Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis , very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

Guanosine triphosphate (GTP) is essential for macromolecular biosynthesis, and its intracellular levels are tightly regulated in bacteria. Loss of the alarmone (p)ppGpp disrupts GTP regulation in Bacillus subtilis , causing cell death in the presence of exogenous guanosine and underscoring the critical importance of GTP homeostasis. To investigate the basis of guanosine toxicity, we performed a genetic selection for spontaneous mutations that suppress this effect, uncovering an unexpected link between GTP synthesis and glycolysis. In particular, we identified suppressor mutations in pyk , which encodes pyruvate kinase, a glycolytic enzyme. Metabolomic analysis revealed that inactivating pyruvate kinase prevents guanosine toxicity by reducing GTP levels. Although traditionally associated with ATP generation via substrate-level phosphorylation, B. subtilis pyruvate kinase in vitro was found to produce GTP and UTP approximately 10 and three times more efficiently than ATP, respectively. This efficient GTP/UTP synthesis extends to Enterococcus faecalis and Listeria monocytogenes , challenging the conventional understanding of pyruvate kinase’s primary role in ATP production. These findings support a model in which glycolysis directly contributes to GTP synthesis, fueling energy-demanding processes, such as protein translation. Finally, we observed a synergistic essentiality of the Δ ndk Δ pyk double mutant specifically on glucose, indicating that pyruvate kinase and nucleoside diphosphate kinase are the major contributors to nucleoside triphosphate production and complement each other during glycolysis. Our work highlights the critical role of nucleotide selectivity in pyruvate kinase and its broader implications in cellular physiology. IMPORTANCE: In this study, we reveal that pyruvate kinase, a key glycolytic enzyme, primarily generates GTP from GDP in Bacillus subtilis , relative to other nucleotide triphosphates, such as ATP. This finding, uncovered through genetic selection for mutants that suppress toxic GTP overaccumulation, challenges the conventional understanding that pyruvate kinase predominantly produces ATP via substrate-level phosphorylation. The substantial role of GTP production by pyruvate kinase suggests a model where glycolysis rapidly and directly supplies GTP as the energy currency to power high GTP-demanding processes such as protein synthesis. Our results underscore the importance of nucleotide selectivity (ATP vs GTP vs UTP) in shaping the physiological state and fate of the cell, prompting further exploration into the mechanisms and broader implications of this selective nucleotide synthesis.

Background: Global awareness of ambient air pollution has heightened due to its detrimental impact on health, particularly in regions with elevated PM_2.5 levels. Chiang Mai has emerged as an area experiencing the highest PM_2.5 levels in Thailand. Objectives: to examine the prevalence of respiratory allergies and assess the impact of air pollution on the health-related quality of life (QoL) among university students in Chiang Mai. Methods: Chiang Mai University (CMU) and Maejo University (MJU) students were recruited. The Global Asthma Network (GAN) questionnaire screened for respiratory allergies (RAs). The disease-specific QoL questionnaire (Rcq-36) was administered twice during low-PM_2.5 and high-PM_2.5 seasons to evaluate air pollution’s impact on health-related QoL. Those showing potential RAs underwent a skin prick test (SPT) to investigate allergic sensitization. Results: Out of 406 participants, 131 (32%) reported respiratory allergies. Among those undergoing SPT, a high rate (82.54%) had positive results. Across both universities, students reported significantly lower QoL in multiple domains, particularly respiratory, eye, sleep, and emotional well-being, during the high-PM_2.5 season. This aligned with their poorer self-reported health on a visual analog scale (VAS; p-value < 0.01). PM_2.5 levels significantly impacted social functioning for CMU students (p-value = 0.001) and role limitations for MJU students (p-value < 0.001). Notably, participants without respiratory allergies (non-RAs) were more significantly affected by PM_2.5 than RA participants in almost all parameters, despite experiencing fewer baseline symptoms. Conclusions: Respiratory allergies, particularly allergic rhinitis, are prevalent among university students in Chiang Mai. This study underscores the substantial negative impact of ambient air pollution on QoL for both allergic and non-allergic students.

Functional fermented fruit juices produced using a combination of non-Saccharomyces yeast and lactic acid bacteria (LAB) are relatively unexplored. The effects of three inoculation protocols, single inoculation with Lactobacillus acidophilus (La), single inoculation with Torulaspora delbrueckii (Td), and co-culture of both La + Td, on the physicochemical, microbiological, sensory properties, and metabolic profile of fermented JA juices after 24 h at 30 °C were investigated. Uninoculated (UI) Japanese apricot (JA) juice was used as a control. The results show significant increases in the color intensity of the fermented-JA juices, whereas an enhancement of total phenolic contents is observed in the fermented JA-juices acquired through the use of La for both single and co-culture inoculations. The colony counts of LAB and yeast in the inoculated JA juices increased by approximately 2.0 and 1.7 log CFU/mL at 24 h, respectively. The antibacterial activity of JA juices against four pathogenic bacteria was detected. All JA juices exhibited antimicrobial activity against the tested pathogenic strains, with strong antibacterial properties of La-fermented juice being recorded against Bacillus cereus at the lowest MIC of 124 µL/mL. Additionally, La + Td-fermented and UI-JA juices demonstrated comparable anticancer activity against HT-29 cells with IC50 values of 823.37 and 754.87 µg/mL, respectively. Furthermore, a total of 995 compounds was identified as differential fermentation metabolites through non-targeted metabolome analysis across different fermentation groups. These findings illustrate the significant potential of using JA juice for La and Td fermentation to develop functional juices.

Helicobacter pylori is a gastric pathogen implicated in peptic ulcer disease and gastric cancer. The increasing prevalence of antibiotic-resistant strains underscores the urgent need for alternative therapeutic strategies. In this study, we investigated the chemical composition and antibacterial activity of an aqueous extract from Caulerpa lentillifera (sea grape), a farm-cultivated edible green seaweed collected from Krabi Province, Thailand. Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) revealed that the extract was enriched in bioactive nucleosides and phenolic compounds. In vitro assays demonstrated dose-dependent inhibition of H. pylori growth following exposure to sea grape extract. Furthermore, untargeted intracellular metabolomic profiling of H. pylori cells treated with the extract uncovered significant perturbations in central carbon and nitrogen metabolism, including pathways associated with the tricarboxylic acid (TCA) cycle, one-carbon metabolism, and alanine, aspartate, and glutamate metabolism. Pyrimidine biosynthesis was selectively upregulated, indicating a potential stress-induced shift toward nucleotide salvage and DNA repair. Of particular note, succinate levels were markedly reduced despite accumulation of other TCA intermediates, suggesting disruption of electron transport-linked respiration. These findings suggest that bioactive metabolites from C. lentillifera impair essential metabolic processes in H. pylori, highlighting its potential as a natural source of antimicrobial agents targeting bacterial physiology.

Honey bees (Apis mellifera L.) are considered highly significant economic insects. It is a source of valuable food and medicinal products such as honey, bee pollen, royal jelly, bee brood, and beeswax, which possess excellent nutritional and pharmacological properties. Nevertheless, honey bee health and productivity were often challenged by various environmental factors. Therefore, bee colony management is of the utmost importance. In this light, bee supplements and gut microbiota are crucial to ensure that bees receive sufficient nutritional value to maintain their health and productivity. In this study, we isolate and characterize lactic acid bacteria from the hindgut of the worker bee. 16S rRNA sequencing revealed that three isolated bacteria were Apilactobacillus kunkeei (AK), Lactiplantibacillus sp. (LP), and Lactobacillus brevis (LB). Three species of lactic acid bacteria were investigated for potential probiotic properties by supplementing 50% (w/w) sucrose syrup in the form of a direct-fed microorganism (DFM). The supplement with DFM had no negative effect on average lifespan. Examination took place of the impact of probiotics on the development of the hypopharyngeal glands (HPGs) in the bee’s head at days 3, 6, and 9 post-treatments. The cage-bees fed by pollen and DFM syrup exhibited acini surface areas ranging from 0.020 to 0.023 mm². The L. brevis (LB) group exhibited enhanced HPG development, with an average acini size of 0.027 ± 0.007 mm² at day 6, while the non-treatment control had an average acini size of 0.023 ± 0.006 mm². The significant size differences were maintained throughout the 9-day period. In addition, the DFM syrup enhanced microbial protein content in the bee head, digestibility, and community complexity compared with the negative control groups. Therefore, the DFM syrup with a potential strain of probiotic may enhance overall honey bee health status.