curve lies between the critical points of CO2 and the trialkoxysilane compounds. The solubility of trimethoxymethylsilane and triethoxymethylsilane in CO2 increased with increasing temperature at constant pressure, following a type-I phase behavior characteristic. The experimentally observed VLE values of the CO2 + trialkoxysilane systems were correlated using the Peng-Robinson equation of state with binary parameters (kij and ηij) in the conventional mixing rule. The model accuracy was validated by calculating the average relative deviation percentage for the pressure of the binary systems, resulting in values of 4.98% for the trimethoxymethylsilane + CO2 system and 3.64% for the triethoxymethylsilane + CO2 system. The estimated variables fell within reasonable limits and showed no significant differences between the predicted and observed VLE data for both systems.

Self-aggregation effect of the ternary system “Alga EPS-DOM-HMs” and the characterization of the self-adaptation metabolic response of microalgae

Heavy metals (HMs) present in the natural aquatic environment can form a ternary aggregate of “EPS-DOM-HMs” with the prevalent microalgae extracellular polymers substances (EPS) and macromolecular dissolved organic matters (DOMs), which show special molecular structure and biological interaction. This study reveals the formation of “EPS-TA-HMs” and the mechanism of their physiological and metabolic effects on Raphidocelis subcapitata. Results indicate that TA-Cr(III) can bind to EPS to form ternary aggregates with substances coexisting large and small hydrodynamic diameters and that the interactions are dominated by hydrophobic interactions of the protein binding to the pyrrole ring of the polyphenol and hydrogen bonding interactions formed by OC–(N R O). The protein structure of EPS has the largest proportion of proline, glycine, aspartic acid, and tryptophan. These interactions promoted the secretion of EPS components and reduced the growth inhibition of Raphidocelis subcapitata by 45.9 % compared with Cr(III) exposure. TEM analysis combined with EDS analysis indicated that Cr(III) was taken intracellularly and TA-Cr(III) was not. In addition, metabolomics analyses revealed that microalgae initiate adaptive mechanisms via the activation of a two-component system (i.e., maintenance of high metabolic activity). This study underscored the morphology of HMs in real aquatic environments and the mechanisms of metabolic effects on aquatic organisms.

Insight into the outer membrane asymmetry of P. aeruginosa and the role of MlaA in modulating the lipidic composition, mechanical, biophysical, and functional membrane properties of the cell envelope.

In Gram-negative bacteria, the outer membrane (OM) is asymmetric, with lipopolysaccharides (LPS) in the outer leaflet and glycerophospholipids (GPLs) in the inner leaflet. The asymmetry is maintained by the Mla system (MlaA-MlaBCDEF), which contributes to lipid homeostasis by removing mislocalized GPLs from the outer leaflet of the OM. Here, we ascribed how Pseudomonas aeruginosa ATCC 27853 coordinately regulates pathways to provide defense against the threats posed by the deletion of mlaA. Especially, we explored (i) the effects on membrane lipid composition including LPS, GPLs, and lysophospholipids, (ii) the biophysical properties of the OM such as stiffness and fluidity, and (iii) the impact of these changes on permeability, antibiotic susceptibility, and membrane vesicles (MVs) generation. Deletion of mlaA induced an increase in total GPLs and a decrease in LPS level while also triggering alterations in lipid A structures (arabinosylation and palmitoylation), likely to be induced by a two-component system (PhoPQ-PmrAB). Altered lipid composition may serve a physiological purpose in regulating the mechanobiological and functional properties of P. aeruginosa. We demonstrated an increase in cell stiffness without alteration of turgor pressure and inner membrane (IM) fluidity in ∆mlaA. In addition, membrane vesiculation increased without any change in OM/IM permeability. An amphiphilic aminoglycoside derivative (3',6-dinonyl neamine) that targets P. aeruginosa membranes induced an opposite effect on ∆mlaA strain with a trend toward a return to the situation observed for the WT strain. Efforts dedicated to understanding the crosstalk between the OM lipid composition, and the mechanical behavior of bacterial envelope, is one needed step for designing new targets or new drugs to fight P. aeruginosa infections.IMPORTANCEPseudomonas aeruginosa is a Gram-negative bacterium responsible for severe hospital-acquired infections. The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore, compromising this structure could increase sensitivity to antibiotics. The OM is asymmetric with the highly packed lipopolysaccharide monolayer at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla pathway resulting in the retrograde transport of glycerophospholipids from the OM to the inner membrane. In this study, we show that deleting mlaA, the membrane component of Mla system located at the OM, affects the mechanical and functional properties of P. aeruginosa cell envelope. Our results provide insights into the role of MlaA, involved in the Mla transport pathway in P. aeruginosa.

Staphylococcus aureus SaeRS impairs macrophage immune functions through bacterial clumps formation in the early stage of infection

The Staphylococcus aureus (S. aureus) SaeRS two-component system (TCS) regulates over 20 virulence factors. While its impact on chronic infection has been thoroughly discussed, its role in the early stage of infection remains elusive. Since macrophages serve as the primary immune defenders at the onset of infection, this study investigates the influence of SaeRS on macrophage functions and elucidates the underlying mechanisms. Macrophage expression of inflammatory and chemotactic factors, phagocytosis, and bactericidal activity against S. aureus were assessed, along with the evaluation of cellular oxidative stress. SaeRS was found to impair macrophage function. Mechanistically, SaeRS inhibited NF-κB pathway activation via toll-like receptor 2 (TLR2). Its immune-modulating effect could partially be explained by the strengthened biofilm formation. More importantly, we found SaeRS compromised macrophage immune functions at early infection stages even prior to biofilm formation. These early immune evasion effects were dependent on bacterial clumping as cytokine secretion, phagocytosis, and bactericidal activity were repaired when clumping was inhibited. We speculate that the bacterial clumping-mediated antigen mask is responsible for SaeRS-mediated immune evasion at the early infection stage. In vivo, ΔsaeRS infection was cleared earlier, accompanied by early pro-inflammatory cytokines production, and increased tissue oxidative stress. Subsequently, macrophages transitioned to an anti-inflammatory state, thereby promoting tissue repair. In summary, our findings underscore the critical role of the SaeRS TCS in S. aureus pathogenicity, particularly during early infection, which is likely initiated by SaeRS-mediated bacterial clumping.

The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of staphylococcus aureus

ABSTRACT The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced haemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and haemolytic activity in S. aureus.

BvgR is important for virulence-related phenotypes in Bordetella bronchiseptica.

Bordetella bronchiseptica is a pathogenic bacterium that causes respiratory infections in mammals. Adhesins, toxins, and secretion systems necessary for infection are regulated by the two-component system BvgAS. When the BvgAS system is inactive, there is no transcription of virulence-activated genes, and virulence-repressed genes (vrg) are expressed. The regulation of some vrgs in B. bronchiseptica is dependent upon the virulence-activated gene bvgR. Although having a regulatory role, no DNA-binding domain is described for BvgR. Instead, it contains an EAL domain, usually found in cyclic-di-GMP (c-di-GMP)-specific phosphodiesterases. c-di-GMP is a bacterial second messenger that regulates multiple phenotypes in bacteria, including B. bronchiseptica. The current study aimed to deepen our knowledge about BvgR. We employed RNA-seq analysis to define the BvgR regulon, and then we investigated the phenotypes in which BvgR regulation might be involved such as biofilm formation, cytotoxicity, and virulence. Our result revealed that BvgR inhibits biofilm formation and flagellin expression in virulent phase. Although BvgR has long been considered a repressor protein, our results show that it also upregulates almost 100 genes. This regulation is likely indirect, as BvgR lacks a DNA-binding domain. Notably, among the upregulated genes, we identified 15 associated with the type three secretion system. Consistent with these findings, a B. bronchiseptica strain deficient in bvgR was less cytotoxic than the wild-type strain, elicited a milder immune response, and was less able to persist in the lower respiratory tract of mice.IMPORTANCEBordetella bronchiseptica is a harmful bacterium responsible for respiratory infections in mammals. Its ability to cause disease is tightly regulated by a system called BvgAS. In this study, we focused on understanding the role of a specific gene called bvgR in regulating B. bronchiseptica's virulence factors. Our findings revealed that BvgR, previously thought to primarily repress gene expression, actually plays a complex role in both activating and inhibiting various genes involved in bacterial virulence. This newfound understanding sheds light on the intricate mechanisms underlying B. bronchiseptica's ability to cause infections, providing valuable insights for developing strategies to combat these infections in humans and animals.

Collision frequencies across collision regimes in two-component systems

Agglomeration is the most important growth process in particle systems but modelling efforts typically assume mono-disperse primary particle distributions for the closure of the collision frequency that determines the growth rates. Real systems such as sooting flames, however, involve poly-disperse primary particle distributions. Also, systems with multiple components, where primary particles are of distinct but different sizes, cannot be treated as mono-disperse. Here, we introduce bi-disperse primary particle distributions and use Langevin dynamics simulations to develop closures for the collision kernels that are applicable over a wide range of agglomerate characteristics. The simulations cover fractal dimensions from 1.4 to 2.2, primary particle diameters from 5 nm to 50 nm, primary particle size ratios from 2 to 10 and agglomerates of up to a size of 200 primary particles with varying particle compositions. The Langevin dynamics simulations cover all collision regimes from ballistic to diffusive and allow to deduce expressions for the respective collision diameters, the hydrodynamic radii and the projected area as functions of particle characteristics. It is shown that existing expressions for the transition regime that were developed for the modelling of the collision kernel of spherical particles continue to hold for collision kernels of agglomerates in two-component systems under the condition that the collision diameters and drag coefficients are modelled accurately. An example ‘a posteriori’ simulation for a particle size ratio of 6 uses the population balance equation and demonstrates that bi-variate kernels are needed for the accurate prediction of the growth rates. Errors in predicted number density at the end of the simulations are less than 12 % while mono-variate kernels developed for mono-disperse primary particle systems overpredict the growth rate by 46%.

Both GacS-regulated lipopeptides and the type three secretion system contribute to Pseudomonas cichorii induced necrosis in lettuce and chicory

Pseudomonas cichorii SF1-54, the causal agent of lettuce midrib rot disease, produces lipopeptides cichofactins and cichopeptins which are important virulence factors. The GacS/GacA two-component system is well known to regulate production of lipopeptides in pseudomonads. Additionally, the functions of the type three secretion system (T3SS) in P. cichorii-plant interactions are not clarified. In this study, we investigated the role of the GacS-regulated lipopeptides and the T3SS in pathogenicity of P. cichorii SF1-54 on two host plants, chicory and lettuce, by constructing mutants in hrpL, which encodes the key sigma factor to control T3SS expression, and gacS. Compared with the wildtype, the hrpL mutant produced lipopeptides at a similar level but the gacS mutant was strongly impaired in lipopeptide production. The mutant deficient in hrpL did not significantly differ from the wildtype in virulence on chicory and lettuce. The gacS mutant exhibited significantly less symptoms on both host plants compared to the wildtype and the hrpL mutant. Intriguingly, the gacS hrpL-double mutant no longer produced lipopeptides, lost virulence and showed impaired colonization on chicory, but was still weakly virulent on lettuce. Thus, contribution of both the GacS-regulated lipopeptides and T3SS to virulence of P. cichorii SF1-54 is host plant dependent.

Analysis of antibiotic response in Clinical Wound Pseudomonas aeruginosa isolates: Unveiling Proteome Dynamics of tobramycin tolerant phenotype

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen, causing serious chronic infections. P. aeruginosa can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound P. aeruginosa isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and applied proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the P. aeruginosa genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.

Novel and reusable magnetic MOF nanocomposite coupled ionic liquid- promoted efficient chemical fixation of CO2 into α-alkylidene cyclic carbonates

Carbon dioxide as a C1 building block to synthesize α-alkylidene cyclic carbonates is an environmental and sustainable approach. In this work, we designed and synthesized a type of multifunctional magnetic MOF nanocomposite catalysts, which could realize the carboxylic cyclization of CO2 and propargylic alcohols into α-alkylidene cyclic carbonates under solvent-free conditions. Among all the prepared nanocomposites, the MnFe2O4@SiO2@Cu-MOF nanocomposite is the best in catalytic activity combined with the tetrabutylphosphonium acetate ([Bu4P]OAc) ionic liquid cocatalyst. The catalytic system MnFe2O4@SiO2@Cu-MOF/[Bu4P]OAc displayed excellent performance in catalyzing the carboxylic cyclization of CO2 and different propargylic alcohols, and a series of α-alkylidene cyclic carbonates were obtained in high to excellent yields (88 ∼ 98 %) under mild reaction conditions (0.2 MPa, 35 °C). In addition, the two-component catalytic system had high stability and reusability, and can be easily separated and reused up to six consecutive cycles without considerable decrease in catalytic activity. Moreover, using the ionic liquid [Bu4P]OAc as the cocatalyst, the nanocomposite had good substrate adaptability for the catalytic carboxylic cyclization, which opens interesting prospects for the development of new magnetic MOF nanocomposites as efficient heterogeneous catalysts for the chemical transformation of CO2 into value-added chemicals.

Unveiling the Coordinated Action of DesK/DesR and YvfT/YvfU to Control the Expression of an ABC Transporter in Bacillus subtilis.

Two-component systems (TCSs) are vital signal transduction pathways ubiquitous among bacteria, facilitating their responses to diverse environmental stimuli. In Bacillus subtilis, the DesK histidine kinase thermosensor, together with the response regulator DesR, constitute a TCS dedicated to membrane lipid homeostasis maintenance. This TCS orchestrates the transcriptional regulation of the des gene, encoding the sole desaturase in these bacteria, Δ5-Des. Additionally, B. subtilis possesses a paralog TCS, YvfT/YvfU, with unknown target gene(s). In this work, we show that YvfT/YvfU controls the expression of the yvfRS operon that codes for an ABC transporter. Interestingly, we found that this regulation also involves the action of DesK/DesR. Notably, opposite to des, yvfRS transcription is induced at 37°C and not at 25°C. Our invivo and invitro experiments demonstrate that both YvfU and DesR directly bind to the operon promoter region, with DesR exerting its control over yvfRS expression in its unphosphorylated state. Our study uncovers an intriguing case of cross-regulation where two homologous TCSs interact closely to finely tune gene expression in response to environmental cues. These findings shed light on the complexity of bacterial signal transduction systems and their critical role in bacterial adaptability.

It Takes Two to Make a Thing Go Right: Epistasis, Two-Component Response Systems, and Bacterial Adaptation

Understanding the interplay between genotype and fitness is a core question in evolutionary biology. Here, we address this challenge in the context of microbial adaptation to environmental stressors. This study explores the role of epistasis in bacterial adaptation by examining genetic and phenotypic changes in silver-adapted Escherichia coli populations, focusing on the role of beneficial mutations in two-component response systems (TCRS). To do this, we measured 24-hour growth assays and conducted whole-genome DNA and RNA sequencing on E. coli mutants that confer resistance to ionic silver. We showed recently that the R15L cusS mutation is central to silver resistance, primarily through upregulation of the cus efflux system. However, here we show that this mutation’s effectiveness is significantly enhanced by epistatic interactions with additional mutations in regulatory genes such as ompR, rho, and fur. These interactions reconfigure global stress response networks, resulting in robust and varied resistance strategies across different populations. This study underscores the critical role of epistasis in bacterial adaptation, illustrating how interactions between multiple mutations and how genetic backgrounds shape the resistance phenotypes of E. coli populations. This work also allowed for refinement of our model describing the role TCRS genes play in bacterial adaptation by now emphasizing that adaptation to environmental stressors is a complex, context-dependent process, driven by the dynamic interplay between genetic and environmental factors. These findings have broader implications for understanding microbial evolution and developing strategies to combat antimicrobial resistance.

Dependence on initial data for a stochastic modified two-component Camassa-Holm system

We study a stochastic modified two-component Camassa-Holm equation on R. We establish a local well-posedness result in the sense of Hadamard, i.e. existence, uniqueness and continuous dependence on initial data, as well as blow-up criteria for pathwise solutions in the Sobolev spaces Hs with s>3/2. Motivated by the work of Miao et al. (2024) [29], we show that the solution map y0↦y(t) defined by the corresponding Cauchy problem is weakly unstable, due to either a lack of strong stability in the exiting time or the absence of uniformly continuous dependence on the initial data.

Association of CovRS Two-Component Regulatory System with NADase Induction by Clindamycin Treatment in Streptococcus pyogenes.

Administration of high-dose clindamycin (CLI) and penicillin is recommended for the treatment of streptococcal toxic shock syndrome (STSS). However, CLI-resistant strains have been identified worldwide. In this study, some CLI-resistant strains demonstrated increased extracellular activity of the NAD-glycohydrolase (NADase) exotoxin following CLI treatment. These results support our previous conclusion that CLI-susceptible and CLI-resistant Streptococcus pyogenes strains exhibit CLI-dependent NADase induction. Furthermore, we investigated the mechanism of this phenomenon using 13 types of two-component sensor knockout strains derived from the CLI-susceptible strain 1529 that has a CLI-dependent NADase induction phenotype. Among the knockout strains, only 1529ΔcovS lost the phenotype. Additionally, 1529ΔspeB, 1529Δmga, and 1529Δrgg retained the CLI-dependent NADase induction phenotype. These findings indicate that CovS is related to this phenotype in a SpeB-independent manner.

PhoPQ-mediated lipopolysaccharide modification governs intrinsic resistance to tetracycline and glycylcycline antibiotics in Escherichia coli.

Elucidating the resistance mechanisms of clinically important antibiotics helps in maintaining the clinical efficacy of antibiotics and in the prescription of adequate antibiotic therapy. Although tetracycline and glycylcycline antibiotics are clinically important in combating multidrug-resistant Gram-negative bacterial infections, their mechanisms of resistance are not fully understood. Our research demonstrates that the E. coli PhoPQ two-component system affects resistance to tetracycline and glycylcycline antibiotics by controlling the expression of phosphoethanolamine transferase EptB, which catalyzes the modification of the inner core residue of lipopolysaccharide (LPS). Therefore, our findings highlight a novel resistance mechanism to tetracycline and glycylcycline antibiotics and the physiological significance of LPS core modification in E. coli.

Preparation and Characterization of Solid Dispersion of Sorafenib Tosylate for Enhancement of Bioavailability.

Solid dispersions have garnered significant attention as an effective strategy for enhancing the solubility and, consequently, the bioavailability of poorly water-soluble drugs. By forming solid dispersions of these drugs with water-soluble carriers, issues related to limited solubility have been mitigated, leading to improved dissolution rates. The core focus of solid dispersion technology revolves around the creation of two-component systems involving the drug and a polymer, where the successful dispersion and stabilization of the drug play pivotal roles in the formulation development process. As a result, this technology is widely acknowledged as a highly valuable approach for enhancing the dissolution properties of poorly water-soluble drugs. In recent years, a substantial body of knowledge has been amassed concerning solid dispersions. Nonetheless, their practical implementation in the commercial sphere remains somewhat limited. In the current investigation, we embarked on the preparation of a solid dispersion of sorafenib tosylate, with the primary objective of achieving the maximum release of the drug within the shortest possible timeframe. Our aim was to develop and optimize the solid dispersions of sorafenib tosylate in a manner that ensures the successful design and formulation of this system.

Imaging and proteomics toolkits for studying organelle contact sites.

Organelle contact sites are regions where two heterologous membranes are juxtaposed by molecular tethering complexes. These contact sites are important in inter-organelle communication and cellular functional integration. However, visualizing these minute foci and identifying contact site proteomes have been challenging. In recent years, fluorescence-based methods have been developed to visualize the dynamic physical interaction of organelles while proximity labeling approaches facilitate the profiling of proteomes at contact sites. In this review, we explain the design principle for these contact site reporters: a dual-organelle interaction mechanism based on how endogenous tethers and/or tethering complexes localize to contact sites. We classify the contact site reporters into three categories: (i) single-protein systems, (ii) two-component systems with activated reporter signal upon organelle proximity, and (iii) reporters for contact site proteomes. We also highlight advanced imaging analysis with high temporal-spatial resolution and the use of machine-learning algorithms for detecting contact sites.

A conserved two-component system senses intracellular iron levels and regulates redox balance in Mycobacterium spp.

For bacteria, an intricate coordination between sensing and regulating iron levels and managing oxidative stress is required as their levels are tightly interlinked. While various oxidative stress and heme-based redox sensors have been reported for both pathogenic and non-pathogenic bacteria, the mechanisms governing the modulation of intracellular iron levels in response to changes in redox status remain unclear. In this study, a gene-inactivated strain of mycobacterial sensor kinase PdtaS showed dysregulated expression of genes associated with iron metabolism, including Fe-S clusters, NADH dehydrogenases, and iron uptake. The strain showed poor growth in nutrient-limiting conditions, a defect rescuable by heme but not by Fe3+ supplementation. This observation was associated with the PAS domain of the PdtaS sensor kinase. Biochemical and biophysical experiments established heme-binding to the PAS domain and its inhibitory effect on PdtaS auto-kinase activity, suggesting that the absence of heme induces activation of this sensor kinase. Interestingly, despite having an endogenous heme biosynthetic pathway or even external heme supplementation, the ∆pdtaS mutant exhibited persistent low intracellular iron levels concomitant with elevated oxidative stress. Antioxidant supplementation mitigated growth defects, emphasizing the link between oxidative stress, intracellular iron levels, and PdtaS activity. RNA-IP identified key targets associated with redox homeostasis and iron metabolism as targets of the PdtaR response regulator. The study proposes a novel role for the PdtaS-PdtaR TCS in sensing heme, regulation of intracellular iron levels, and redox balance.IMPORTANCEThe research article investigates the intricate interplay between bacteria's ability to take and utilize iron without inducing excess iron's toxic effects, including oxidative stress. The study shows that bacteria achieve this by sensing intracellular iron available as heme through a sensory protein PdtaS, which turns off when heme is in excess and prevents iron uptake and iron efflux. The process shields bacteria from generating Fe-dependent free radicals and allows it to maintain viability. The absence of sensor kinase abrogates all these processes, increasing bacteria susceptibility to ROS and thereby slowing growth. This feature of the sensor kinase PdtaS makes it an attractive co-therapeutic target for tuberculosis therapy, where its inhibition will prevent iron uptake, even in the presence of low iron, thereby halting bacterial proliferation.

Atypical Mycobacterium abscessus BlaRI Ortholog Mediates Regulation of Energy Metabolism but Not β-Lactam Resistance.

Mycobacterium abscessus (Mab) is highly drug resistant, and understanding regulation of antibiotic resistance is critical to future antibiotic development. Regulatory mechanisms controlling Mab's β-lactamase (BlaMab) that mediates β-lactam resistance remain unknown. S. aureus encodes a prototypical protease-mediated two-component system BlaRI regulating the β-lactamase BlaZ. BlaR binds extracellular β-lactams, activating an intracellular peptidase domain which cleaves BlaI to derepress blaZ. Mycobacterium tuberculosis (Mtb) encodes homologs of BlaRI (which we will denote as BlaIR to reflect the inverted gene order in mycobacteria) that regulate not only the Mtb β-lactamase, blaC, but also additional genes related to respiration. We identified orthologs of blaIRMtb in Mab and hypothesized that they regulate blaMab. Surprisingly, neither deletion of blaIRMab nor overexpression of only blaIMab altered blaMab expression or β-lactam susceptibility. However, BlaIMab did bind to conserved motifs upstream of several Mab genes involved in respiration, yielding a putative regulon that partially overlapped with BlaIMtb. Prompted by evidence that respiration inhibitors including clofazimine induce the BlaI regulon in Mtb, we found that clofazimine triggers induction of blaIRMab and its downstream regulon. Highlighting an important role for BlaIRMab in adapting to disruptions in energy metabolism, constitutive repression of the BlaIMab regulon rendered Mab highly susceptible to clofazimine. In addition to our unexpected findings that BlaIRMab does not regulate β-lactam resistance, this study highlights the novel role of mycobacterial BlaRI-type regulators in regulating electron transport and respiration.

The PhoBR two-component system upregulates virulence in Aeromonas dhakensis C4–1

The PhoBR two-component system is a critical regulator of virulence in many bacterial pathogens, but its role in Aeromonas dhakensis remains poorly characterized. In this study, three markerless knockout mutants (ΔphoB, ΔphoR and ΔphoBR) were constructed to explore the function of PhoBR. Under low phosphate conditions (0.2 mM Pi), physiological analyses showed that all mutants exhibited defects in growth, motility, biofilm formation and resistance to acid, with ΔphoBR also showing resistance to H2O2, while no significant changes were observed in extracellular protease secretion. Cell adhesion and zebrafish infection assays indicated that PhoBR is crucial for A. dhakensis adhesion to EPC cells. The ΔphoBR mutant had the highest LD50 value (1.77 × 107 CFU per fish), which was 38.15 times higher than that of the wild type strain. Additionally, ΔphoBR conferred the highest relative percent survival (RPS) of 61.5 % in zebrafish following two immunizations. These findings suggest that PhoBR positively regulates virulence in A. dhakensis and that the ΔphoBR mutant could serve as a potential vaccine candidate for the prevention of aeromoniasis in aquaculture.