Production Of Signaling Molecules Research Articles

Microbial Primer: what is the stringent response and how does it allow bacteria to survive stress?

The stringent response is a conserved bacterial stress response that allows bacteria to alter their activity and survive under nutrient-limiting conditions. Activation of the stringent response is characterized by the production of intracellular signalling molecules, collectively termed (p)ppGpp, which interact with multiple targets inside bacterial cells. Together, these interactions induce a slow growth phenotype to aid bacterial survival by altering the transcriptomic profile of the cell, inhibiting ribosome biosynthesis and targeting enzymes involved in other key metabolic processes.

The LuxO-OpaR quorum-sensing cascade differentially controls Vibriophage VP882 lysis-lysogeny decision making in liquid and on surfaces.

Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to synchronize collective behaviors. QS relies on the production, release, and group-wide detection of extracellular signaling molecules called autoinducers. Vibrios use two QS systems: the LuxO-OpaR circuit and the VqmA-VqmR circuit. Both QS circuits control group behaviors including biofilm formation and surface motility. The Vibrio parahaemolyticus temperate phage φVP882 encodes a VqmA homolog (called VqmAφ). When VqmAφ is produced by φVP882 lysogens, it binds to the host-produced autoinducer called DPO and launches the φVP882 lytic cascade. This activity times induction of lysis with high host cell density and presumably promotes maximal phage transmission to new cells. Here, we explore whether, in addition to induction from lysogeny, QS controls the initial establishment of lysogeny by φVP882 in naïve host cells. Using mutagenesis, phage infection assays, and phenotypic analyses, we show that φVP882 connects its initial lysis-lysogeny decision to both host cell density and whether the host resides in liquid or on a surface. Host cells in the low-cell-density QS state primarily undergo lysogenic conversion. The QS regulator LuxO~P promotes φVP882 lysogenic conversion of low-cell-density planktonic host cells. By contrast, the ScrABC surface-sensing system regulates lysogenic conversion of low-cell-density surface-associated host cells. ScrABC controls the abundance of the second messenger molecule cyclic diguanylate, which in turn, modulates motility. The scrABC operon is only expressed when its QS repressor, OpaR, is absent. Thus, at low cell density, QS-dependent derepression of scrABC drives lysogenic conversion in surface-associated host cells. These results demonstrate that φVP882 integrates cues from multiple sensory pathways into its lifestyle decision making upon infection of a new host cell.

Effects of chlorogenic acid-grafted-chitosan on biofilms, oxidative stress, quorum sensing and c-di-GMP in Pseudomonas fluorescens

This study determined the inhibitory mechanism as well as anti-biofilm activity of chlorogenic acid-grafted-chitosan (CS-g-CA) against Pseudomonas fluorescens (P. fluorescens) in terms of biofilm content, oxidative stress, quorum sensing and cyclic diguanosine monophosphate (c-di-GMP) concentration, and detected the changes in the expression levels of related genes by quantitative real-time PCR (qRT-PCR). Results indicated that treatment with sub-concentrations of CS-g-CA for P. fluorescens led to reduce the biofilm size of large colonies, decrease the content of biofilm and extracellular polymers, weaken the motility and adhesion of P. fluorescens. Moreover, CS-g-CA resulted in higher ROS levels, diminished catalase activity (CAT), and increased superoxide dismutase (SOD) in P. fluorescens. CS-g-CA reduced the production of quorum-sensing signaling molecules (AHLs) and the concentration of c-di-GMP in bacteria. Genes for flagellar synthesis (flgA), the resistance to stress (rpoS and hfq), and pde (phosphodiesterases that degrade c-di-GMP) were significantly down-regulated as determined by RT-PCR. Overall, CS-g-CA leads to the accumulation of ROS in bacteria via P. fluorescens environmental resistance genes and decreases the activity of enzymes in the bacterial antioxidant system, and interferes with the production and reception of quorum-sensing signaling molecules and the synthesis of c-di-GMP in P. fluorescens, which regulates the generation of biofilms.

Effects of exogenous autoinducer-2 precursor on the ability of Lactiplantibacillus plantarum SH7 to degrade biogenic amines: In vitro and in a dry-sausage model

The effects of the exogenous autoinducer-2 (AI-2) precursor, 4,5-dihydroxy-2,3-pentanedione (DPD; 0, 300, 600, and 900 nmol/L), on the degradation capacity of Lactiplantibacillus plantarum SH7 toward biogenic amines (BA) were investigated both in vitro and in a dry-sausage model. L. plantarum SH7 exhibited a significant increase in BA-degradation capacity (putrescine, cadaverine, and histamine) under DPD treatments compared to the absence of DPD treatment in vitro. Additionally, a significant increase in the production of AI-2 signal molecule and diamine oxidase in L. plantarum SH7 was observed with the addition of exogenous DPD (p < 0.05). In the dry-sausage model, the contents of BAs (tryptamine, 2-phenethylamine, putrescine, cadaverine, and histamine) significantly decreased with the addition of DPD (p < 0.05). Meanwhile, the sausage with 600-nM DPD exhibited the lowest cadaverine content (11.13 mg/kg), while the sausage with 900-nM DPD exhibited the lowest tryptamine, putrescine, and histamine contents (12.44, 11.15, and 13.21 mg/kg, respectively). Results suggest that an optimal concentration of exogenous DPD can enhance the capacity of L. plantarum SH7 to degrade BAs and that the degradation of BAs by L. plantarum SH7 may be dependent on the LuxS/AI-2 quorum sensing (QS) system. This study provides the theoretical basis for the development of functional starter cultures of lactic acid bacteria with BA-degradation ability based on targeted LuxS/AI-2 QS system regulation and reducing the accumulation of BAs in fermented meat products.

Different reactions of wheat, maize, and rice plants to putrescine treatment

Polyamines play an important role in growth and differentiation by regulating numerous physiological and biochemical processes at the cellular level. In addition to their roborative effect, their essential role in plant stress responses has been also reported. However, the positive effect may depend on the fine-tuning of polyamine metabolism, which influences the production of free radicals and/or signalling molecules. In the present study, 0.3 mM hydroponic putrescine treatment was tested in wheat, maize, and rice in order to reveal differences in their answers and highlight the relation of these with polyamine metabolism. In the case of wheat, the chlorophyll content and the actual quantum yield increased after putrescine treatment, and no remarkable changes were detected in the stress markers, polyamine contents, or polyamine metabolism-related gene expression. Although, in maize, the actual quantum yield decreased, and the root hydrogen peroxide content increased, no other negative effect was observed after putrescine treatment due to activation of polyamine oxidases at enzyme and gene expression levels. The results also demonstrated that after putrescine treatment, rice with a higher initial polyamine content, the balance of polyamine metabolism was disrupted and a significant amount of putrescine was accumulated, accompanied by a detrimental decrease in the level of higher polyamines. These initial differences and the putrescine-induced shift in polyamine metabolism together with the terminal catabolism or back-conversion-induced release of a substantial quantity of hydrogen peroxide could contribute to oxidative stress observed in rice.

A new approach to understanding the role of gasotransmitters in the development of chronic generalized periodontitis

Relevance. Recent studies investigating the role of the microbial gaseous substances (O2, N2, CO2, CH4, NO, CO, H2S) indicate not only in the regulation of the host's metabolic activity and the functioning of its nervous system, in particular but also their participation the pathogenesis of some diseases. However, there is scarce data in the national and international literature on the production of gas signaling molecules by the oral microbiota (Streptococcus spp. and Staphylococcus spp.) and the changes in the gas composition during the development of chronic inflammatory periodontal diseases.Material and methods. The study included 69 people. The main group included 36 patients aged 35 to 67 years with clinically confirmed moderate chronic generalized periodontitis. The control group included 33 patients aged 27 to 55 without periodontal disease. The samples from the back of the tongue were the study material. The gas chromatography determined the production of gas signaling molecules using the Khromatek-crystal 5000.2 device. The measurement of the amount of released gases was in % (for O2, N2) and ppm (0.001 mg/mL) for other gas molecules (CO2, CH4, NO, CO, H2S).Results. The metabolic activity of streptococci only for the production of NO (p = 0.002) and CO (p = 0.008) appeared to have a statistically significant difference. In periodontal inflammation, there was practically no NO emission by Streptococci spp., and the concentration of CO was ten times higher than in the group of healthy individuals. The difference in the number of other signaling gas molecules was not statistically significant (p &gt; 0.05) in healthy people and patients with chronic generalized periodontitis.In the production of gasotransmitters among Staphylococcus spp., N2 production (p = 0.007, increasing in the comparison group) was statistically significantly different. As in the streptococcal sampling, the amount of CO significantly increased in periodontal inflammation. Certain species of staphylococci showed a significant decrease in the production of the entire gas molecule range in the main group. At the same time, unlike Streptococcus spp., Staphylococcus spp. absorbed a much higher amount of nitric oxide in chronic periodontitis.Conclusion. In patients with chronic generalized periodontitis and inflammation, the oral microbiota is poorly active and produces a low concentration of gasotransmitters, so they cannot participate in inflammatory process reduc-tion, thereby contributing to the progression of the disease.

A brainstem–hypothalamus neuronal circuit reduces feeding upon heat exposure

Empirical evidence suggests that heat exposure reduces food intake. However, the neurocircuit architecture and the signalling mechanisms that form an associative interface between sensory and metabolic modalities remain unknown, despite primary thermoceptive neurons in the pontine parabrachial nucleus becoming well characterized1. Tanycytes are a specialized cell type along the wall of the third ventricle2 that bidirectionally transport hormones and signalling molecules between the brain’s parenchyma and ventricular system3–8. Here we show that tanycytes are activated upon acute thermal challenge and are necessary to reduce food intake afterwards. Virus-mediated gene manipulation and circuit mapping showed that thermosensing glutamatergic neurons of the parabrachial nucleus innervate tanycytes either directly or through second-order hypothalamic neurons. Heat-dependent Fos expression in tanycytes suggested their ability to produce signalling molecules, including vascular endothelial growth factor A (VEGFA). Instead of discharging VEGFA into the cerebrospinal fluid for a systemic effect, VEGFA was released along the parenchymal processes of tanycytes in the arcuate nucleus. VEGFA then increased the spike threshold of Flt1-expressing dopamine and agouti-related peptide (Agrp)-containing neurons, thus priming net anorexigenic output. Indeed, both acute heat and the chemogenetic activation of glutamatergic parabrachial neurons at thermoneutrality reduced food intake for hours, in a manner that is sensitive to both Vegfa loss-of-function and blockage of vesicle-associated membrane protein 2 (VAMP2)-dependent exocytosis from tanycytes. Overall, we define a multimodal neurocircuit in which tanycytes link parabrachial sensory relay to the long-term enforcement of a metabolic code.

Review of quorum-quenching probiotics: A promising non-antibiotic-based strategy for sustainable aquaculture.

The emergence of antibiotic-resistant bacteria (ARBs) and genes (ARGs) in aquaculture underscores the urgent need for alternative veterinary strategies to combat antimicrobial resistance (AMR). These measures are vital to reduce the likelihood of entering a post-antibiotic era. Identifying environmentally friendly biotechnological solutions to prevent and treat bacterial diseases is crucial for the sustainability of aquaculture and for minimizing the use of antimicrobials, especially antibiotics. The development of probiotics with quorum-quenching (QQ) capabilities presents a promising non-antibiotic strategy for sustainable aquaculture. Recent research has demonstrated the effectiveness of QQ probiotics (QQPs) against a range of significant fish pathogens in aquaculture. QQ disrupts microbial communication (quorum sensing, QS) by inhibiting the production, replication, and detection of signalling molecules, thereby reducing bacterial virulence factors. With their targeted anti-virulence approach, QQPs have substantial promise as a potential alternative to antibiotics. The application of QQPs in aquaculture, however, is still in its early stages and requires additional research. Key challenges include determining the optimal dosage and treatment regimens, understanding the long-term effects, and integrating QQPs with other disease control methods in diverse aquaculture systems. This review scrutinizes the current literature on antibiotic usage, AMR prevalence in aquaculture, QQ mechanisms and the application of QQPs as a sustainable alternative to antibiotics.

N- acyl homoserine lactones (AHLs) type signal molecules produced by rhizobacteria associated with plants that growing in a metal(oids) contaminated soil: A catalyst for plant growth

The present study explores the potential of rhizobacteria isolated from Baccharis linearis and Solidago chilensis in metal(loid)-contaminated soil for producing N-acyl-homoserine lactones (AHLs)-type signal molecules and promoting plant growth. A total of 42 strains were isolated, four demonstrating the production of AHL-type signal molecules. Based on 16S rRNA gene sequencing analyses and MALDI-TOF analyses, these four isolates were identified as belonging to the Pseudomonas genus, specifically P. brassicacearum, P. frederickberguensis, P. koreensis, and P. orientalis. The four AHL-producing strains were evaluated for metal(loid)s tolerance, their plant growth promotion traits, AHL quantification, and their impact on in vitro Lactuca sativa plant growth.The study found that four strains exhibited high tolerance to metal(loid)s, particularly As, Cu, and Zn. Additionally, plant growth-promoting traits were detected in AHL-producing bacteria, such as siderophore production, ammonia production, ACC deaminase activity, and P solubilization. Notably, AHL production varied among strains isolated from B. linearis, where C7-HSL and C9-HSL signal molecules were detected, and S. chilensis, where only C7-HSL signal molecules were observed. In the presence of copper, the production of C7-HSL and C9-HSL significantly decreased in B. linearis isolates, while in S. chilensis isolates, C7-HSL production was inhibited. Further, when these strains were inoculated on lettuce seeds and in vitro plants, a significant increase in germination and plant growth was observed. Mainly, the inoculation of P. brassicacearum and P. frederickberguensis led to extensive root hair development, significantly increasing length and root dry weight.Our results demonstrate that rhizospheric strains produce AHL molecules and stimulate plant growth, primarily through root development. However, the presence of copper reduces the production of these molecules, potentially affecting the root development of non-metalloid tolerant plants such as S. chilensis, which would explain its low population in this hostile environment.

Phenolic Compounds from Ocimum basilicum Revealed as Antibacterial by Experimental and Computational Screening‐Based Studies against Oral Infections

Dental infections are initiated by Streptococcus sanguinis and Streptococcus mutans through the biofilm formation. In streptococcal, the ComA enzyme catalyzes the first step to produce signalling molecules in quorum sensing that influence biofilm formation. Ocimum basilicum is one of the natural sources which is potential for antibacterial agent. This study is aimed to isolate phenolic compounds from O. basilicum leaves with antibacterial activity against oral pathogens by experimental and computational screening studies. Isolation of phenolic compounds 1–4 was carried out by bioactivity guided using column chromatography. Methylparaben (1), methyl protocatechuate (2), caffeic acid (3), and methyl caffeate (4) were characterized by 1D and 2D‐NMR. The antibacterial activity was tested by disk diffusion, microdilution methods. 1 (625 μg·mL−1) provided moderate activity, and 2 (156.3 μg·mL−1) provided strong activity against S. mutans. 3 (125 μg·mL−1) and 4 (312.5 μg·mL−1) provided strong activity against S. sanguinis. In addition, phenolic compounds and their derivatives were predicted against ComA using Autodock 4.0 tools. The activity correlated positively with in silico tests against ComA where 2 (−7.69 kcal·mol−1) had a stronger binding affinity than 1 (−6.64 kcal·mol−1). 3 (−6.86 kcal·mol−1) provided a slightly stronger bond strength than 4 (−6.65 kcal·mol−1). The strongest binding affinity of hydroxybenzoic acid derivatives is 3,5‐dihydroxybenzoic acid (−8.10 kcal·mol−1). In addition, the strongest binding affinity of hydroxycinnamic acid derivatives is chlorogenic acid (−9.16 kcal·mol−1). The pharmacokinetics were predicted by ADMET analysis, and methyl caffeate (4) showed the best potential as a drug compound. The conclusion is that phenolic compounds isolated from O. basilicum have potential as natural antibacterials. In silico tests also showed the relationship between the structure and activity by the presence of the hydroxyl group in hydroxybenzoic acid derivatives and the presence of an alkyl group in the unsaturated side chain in hydroxycinnamic acid derivatives.

Fatty Acid Metabolism: A New Perspective in Breast Cancer Precision Therapy.

Breast cancer has a special tumor microenvironment compared to other solid tumors, which is usually surrounded by a large number of adipocytes that can produce and secrete fatty acids and adipokines. Adipocytes have a remodeling effect on breast cancer lipid metabolism, while fatty acids and lipid droplets can make breast cancer cells more aggressive. Lipid metabolism, especially the synthesis of fatty acids, is an important cellular process for membrane biosynthesis, energy storage, and signal molecule production. Therefore, blocking the lipid supply to cancer cells or changing the lipid composition has an important impact on the signal transmission and cell proliferation of cancer cells. Alterations in lipid availability can also affect cancer cell migration, induction of angiogenesis, metabolic symbiosis, evasion of immune surveillance, and cancer drug resistance. Fatty acid synthesis and metabolism have received extensive attention as potential targets for cancer therapy, and studies on modulating the tumor lipid microenvironment to improve the sensitivity of antitumor drugs have also been discussed; however, strategies to target these processes have not been translated into clinical practice.

Modeling downstream impact of a quorum sensing system of Pseudomonas aeruginosa in colony spreading

A single bacterial colony typically consists of two or more individual cells. To further growth, the cells within the colony need to coordinate through Quorum Sensing (QS), which serves as a communication mechanism between cells. The Quorum Sensing signalling system in Pseudomonas aeruginosa is known to involve multiple control components, notably the las and rhl systems. The las system controls the rhl system in a hierarchical signalling cascade, and the rhl system regulates certain gene expressions, including the rhlA and rhlB genes. Transcription of rhlA and rhlB results in the production of the enzyme rhamnosyltransferase, which leads to the biosurfactant production identified as rhamnolipids. In this paper, we investigate how the las system controls the rhl system to influence rhamnolipids production. We illustrate how the pulsed production of signal molecules in the las system affects the rhl system, leading to an increase in rhamnolipids concentration. Rhamnolipids play a crucial role in bacterial spreading. We employ a simple symmetric diffusion model to simulate colony spreading and demonstrate how bacteria spread from the centre of the colony. Our findings shed light on the interaction between the las and rhl systems, which may enable cells to trigger rhamnolipids production at the edge of the bacterial colony.

From Pain Relief to Cancer Defense: The Promise of NSAIDs

NSAIDs (Non-Steroidal Anti-inflammatory Drugs) are commonly used to treat various types of pain and inflammation. In recent decades, extensive scientific research has been conducted to examine the use of NSAIDs in the treatment and prevention of cancer. Chronic inflammation has been linked to various cancer types, suggesting that prolonged inflammation can promote genetic mutations and accelerate their accumulation within cells. The COX pathway, short for Cyclooxygenase pathway, is a critical biochemical pathway in the human body involved in the production of important signaling molecules called prostaglandins. Key factors like COX enzymes and cytokines are important in the development and progression of inflammation-induced cancer. Angiogenesis occurs during inflammation, and it plays a major role in cancer development and metastasis. NSAIDs inhibit this process, which may also contribute to their anticancer effects. This review highlights the potential of NSAIDs, particularly aspirin, ibuprofen, and celecoxib, to influence various aspects of tumor behavior. Although promising, further rigorous studies are needed to establish their clinical efficacy and safety in diverse cancer scenarios. The use of NSAIDs as adjunctive therapies along with conventional treatments presents a promising avenue for enhancing cancer management strategies.

Paradigm shift: Bacterial quorum sensing and possible control targets

QS Designates a cell-to-cell communication process that enables bacteria to collectively modify their behaviour in response to changes in the cell density and species composition of the surrounding microbial community. These processes involve the production, release and group-wide detection of extracellular signalling molecules, which are generically called Autoinducers (AIs). It controls various genes which are responsible for various phenotypes like bioluminescence, the secretion of virulence factors, and the formation of biofilms in bacteria. Quorum quenching inhibits QS and the substances that inhibit it are called quorum sensing inhibitors. Several chemical compounds and enzymes mediate inhibition of QS, such as, lactonases, acylases and oxidoreductases. Other than these, there are some non-enzymatic methods for quorum quenching and also some plant phytochemicals have been found to inhibit it. Blocking of QS by QS Inhibition (QSI) may play an important role to disrupt biofilm formation in a device associated infection and chronic drug resistant infection. More researches are required in this area related with QS and QSI. However, some chemicals have been found to be mimicking the quorum sensing AIs activities like serotonin and rosmaric acid.

Ultrabright organic fluorescent probe for quantifying the dynamics of cytosolic/nuclear lipid droplets

Lipid droplets (LDs) are extremely active organelles that play a crucial role in energy metabolism, membrane formation, and the production of lipid-derived signaling molecules by regulating lipid storage and release. Nevertheless, directly limited by the lack of superior fluorescent probes, studies of LDs dynamic motion velocity have been rarely reported, especially for nuclear LDs. Herein, a novel organic fluorescent probe Lipi-Bright has been rationally developed based on bridged cyclization of distyrylbenzene. The fully ring-fused molecule structure endows the probe with high photostability. Moreover, this new fluorescent probe displays the features of excellent LDs staining specificity as well as ultrahigh fluorescence brightness. Lipi-Bright labeled LDs was dozens of times brighter than representative probes BODIPY 493/503 or Nile Red. Consequently, by in-situ time-lapse fluorescence imaging, the dynamics of LDs have been quantitatively studied. For instance, the velocities of cytosolic LDs (37 ± 15 nm/s) are found to be obviously faster than those of nuclear LDs (24 ± 4 nm/s), and both the cytosolic LDs and the nuclear LDs would be moved faster or slower depend on the various stimulations. Overall, this work providing plentiful information on LDs dynamics will greatly facilitate the in-depth investigation of lipid metabolism.

Natural silencing of quorum-sensing activity protects Vibrio parahaemolyticus from lysis by an autoinducer-detecting phage.

Quorum sensing (QS) is a chemical communication process that bacteria use to track population density and orchestrate collective behaviors. QS relies on the production, accumulation, and group-wide detection of extracellular signal molecules called autoinducers. Vibriophage 882 (phage VP882), a bacterial virus, encodes a homolog of the Vibrio QS receptor-transcription factor, called VqmA, that monitors the Vibrio QS autoinducer DPO. Phage VqmA binds DPO at high host-cell density and activates transcription of the phage gene qtip. Qtip, an antirepressor, launches the phage lysis program. Phage-encoded VqmA when bound to DPO also manipulates host QS by activating transcription of the host gene vqmR. VqmR is a small RNA that controls downstream QS target genes. Here, we sequence Vibrio parahaemolyticus strain O3:K6 882, the strain from which phage VP882 was initially isolated. The chromosomal region normally encoding vqmR and vqmA harbors a deletion encompassing vqmR and a portion of the vqmA promoter, inactivating that QS system. We discover that V. parahaemolyticus strain O3:K6 882 is also defective in its other QS systems, due to a mutation in luxO, encoding the central QS transcriptional regulator LuxO. Both the vqmR-vqmA and luxO mutations lock V. parahaemolyticus strain O3:K6 882 into the low-cell density QS state. Reparation of the QS defects in V. parahaemolyticus strain O3:K6 882 promotes activation of phage VP882 lytic gene expression and LuxO is primarily responsible for this effect. Phage VP882-infected QS-competent V. parahaemolyticus strain O3:K6 882 cells lyse more rapidly and produce more viral particles than the QS-deficient parent strain. We propose that, in V. parahaemolyticus strain O3:K6 882, constitutive maintenance of the low-cell density QS state suppresses the launch of the phage VP882 lytic cascade, thereby protecting the bacterial host from phage-mediated lysis.

Cinnamaldehyde reduces Yersinia enterocolitica activity and biofilm formation by altering membrane permeability and signalling

Yersinia enterocolitica is a foodborne pathogen that can infect food not only in its planktonic state but also as a biofilm on contact surfaces, making recurrent infections difficult to control. This article discusses various ways in which cinnamaldehyde can combat Y. enterocolitica. These include its effects on the permeability, integrity, and amino acid structure of cell membranes as well as on intracellular energy metabolism. We also investigated the effects of cinnamaldehyde on the formation of biofilms by Y. enterocolitica, including interference with quorum-sensing pathways, bacterial motility and adhesion, extracellular polymer production, and metabolic activity within the biofilm. Our results demonstrated that the minimum concentration of cinnamaldehyde required to effectively inhibit Y. enterocolitica is 0.625 mg/mL. It causes cell death by damaging the membrane, disrupting oxidative respiratory processes, interfering with cellular homeostasis, and disrupting cell function. Correspondingly, a concentration of 0.078 mg/mL cinnamaldehyde can suppress the production of biofilm and signalling molecules. This research aims to explore the mechanism by which cinnamaldehyde inhibits the activity of Y. enterocolitica and its biofilm, with the goal of providing more insight into whether cinnamaldehyde can be used as a natural, plant-based preservative and anti-biofilm agent.

Functional Characterization of Allelic Variations of Human Cytochrome P450 2C8 (V181I, I244V, I331T, and L361F).

The human cytochrome P450 2C8 is responsible for the metabolism of various clinical drugs as well as endogenous fatty acids. Allelic variations can significantly influence the metabolic outcomes. In this study, we characterize the functional effects of four nonsynonymous single nucleotide polymorphisms *15, *16, *17, and *18 alleles recently identified in cytochrome P450 2C8. The recombinant allelic variant enzymes V181I, I244V, I331T, and L361F were successfully expressed in Escherichia coli and purified. The steady-state kinetic analysis of paclitaxel 6-hydroxylation revealed a significant reduction in the catalytic activities of the V181I, I244V, and L361F variants. The calculated catalytic efficiency (kcat/Km) of these variants was 5-26% of that of the wild-type enzyme. The reduced activities were due to both decreased kcat values and increased Km values of the variants. The epoxidation of arachidonic acid by the variants was analyzed. The L361F variant only exhibited 4-6% of the wild-type catalytic efficiency in ω-9- and ω-6-epoxidation reactions to produce 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET, respectively. These reductions were mainly due to a decrease in the kcat value of the L361F variant. The binding titration analysis of paclitaxel and arachidonic acid showed that all variants had similar affinities to those of the wild-type (10-14 μM for paclitaxel and 20-49 μM for arachidonic acid). The constructed paclitaxel docking model of the variant enzyme suggests that the L361F substitution leads to the incorrect orientation of paclitaxel in the active site, with the 6'C of paclitaxel displaced from the productive catalytic location. This study suggests that individuals carrying the newly identified P450 2C8 allelic variations are likely to have an altered metabolism of clinical medicines and production of fatty acid-derived signal molecules.

Effects of deletion of siderophore biosynthesis gene in Pseudomonas fragi on quorum sensing and spoilage ability

Siderophores are important factors in the spoilage process of Pseudomonas fragi, considered to be one of the main spoilage bacterium of tuna, and the secretion of siderophores is regulated by quorum sensing (QS). This study aimed to construct a mutant with the deletion of the siderophore synthetase gene of P. fragi (MS-10), and to explore its effects on the growth, QS, and spoilage potential of P. fragi. The results showed that the deletion of the siderophore biosynthesis gene slowed down the growth rate of the strain. The apoptosis rate increased by 27.7 % compared with that of the wild-type strain at 4 °C for 48 h. Biofilm formation, extracellular protease expression, and signal molecule production were all significantly lower in the mutant strain compared with the wild-type strain. The total viable count and the histamine content showed that the tuna sterile fish block inoculated with the wild-type strain exceeded the acceptable standards by 5 days and was completely spoiled by 7 days, whereas the mutant strain exceeded the acceptable standards by 6 days and was completely spoiled by 9 days. The pH, texture, and other indicators showed that the variation range of the mutant strain was lower than that of the wild-type strain. The deletion of the siderophore biosynthesis gene reduced the spoilage ability of P. fragi. Based on the results, the development of novel preservation agents targeting the control of the siderophore biosynthesis gene could be a new idea for the preservation of aquatic products.

SCREENING OF MARINE BACTERIUM VIBRIO ALGINOLYTICUS STRAIN AS05 FOR THE PRODUCTION OF N-ACYL HOMOSERINE LACTONE-BASED QUORUM SENSING SIGNALING MOLECULES

Marine environments, including aquatic and coastal environments, are highly prevalent of marine bacteria with the highest levels of Vibrio species. Vibrios play a vital role in marine ecology associated with carbon and energy acquisition. The density-dependent quorum sensing (QS) system may regulate certain biological activities in marine bacteria. QS is a conversation system utilized by many bacterial communities to communicate and coordinate through different signalling molecules. N-acyl homoserine lactones (AHLs) are the most important QS signalling molecules widely produced by Gram-negative bacteria. This study aimed to investigate the detection and Identification of AHL-based QS signalling molecules produced by marine bacterium V. alginolyticus strain AS05 isolated from marine water of the Arabian Sea, Karachi, Pakistan. Marine medium Zobell-2216 was used to isolate bacterial strains. Moreover, 16S rRNA analysis was applied to identify AS05 strain. Agar plate bioassay was used to detect the production of AHL signalling molecules using Chromobacterium violaceum CV026 as a biosensor. The Identification of AHLs was made by reversed-phase thin-layer chromatography (RP-TLC) analysis. The NCBI-blast results revealed the identification of the isolated bacterial strain as Vibrio alginolyticus strain AS05 (OQ130030) member of the family of Vibrionaceae under the class of Gammaproteobacteria. The results of agar plate bioassay using CV026 as a biosensor strain revealed highly positive reactions for producing AHL signalling molecules. Moreover, two AHL molecules produced by AS05 bacterial strain were identified as C6-HSL and C-8HSL based on TLC analysis. This study reveals the detection and Identification of two different AHL signalling molecules produced by V. alginolyticus AS05 isolated from marine water of the Arabian Sea, Karachi, Pakistan. This study provides insight into investigating quorum-sensing signalling molecules in the Arabian Sea, Karachi, Pakistan, and marine bacterial species.