ABSTRACT Glyceraldehyde-3-phosphate dehydrogenases (GapDHs) are key enzymes in central carbon metabolism, yet their functional diversity extends beyond canonical glycolysis and gluconeogenesis. The soil bacterium Pseudomonas putida KT2440 encodes four GapDH isoenzymes (i.e., GapA, GapB, GapC, and GapD). GapA and GapB have been recently described as gatekeepers of metabolic segregation, with GapA driving glycolysis and GapB associated with gluconeogenesis. However, the physiological roles of GapC and GapD remain poorly defined. In this work, we combined targeted mutagenesis, growth phenotyping, in vitro biochemical analysis, proteomics, and metabolomics to dissect the functions of these lesser-studied GapDH isoforms. GapC and GapD have distinct structural features, diverging from the canonical GapA and GapB enzymes. Physiological characterization of gap -deletion mutants of P. putida across multiple glycolytic and gluconeogenic substrates showed that GapC and GapD exert substrate-specific effects and contribute to network-wide redox balancing. Proteomic and metabolomic profiling further indicated that GapC and GapD are integrated into specialized metabolic modules, supporting auxiliary functions that complement the dominant roles of GapA and GapB. This case of metabolic redundancy at the GapDH node highlights the versatility of central carbon metabolism in bacteria adapted to changing environmental conditions. IMPORTANCE Bacteria often carry multiple isoenzymes for the same metabolic step, yet the reasons for such redundancy remain poorly understood. By dissecting the roles of GapDH isoforms in P. putida , we show that these enzymes are not functionally equivalent but rather partition their activity across glycolytic, gluconeogenic, and auxiliary functions. GapC and GapD provide secondary capacity under specific conditions that complement the known roles of GapA and GapB, which dominate glycolysis and gluconeogenesis, respectively. Growth phenotyping, together with proteomic and metabolomic analyses, revealed that isoenzyme specialization is reflected in both substrate-dependent abundance patterns and metabolite profiles. This study underscores the evolutionary diversification of central metabolic enzymes in environmental bacteria and their coordinated regulation across nutritional states. Furthermore, our findings provide a framework for engineering carbon flux in Pseudomonas and related bacteria, with implications for metabolic engineering applications targeting diverse feedstocks.
ABSTRACT Burkholderia pseudomallei , the causative agent of melioidosis, is ubiquitous in Southeast Asia but remains understudied in many endemic countries. The species’ high genetic diversity necessitates large-scale sampling to resolve fine-scale population structure. In countries bordering the Mekong River, the geographic range of B. pseudomallei strains may be increased by water-mediated dispersal. Between 2021 and 2023, we collected and sequenced 253 clinical B. pseudomallei isolates from patients admitted to six Cambodian hospitals and geotagged isolates by patient village. Isolates were genetically diverse and grouped into 11 clades, including one characterized by a chromosomal genomic island containing unique genes absent from other isolates, consistent with local evolution. Comparison with 3,234 publicly available global genomes revealed intermixing between Cambodian and other Southeast Asian isolates but clear distinction from Australian strains. Phylogeographic analyses further demonstrated spatial localization along major waterways, suggesting that river systems, including the Mekong, may contribute to bacterial dissemination in Cambodia. Taken together, these findings demonstrate the importance of localized data sets for contextualizing the population structure and evolution of this diverse pathogen in Southeast Asia. IMPORTANCE Burkholderia pseudomallei is a highly diverse bacterial species with genomic features that vary based on geographic sampling location. Existing data predominantly from Thailand show that isolates from Southeast Asia may be closely related to each other. Here, we sequenced isolates associated with human disease in Cambodia and compared these against genomes from public sequence repositories. We identified many distinct bacterial strains and found that some of these were localized either to the south or in the north of the country. We identified a unique group of genes in one cohort strain with putative roles in bacterial metabolism and genomic recombination. Finally, in a global phylogeny constructed from publicly available genomes, we found that Cambodian isolates intermixed with other Southeast Asian isolates. Our study is the largest genomic study of B. pseudomallei in Cambodia to date, with findings that contribute to the global understanding of the bacterium’s diversity and evolution.
ABSTRACT Understanding the environmental dissemination of antimicrobial resistance (AMR) is crucial for mitigating its spread and reducing the clinical burden of resistant infections. In a previous study, we demonstrated that copiotrophic enrichment in a medium that stimulates the growth of gut-associated bacteria can reveal clinically associated bacterial populations and antibiotic resistance genes (ARGs) that are not detected through direct environmental sampling. In the present study, we applied this enrichment approach to poultry manure to examine how β-lactamase-resistant antibiotics (cefotaxime and meropenem) shape the composition and resistance profiles of copiotrophic, gut-like microbial community relative to an earlier β-lactam (ampicillin) and to antibiotic-free controls. Our results show that extended-spectrum and last-resort β-lactams induced more pronounced shifts in microbial diversity, resistome, and mobilome profiles compared to ampicillin. When focusing on specific taxa, such as clinically relevant enteric bacteria and both susceptible and β-lactam-resistant coliforms, differential responses were observed correlating to the antimicrobial range of action. Moreover, cefotaxime and meropenem exerted different effects on the relative abundance of enteric bacteria and plasmids harboring multiple ARGs. Together, these findings underline how the diversity of pathogen and AMR indicators in gut microbiomes may be shaped by different selective pressures imposed by β-lactamase-resistant antibiotics. IMPORTANCE This study explores how different generations of β-lactam antibiotics shape microbial communities and antibiotic resistance in a gut-like in vitro system. Using a metabolically driven synthetic community derived from manure, we found that advanced β-lactams such as cefotaxime and meropenem led to stronger and more specific shifts in both community composition and resistance gene profiles compared to ampicillin. These results emphasize that the ecological impact of antibiotics depends not only on whether bacteria are inhibited but also on the spectrum of activity of each compound. In addition, the system we used provides a simple and controllable way to study interactions among antibiotics, microbial communities, and the resistome. Overall, this work offers a clearer ecological view of how clinically important antibiotics may influence microbiomes and the spread of antibiotic resistance genes.
ABSTRACT Dark septate endophytes (DSEs) are ubiquitous root-colonizing fungi characterized by melanized, septate hyphae and a frequent association with plants in stressful or nutrient-poor environments. Although DSEs are increasingly recognized for context-dependent effects on host stress resilience, the specialized metabolic capacity of many DSE lineages remains poorly resolved. Hyaloscypha finlandica is a recurrent root-associated fungus of Populus and other hosts, making it a useful system for understanding symbiotic mechanisms using multi-omics analyses. Here, we combined comparative biosynthetic gene cluster (BGC) prediction across 41 ascomycete genomes with untargeted metabolomics of H. finlandica PMI 746 grown in a defined minimal medium and a complex plant-derived medium. Benchmarking antiSMASH, FunBGCeX, and BGC-Prophet revealed broad conservation of PKS-, NRPS-, and terpene-associated biosynthetic capacity among DSEs. LC-MS/MS profiling detected strong medium-associated shifts in the extracellular chemical footprint of H. finlandica and identified putative terpenoid-, polyketide-, peptide-, siderophore-, and DHN-melanin-related features through spectral matching and in silico chemical-class inference. Authentic-standard comparison supported a probable assignment of 7-oxodehydroabietic acid, whereas most other annotations remain putative or class-level assignments. Integrating genomic and metabolomic evidence identified representative candidate intersections between encoded biosynthetic capacity and observed chemistry, but definitive BGC-product links remain targets for future genetic and structural validation. Together, these results provide a public paired-omics resource for a core Populus-associated DSE and a conservative framework for interpreting fungal multi-omics in non-model endophytes.IMPORTANCEDark septate endophytes (DSEs) are widespread fungal symbionts that influence plant performance, yet their encoded and expressed metabolic capacities remain largely uncharacterized. This study provides a paired comparative genome-mining and untargeted metabolomics resource for Hyaloscypha finlandica, a recurrent Populus-associated DSE. By explicitly distinguishing biosynthetic gene cluster (BGC) prediction, spectral annotation, authentic-standard support, and unresolved BGC-Prophet assignment, this work provides a transparent reference for future studies of DSE specialized metabolism. The resulting data sets support comparative analyses across fungal endophytes and help prioritize candidate metabolites and gene clusters for targeted validation.
ABSTRACT Pseudomonas aeruginosa (P.a) is a clinically important opportunistic pathogen capable of adapting and surviving a wide range of environments and stress conditions. A central regulator of this adaptability is the stringent response, mediated by the alarmones (p)ppGpp. Activation of the stringent response triggers a global physiological shift away from active metabolism toward stress management and cellular quiescence, a key transition to stationary phase. To date, few studies have investigated the protein-level processes under stringent response control. In this study, we performed shotgun proteomic profiling (LC-MS/MS) of a (p)ppGpp-null relA spoT double knockout mutant (ΔSR) and its isogenic PAO1 parent during late exponential and stationary phase planktonic growth in rich medium. In this first characterization of the stringent response proteome in P.a, we identified 146 differentially abundant proteins (DAP) in late exponential phase ΔSR cells, and 2.3-fold greater DAP (339) in stationary-phase cells. Functional characterization and system-level analysis of the differentially abundant stationary phase proteome highlighted the key role of (p)ppGpp in shutting down a wide range of metabolic processes while inducing adaptive processes such as responses to oxidative stress. Comparative RNA-seq analysis also uncovered incongruent protein and mRNA responses in the ΔSR relative to wild-type cells in several processes, including iron homeostasis and antioxidant defenses. Transcriptomic analysis of the ΔSR also revealed differential expression of numerous sRNAs, including PrrF1/2, raising the possibility that sRNAs may mediate certain stringent-response-dependent post-transcriptional control. Phenotypic validation of the ΔSR proteome confirmed multiple defects in antioxidant defenses and dysregulated pyoverdine biosynthesis.IMPORTANCEPseudomonas aeruginosa is an important opportunistic human pathogen that causes difficult-to-treat infections and is highly adaptable to stressful environments. A key orchestrator of this adaptability is the stringent response, mediated by the signaling molecule (p)ppGpp. In this study, we characterized the extensive proteome remodeling in the (p)ppGpp-null relA spoT mutant during late exponential and stationary phase and provided the first protein-level analysis of the stringent response in P. aeruginosa. These data highlight the role of (p)ppGpp in shutting down broad metabolic functions and activating adaptive pathways, including stress-resistance responses, particularly during stationary phase. Using comparative proteomics and RNA-seq, we also find that several processes—such as iron regulation and oxidative stress responses—show mismatched RNA and protein changes, suggesting additional layers of post-transcriptional control, potentially involving small regulatory RNAs.
