Quorum sensing (QS) can regulate the production of multiple functional factors in bacteria, but the process of identifying its regulatory targets is very complex and labor-intensive. In this study, an efficient and rapid method to find QS targets through prediction was used. The genome of Lactiplantibacillus plantarum (L. plantarum) L3 was sequenced and characterized, and then linked the L. plantarum L3 genome to the STRING database for QS system regulatory target prediction. A total of 3,167,484 base pairs (bps) were examined from the genome of L. plantarum L3, and 30 QS-related genes were discovered (including luxS). The STRING database prediction indicated that the 30 QS-related genes are mainly involved in the regulation of nine metabolic pathways. Furthermore, metE, metK, aroB, cysE, and birA1 were predicted to be regulatory targets of the LuxS/AI-2 QS system, and these five targets were validated based on quantitative real-time PCR and content determination. Successful elucidation of the LuxS/AI-2 QS system\'s key targets and regulation mechanism in L. plantarum L3 demonstrated the effectiveness of the new approach for predicting QS targets and provides a scientific basis for future work on improving regulation of functional factor production.

OBJECTIVE: Doxorubicin is first-line therapy for soft-tissue sarcoma, but patients can develop resistance which is usually fatal. As a novel therapeutic strategy, the present study aimed to determine the synergy of recombinant methioninase (rMETase) and doxorubicin against HT1080 fibrosarcoma cells compared to Hs27 normal fibroblasts, and rMETase efficacy against doxorubicin-resistant HT1080 cells in vitro.
METHODS: The 50% inhibitory concentrations (IC50) of doxorubicin and rMETase, as well as their combination efficacy, against HT1080 human fibrosarcoma cells, Hs27 normal human fibroblasts and doxorubicin-resistant HT1080 (DR-HT1080) cells were determined. Dual-color HT1080 cells which expressed red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize nuclear fragmentation during treatment. Nuclear fragmentation was observed with an IX71 fluorescence microscope.
RESULTS: The IC50 for doxorubicin was 3.3 μM for HT1080 cells, 12.4 μM for DR-HT1080 cells, and 7.25 μM for Hs27 cells. The IC50 for rMETase was 0.75 U/ml for HT1080 cells, 0.42 U/ml for DR-HT1080 cells, and 0.93 U/ml for Hs27 cells. The combination of rMETase and doxorubicin was synergistic against fibrosarcoma cells but not against normal fibroblasts. The combination of doxorubicin plus rMETase also caused more fragmented nuclei than either treatment alone in HT1080 cells. rMETase alone was highly effective against the DR-HT1080 cells as well as the parental HT1080 cells.
CONCLUSIONS: The present results indicate the future clinical potential of rMETase in combination with doxorubicin for fibrosarcoma, including doxorubicin-resistant fibrosarcoma.

In recent years, as the paradigm of communication between cells has been clarified, the ability of bacteria to change their gene expression patterns in response to various extracellular signals has attracted great interest. In particular, intracellular and intercellular communication between bacterial populations, called quorum sensing (QS), is essential for coordinating physiological and genetic activities. QS studies are critical, particularly in elucidating the regulatory mechanisms of infectious processes in food-borne pathogens. Elucidating the QS mechanisms in Salmonella is effective in silencing the virulence factors in the fight against this bacterium. The aims of this study were; to create luxS gene mutants that play a vital role in the QS activity of Salmonella and to determine the effect of this mutation on the expression of virulence genes in the bacteria and to determine the impact of synthetic N-hexanoyl-homoserine lactone (C6HSL) on biofilm formation and AI-2 signaling pathway of Salmonella wild strain and luxS gene mutants. luxS gene mutants were constructed by recombining the gene region with the chloramphenicol gene cassette based on homologous region recombination. In the luxS mutants obtained in this way, the expression of eight different virulence genes (hilA, invA, inv, glgC, fimF, fliF, lpfA, gyrA), which have essential roles in Salmonella pathogenicity, was determined by quantitative real-time reverse transcriptase polymerase chain reaction (rRT-qPCR) method and compared with natural strains. As a result of these studies, it was determined that the expression of each gene examined was significantly reduced in luxS mutant strains. The relative AI-2 activities of Salmonella strains were analyzed depending on time. It was determined that the highest activity occurred at the fourth hour and the AI-2 activities of luxS mutants were reduced compared to the wild strain. Finally, it was determined that C6HSL increased the biofilm activity of Salmonella Typhimurium DMC4, SL1344 wild strains, and mutants, mainly at the 72nd hour. In conclusion, our results proved that C6HSL stimulated QS communication in all strains and increased biofilm of Salmonella formation and autoinducer activity. This situation determines that Salmonella responds to external signals by using QS systems. In addition, this research contributed to provide additional information on interspecies communication mechanisms to develop strategies to prevent biofilm formation of this pathogen.

OBJECTIVE: It has been recently demonstrated that a methionine-restricted diet increases the response to immune checkpoint inhibitors (ICIs) via an increase in PD-L1 in a syngeneic mouse colorectal-cancer model. Our laboratory has developed recombinant methioninase (rMETase) to restrict methionine. The aim of the present study was to determine if rMETase can increase PD-L1 expression in a human colorectal cancer cell line in vitro.
METHODS: We evaluated the half-maximal inhibitory concentration (IC50) value of rMETase on HCT-116 human colorectal cancer cells. HCT-116 cells were treated with rMETase at the IC50 Western immunoblotting was used to compare PD-L1 expression in HCT-116 cells treated with and without rMETase.
RESULTS: The IC50 value of rMETase on HCT-116 was 0.79 U/ml. Methionine restriction using rMETase increased PD-L1 expression compared to the untreated control (p<0.05).
CONCLUSIONS: Methionine restriction with rMETase up-regulates PD-L1 expression in human colorectal cancer cells and the combination of rMETase and ICIs may have the potential to improve immunotherapy in human colorectal cancer.

β C-S lyases (β-CSLs; EC 4.4.1.8) are enzymes catalyzing the dissociation of β carbon-sulfur bonds of cysteine S-conjugates to produce odorant metabolites with a free thiol group. These enzymes are increasingly studied for their role in flavor generation in a variety of food products, whether these processes occur directly in plants, by microbial β-CSLs during fermentation, or in the mouth under the action of the oral microbiota. Microbial β-CSLs react with sulfur aroma precursors present in beverages, vegetables, fruits, or aromatic herbs like hop but also potentially with some precursors formed through Maillard reactions in cooked foods such as meat or coffee. β-CSLs from microorganisms like yeasts and lactic acid bacteria have been studied for their role in the release of polyfunctional thiols in wine and beer during fermentation. In addition, β-CSLs from microorganisms of the human oral cavity were shown to metabolize similar precursors and to produce aroma in the mouth with an impact on retro-olfaction. This review summarizes the current knowledge on β-CSLs involved in flavor generation with a focus on enzymes from microbial species present either in the fermentative processes or in the oral cavity. This paper highlights the importance of this enzyme family in the food continuum, from production to consumption, and offers new perspectives concerning the utilization of β-CSLs as a flavor enhancer.

All sulfur transfer pathways have generally a l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of numerous sulfur-containing biomolecules in the cell. In Escherichia coli, the housekeeping l-cysteine desulfurase IscS has several interaction partners, which bind at different sites of the protein. So far, the interaction sites of IscU, Fdx, CyaY, and IscX involved in iron-sulfur (Fe-S) cluster assembly have been mapped, in addition to TusA, which is required for molybdenum cofactor biosynthesis and mnm5s2U34 tRNA modifications, and ThiI, which is involved in thiamine biosynthesis and s4U8 tRNA modifications. Previous studies predicted that the sulfur acceptor proteins bind to IscS one at a time. E. coli TusA has, however, been suggested to be involved in Fe-S cluster assembly, as fewer Fe-S clusters were detected in a ∆tusA mutant. The basis for this reduction in Fe-S cluster content is unknown. In this work, we investigated the role of TusA in iron-sulfur cluster assembly and iron homeostasis. We show that the absence of TusA reduces the translation of fur, thereby leading to pleiotropic cellular effects, which we dissect in detail in this study.IMPORTANCEIron-sulfur clusters are evolutionarily ancient prosthetic groups. The ferric uptake regulator plays a major role in controlling the expression of iron homeostasis genes in bacteria. We show that a ∆tusA mutant is impaired in the assembly of Fe-S clusters and accumulates iron. TusA, therefore, reduces fur mRNA translation leading to pleiotropic cellular effects.

The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.

OBJECTIVE: The alkylating agent trabectedin, which binds the minor groove of DNA, is second-line therapy for soft-tissue sarcoma but has only moderate efficacy. The aim of the present study was to determine the synergistic efficacy of recombinant methioninase (rMETase) and trabectedin on fibrosarcoma cells in vitro, compared with normal fibroblasts.
METHODS: HT1080 human fibrosarcoma cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and Hs27 normal human fibroblasts, were used. Each cell line was cultured in vitro and divided into four groups: no-treatment control; trabectedin treated; rMETase treated; and trabectedin plus rMETase treated. The dual-color HT1080 cells were used to quantitate nuclear fragmentation in each treatment group.
RESULTS: The combination of rMETase and trabectedin was highly synergistic to decrease HT1080 cell viability. In contrast, there was no synergy on Hs27 cells. Moreover, nuclear fragmentation occurred synergistically with the combination of trabectedin and rMETase on dual-color HT1080 cells.
CONCLUSIONS: The combination treatment of trabectedin plus rMETase was highly synergistic on fibrosarcoma cells in vitro suggesting that the combination can improve the outcome of trabectedin alone in future clinical studies. The lack of synergy of rMETase and trabectedin on normal fibroblasts suggests the combination is not toxic to normal cells. Synergy of the two drugs may be due to the high rate of nuclear fragmentation on treated HT1080 cells, and the late-S/G2 cell-cycle block of cancer cells by rMETase, which is a target for trabectedin. The results of the present study suggest the future clinical potential of the combination of rMETase and trabectedin for soft-tissue sarcoma.

The Escherichia coli cysteine desulfurase SufS (EcSufS) is a dimeric, PLP-dependent enzyme responsible for sulfur mobilization in the SUF Fe-S cluster bioassembly pathway. The enzyme uses cysteine as a sulfur source and generates alanine and a covalent persulfide located on an active site of cysteine. Optimal in vitro activity of EcSufS requires the presence of the transpersulfurase protein, EcSufE, and a strong reductant. Here, presteady-state and single-turnover kinetics are used to investigate the mechanism of EcSufS activation by EcSufE. In the absence of EcSufE, EcSufS exhibits a presteady-state burst of product production with an amplitude of ∼0.4 active site equivalents, consistent with a half-sites reactivity. KinTek Explorer was used to isolate the first turnover of alanine formation and fit the data with a simplified kinetic mechanism with steps for alanine formation (k3) and a net rate constant for the downstream steps (k5). Using this treatment, microscopic rate constants of 2.3 ± 0.5 s-1 and 0.10 ± 0.01 s-1 were determined for k3 and k5, respectively. The inclusion of EcSufE in the reaction results in a similar rate constant for k3 but induces a 10-fold enhancement of k5 to 1.1 ± 0.2 s-1, such that both steps are partially rate-determining. The most likely downstream step where EcSufE could exert influence on EcSufS activity is the removal of the persulfide intermediate. Importantly, this step appears to serve as a limiting feature in the half-sites activity such that activating persulfide transfer allows for rapid shifting between active sites. Single-turnover assays show that the presence of EcSufE slightly slowed the rates of alanine-forming steps, suggesting it does not activate steps in the desulfurase half reaction.

Industrial wastewater is a major environmental concern due to its high copper content, which poses significant toxicity to microbial life. Autoinducer-2 (AI-2) can participate in the inter- and intra-species communication and regulate the physiological functions of different bacterial species by producing AI-2 signal molecules. However, there are few research reports on the luxS gene and lsr operon functions for AI-2 in bacteria with a certain tolerance to copper. This study delves into the potential of quorum sensing mechanisms, particularly the AI-2 system, for enhancing microbial resistance to copper toxicity in Klebsiella michiganensis (KM). We detail the critical roles of the luxS gene in AI-2 synthesis and the lsr operon in AI-2 uptake, demonstrating their collective impact on enhancing copper resistance. Our findings show that mutations in the lsr operon, alongside the knockout of the luxS gene in KM strain (KMΔluxSΔlsr), significantly impair the strain\'s motility (p < 0.0001) and biofilm formation (p < 0.01), underscoring the operon\'s role in AI-2 transport. These genetic insights are pivotal for developing bioremediation strategies aimed at mitigating copper pollution in wastewater. By elucidating the mechanisms through which KM modulates copper resistance, this study highlights the broader ecological significance of leveraging microbial quorum sensing pathways for sustainable wastewater management.