In natural environments, bacteria often encounter low concentrations of nutrient mixtures that are continuously replenished by physical processes such as fluid flow. Studying bacterial physiology under such conditions is experimentally challenging because it is difficult to maintain steady, low nutrient concentrations with rapid renewal. Most studies on nutrient limitation have used approaches such as the chemostat, which rely on long renewal times to sustain low concentrations. We developed a Millifluidic Continuous Culture Device (MCCD), inspired by microfluidics, that enables bacterial cultivation in nutrient mixtures at low micromolar concentrations with rapid renewal driven by fluid flow. Unlike microfluidic systems, the MCCD retains sufficient culture volume to support batch-scale 'omic analyses. Using the MCCD, we cultured Escherichia coli in a mixture of amino acids and nucleobases at three concentration ranges spanning a fivefold difference in growth rates. Surprisingly, at the lowest concentration range, cells exhibited proteomic signatures of iron limitation despite equal total ferrous iron across conditions. Uptake experiments with labeled iron-histidine and iron-cysteine complexes confirmed that amino acids facilitated ferrous iron acquisition. Under continuous flow, siderophores were washed out, rendering this pathway ineffective and revealing a previously unrecognized mechanism of iron acquisition via soluble ferrous iron-amino acid complexes. These findings highlight the importance of studying bacterial physiology at low nutrient concentrations and also suggest a broader role for other organic substrates capable of complexing iron as potential iron sources in environments with rapid renewal.

Resource competition strongly shapes microbial community dynamics and functionality. In polysaccharide-degrading communities, primary degraders release hydrolytic enzymes, whereas exploiters consume released products without producing enzyme themselves. We investigate the competitive strategies employed by marine chitin degraders and N-acetylglucosamine (GlcNAc) exploiters, revealing various mechanisms that impact community viability and growth dynamics. In addition to direct competition strategies such as antibiotic secretion or cell aggregation on chitin particles (which helps monopolize enzyme access), exploiters also inhibit degraders by diverting limiting GlcNAc flux during the early stages of particle degradation. This critical phase requires degraders to overcome the diffusive loss of GlcNAc to sustain their chitinase production. Through quantitative measurements and modeling, we demonstrate that nutrient competition among species and nutrient loss through diffusion during the initial stages of community dynamics strongly influence the long-term success of the community. The initial community composition dictates the former mechanisms, while the latter is closely related to particle size, both of which have profound implications for environmental carbon cycling. The resulting hypersensitivity of the community is analogous to the Allee effect observed in population biology, where the outcomes-in our case polymer degradation-are heavily dependent on starting conditions. This study sheds light on how metabolic competition in the early phases of particle degradation governs species interactions, resource partitioning, and overall community viability, even under identical environmental and genetic conditions.

Chitin, a major structural component of arthropod exoskeletons, is an abundant carbon and nitrogen source in marine ecosystems. While its degradation is well studied in oxic waters, the microbial processes and interactions that mediate its anaerobic breakdown in deep-sea sediments remain poorly understood. Iron oxides are predicted to be energetically favorable electron acceptors for anaerobic chitin degradation, yet the spatial separation of insoluble substrates and the required microbial partnerships in sediments are not well defined. Here, we used potentiostatically controlled bioelectrochemical reactors poised at +0.22 V vs. Standard Hydrogen Electrode, mimicking iron-reducing conditions, to enrich and characterize a chitin-degrading, metal-reducing microbial community from an anoxic deep-sea whale-fall sediment. Amendment with crystalline chitin generated stable anodic currents, which increased upon addition of chitin-associated metabolites (N-acetylglucosamine, glucose, acetate). 16S rRNA gene sequencing revealed a deep-sea affiliated assemblage dominated by Firmicutes (Vallitalea), Spirochaetota, Gammaproteobacteria, and Desulfobacterota (Trichloromonas). Exoenzyme assays, metabolite profiling, and current measurements confirmed that active chitin degradation provided substrate(s) for extracellular electron transfer (EET). Single-cell analyses using FISH-BONCAT and nanoSIMS showed that Vallitalea (primary degrader) and electrode-respiring Desulfobacterota exhibited highest activity within the electrode biofilm, particularly within ca.10 μm of the surface. We isolated a chitin-degrading Vallitalea sp. and an iron-reducing, electrogenic Trichloromonas sp., and demonstrated that, when reconstituted in co-culture, they cooperatively degrade chitin via acetate cross-feeding coupled to EET. This integrated electrochemical and ecophysiological study reveals microbial interactions linking chitin degradation with iron-oxide respiration in deep-sea sediments and provides a defined electrogenic model community for future syntrophy research.

Abstract Monosaccharides support Salmonella enterica serovar Typhimurium colonization of the gut, yet the role of their oxidized derivatives remains understudied. Sugar acids are largely diet-independent carbon sources generated by host-driven oxidative processes, but their contribution during infection - particularly that of less oxidized aldonic and uronic acids - has not been defined. Here, we systematically assess the role of sugar acids derived from D-glucose and D-galactose in S . Typhimurium SL1344 colonization. Among D-glucose-derived acids, D-gluconate accumulated to the highest levels and was the dominant substrate supporting luminal expansion in streptomycin-pretreated mice, exceeding the more oxidized acids D-glucuronate and D-glucarate. During chronic infection, D-glucose-derived sugar acids became increasingly important for pathogen persistence. Ecological niche invasion assays identified these compounds as a principal metabolic niche, whereas D-galactose-derived acids contributed minimally. Consistent with a transient, inflammation-linked nutrient niche, sugar acid utilization pathways were similarly prevalent in Escherichia coli from individuals with and without inflammatory bowel disease. Together, these findings identify D-gluconate as a key inflammation-dependent nutrient source that fuels Enterobacteriaceae expansion in the inflamed gut.

Metabolic enzymes have traditionally been regarded as highly specific catalysts; however, many can catalyze multiple reactions. To systematically investigate the prevalence of such enzyme promiscuity, we used nontargeted metabolomics to measure dynamic metabolite ion profiles in in vitro assays with 667 successfully purified Escherichia coli enzymes in a natural intracellular metabolome extract. Notably, nearly half of these enzymes elicited significant changes in ion traces. Using a machine learning-derived multivariate classifier at a false-discovery rate of 33%, we identified unexpected changes in 135 putatively annotated metabolite ion traces, indicating the presence of so far unknown promiscuous activities in 11% of the tested enzymes, most of which have yet to be recognized for their ability to catalyze multiple reactions. Notably, we found that nucleotide-related substrates or cofactors were enriched among the newly identified reactants. For 11 promiscuous enzymes, we successfully reconstructed 22 complete reaction stoichiometries, four of which were validated experimentally. Key findings include the nucleoside phosphorylase DeoA, for which we expanded the substrate range to include pyrimidines relevant to carbon and energy utilization, and the N-acetylmannosamine kinase (NanK), which displayed both cofactor and sugar substrate promiscuity. Additionally, CobC, a putative adenosylcobalamin/α-ribazole phosphatase, catalyzes flavin mononucleotide dephosphorylation, suggesting a generalist role in vitamin biosynthesis pathways. Beyond specific examples, the results suggest that metabolism harbors a wealth of underexplored catalytic flexibility, relevant for functional annotation, evolution, and genome-scale metabolic models.

Fucoidans, a class of complex polysaccharides produced by brown algae and diatoms, contribute to long-term carbon sequestration owing to their resistance to microbial degradation1,2. Although individual microorganisms can break down portions of these polysaccharides3-5, it remains unclear whether complete breakdown is possible in nature and, if so, by what mechanisms. Here we show that fucoidans are degraded through synergistic interactions between specialized bacteria with complementary metabolic functions. Using metabolomic analysis of a reconstructed marine consortium, we uncovered metabolic guilds of bacteria that preferentially degrade either the sulfated fucose backbone or the side branches of rare monomers. This functional division of labour leads to an unexpectedly high number of synergistic interactions between different degraders that enhanced degradation efficiency up to 97.1%. Despite varying fucoidan structures across different types of algae6, the metabolic functions of degraders remained conserved, enabling quantitative prediction of degradation outcomes based on community and substrate composition. The frequent co-occurrence of functionally complementary fucoidan degraders in ocean metagenomes suggests that synergistic degradation is a globally relevant strategy. Our findings suggest that the environmental turnover of complex biopolymers depends not only on individual metabolic capabilities of degraders but also on ecological interactions shaped by substrate architecture. This work provides a mechanistic framework for understanding carbon cycling in the ocean and for engineering synthetic microbial consortia to degrade recalcitrant polysaccharides.

Abstract Bacterial chitin degradation contributes to global carbon cycling, particularly in marine environments where it is a highly abundant polysaccharide. Despite the taxonomic diversity of co-occurring chitin-degrading bacteria, the influence of individual traits on interactions between them remains poorly understood. Here, we measured key physiological traits of seven chitin degraders and investigated how these traits shape interaction outcomes and chitin degradation in pairwise cocultures. We found mainly negative and neutral interactions among degraders, contrasting with the synergistic dynamics observed with other complex polysaccharides. However, chitin degradation was not consistently diminished. These interaction types could be attributed to the limited partitioning of degradation products, alongside variations in enzyme repertoires and attachment behaviours that help some degraders to prevail over others. Further, we showed that one degrader can strongly inhibit the growth of others, even those possessing favourable physiological traits, likely due to the secretion of inhibitory compounds. These findings extend our understanding of the breadth of interactions among primary polysaccharide degraders and their implications for the degradation process. One-sentence Summary The physiological traits of bacteria that degrade chitin, a highly abundant biopolymer in marine environments, promote a range of neutral and negative interactions among them.

Identifying genomic regions bound by individual proteins such as transcription factors is essential to understanding bacterial gene regulation; however, comprehensive understanding of the effect of protein occupancy on gene regulation would be prohibitively laborious and expensive to achieve using methods such as chromatin immunoprecipitation with sequencing (ChIP-seq) and ChIP with exonuclease treatment (ChIP-exo) for every protein and condition of interest. Here we describe a protocol for performing in vivo protein occupancy display-high resolution (IPOD-HR), a powerful method for genome-wide profiling of protein-bound DNA in prokaryotic systems. Although assay for transposase-accessible chromatin with sequencing (ATAC-seq) is the method of choice for assaying general protein occupancy in eukaryotic systems, bacterial nucleoid-associated proteins can affect ATAC-seq, rendering it unsuitable for use in bacteria. In contrast, IPOD-HR can be used to identify regions of bacterial genomes that are highly bound by proteins, regardless of the identity of the proteins bound, allowing the identification of condition- and genotype-dependent changes in protein occupancy associated with changes in gene regulation. The technique is coupled to RNA polymerase ChIP, followed by sequencing of the extracted samples and downstream analysis using open-source, automated software that we provide and actively maintain. Once cross-linked samples are obtained, the core DNA selection portion of the IPOD-HR protocol takes 3 calendar days to perform. The resulting DNA extracts are subjected to high-throughput sequencing, resulting in sequencing data that are analyzed, which typically requires a few additional days, depending on the number of samples and computing resources. The IPOD-HR experimental method requires familiarity with standard molecular biology techniques suitable for preparing Illumina sequencing inputs, and the computational post-processing pipeline requires basic knowledge of the Linux command line environment.

The type VI secretion system (T6SS) is a macromolecular protein complex found in Gram-negative bacteria that mediates intercellular antagonism and gut Bacteroidales use this system to increase their competitiveness. Furthermore, T6SS is a recognized gut colonization factor that influences gut biodiversity. Bacteroidales T6SSs are divergent from their well-characterized Pseudomonadota counterparts. One conserved feature in many Bacteroidales T6SSs is the presence of multiple Hcp proteins with low sequence similarity, encoded within the secretion system's locus and adjacent to variable effector cassettes. Here we provide evidence that in the Bacteroides fragilis T6SS, these Hcp subunits, similar to other protein export systems function as high and low abundance major and minor subunits. First, we showed that these minor Hcp proteins form a network of interactions. Biochemical characterization of one of the minor Hcp complexes formed between Hcp1 and Hcp2 showed that they form hetero-hexamers with variable stoichiometry. Furthermore, we showed that this complex recognizes the secreted effector Bte1. Our results suggest that these minor subunits function as recognition particles for effectors to mediate secretion and that this is a conserved feature in Bacteroidales T6SS. The functionality of these Hcp-effector modules is encoded in a high-synteny region within T6SS loci, and bioinformatic analyses imply that this arrangement participates in establishing effector variability. Exploiting this feature could pave the way for characterizing numerous Bacteroidales effectors with unknown functions through copurification with their cognate Hcps, unlocking new insights into bacterial interactions and into mechanisms of gut biodiversity establishment. ### Competing Interest Statement The authors have declared no competing interest.

AbstractThe activity of bacterial transcription factors (TFs) is typically modulated through direct interactions with small molecules. However, these input signals remain unknown for most TFs, even in well-studied model bacteria. Identifying these signals typically requires tedious experiments for each TF. Here, we develop a systematic workflow for the identification of TF input signals in bacteria based on metabolomics and transcriptomics data. We inferred the activity of 173 TFs from published transcriptomics data and determined the abundance of 279 metabolites across 40 matched experimental conditions inEscherichia coli. By correlating TF activities with metabolites abundances, we successfully identified previously known TF-metabolite interactions and predicted novel TF effector metabolites for 41 TFs. To validate our predictions, we conductedin vitroassays and confirmed a predicted effector metabolite for LeuO. As a result, we established a network of 80 regulatory interactions between 71 metabolites and 41E. coliTFs. This network includes 76 novel interactions that encompass a diverse range of chemical classes and regulatory patterns, bringing us closer to a comprehensive TF regulatory network inE. coli.

Fucoidans—complex polysaccharides produced by brown algae and diatoms—contribute to long-term carbon sequestration due to their resistance to microbial degradation[1][1],[2][2]. While individual microbes can break down portions of these polymers[3][3]–[5][4], it remains unclear whether complete breakdown is possible in nature and, if so, by what mechanisms. Here we show that fucoidans are degraded through synergistic interactions between specialized bacteria with conserved metabolic functions. Using metabolomic analysis of a reconstructed marine consortium, we uncovered functional guilds of bacteria that target either the sulfated fucose backbone or the side-branches of rare monomers. This division of labor leads to an unexpectedly high number of positive interactions between different degraders that enhanced degradation efficiency up to 97.1%. Despite variation in fucoidan structure across different types of algae, the metabolic functions of degraders remained conserved, enabling quantitative prediction of degradation outcomes based on community composition. Our findings suggest that the environmental turnover of complex macromolecules depends not only on individual metabolic capabilities but also on ecological interactions shaped by substrate architecture. This work provides a mechanistic framework for understanding carbon cycling in the ocean and for engineering synthetic microbial consortia to degrade recalcitrant polysaccharides. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-5

Host-microbiota mutualism is rooted in the exchange of dietary and metabolic molecules. Microbial diversity broadens the metabolite pool, with each taxon contributing distinct compounds in varying proportions. In the human microbiome, high variability in consortial composition is largely compensated by similar metabolic functions across different taxa. However, the extent of compensation in lower diversity mouse models, and whether vivaria are metabolically equivalent, is unknown. We provide a searchable resource of microbiome composition variability across 51 murine vivaria and 12 wild mouse colonies worldwide, with vivarium-specific variants mapped according to predicted 3D structures for each microbial species. Our matched metabolomics data show that realized metabolic potential has relatively low variability, providing functional evidence for metabolic compensation. Additionally, variability is related to taxonomic composition rather than vivarium, revealing taxa-metabolite associations that are potentially relevant to phenotypic differences between vivaria. Collectively, this resource offers tools to strengthen microbiome studies and collaborative science.

Metabolic cross-feeding networks are central to shaping microbial community dynamics in environments ranging from the rhizosphere, gut, and marine carbon cycling. Yet cross-feeding has predominantly been viewed by examining exchanged small metabolites. In contrast, the role of extracellular polymeric substance (EPS)-a complex mixture of proteins, polysaccharides, DNA, and humic-like compounds-in cross-feeding remains poorly understood, mainly due to technical challenges in measuring their secretion relative to small metabolites. Using chitin-degrading microbes as a model system, we used a bicarbonate-buffered bioreactor coupled with elemental analysis, which allowed us to quantify both EPS and small metabolite secretion. This revealed that ~25% of carbon exuded by a chitin degrader is in the form of EPS. EPS was produced at similar levels across marine chitin-degrading isolates and seawater communities, underscoring its importance relative to small metabolites. Notably, different sources of EPS were found to select for distinct and diverse microbial communities. Combining in vitro enzyme assays and untargeted metabolomics, we show that EPS undergoes sequential degradation-from large oligomers to smaller, broadly accessible monomers. This sequential breakdown creates a temporal succession of metabolic niches, potentially fueling a shift from specialist species degrading complex substrates to a more diverse community of generalists using simpler monomers. By identifying EPS as a major and dynamic contributor to cross-feeding networks, our findings reveal a hidden layer of complexity in how microbial communities assemble and function across ecosystems.

Polysaccharide quantification plays a vital role in understanding ecological and nutritional processes in microbes, plants, and animals. Traditional methods typically hydrolyze these large molecules into monomers using chemical methods, but such approaches do not work for all polysaccharides. Enzymatic degradation is a promising alternative but typically requires the use of characterized recombinant enzymes or characterized microbial isolates that secrete enzymes. In this study, we introduce a versatile method that employs undefined enzyme cocktails secreted by individual microbes or complex environmental microbial communities for the hydrolysis of polysaccharides. We focus on colloidal chitin and laminarin as representative polysaccharides of ecological relevance. Our results demonstrate that colloidal chitin can be effectively digested with an enzyme cocktail derived from a chitin-degrading Psychromonas sp. isolate. Utilizing a 3,5-dinitrosalicylic acid reducing sugar assay or liquid chromatography-mass spectrometry for monomer and oligomer detection, we successfully determined chitin concentrations as low as 62 and 15 mg/l, respectively. This allows for effective monitoring of microbial chitin degradation. To extend the applicability of our method, we also leveraged complex, undefined microbial communities as sources of enzyme cocktails capable of degrading laminarin. With this approach, we achieved a detection limit of 30 mg/l laminarin through the reducing sugar assay. Our findings highlight the potential of utilizing enzyme cocktails from both individual microbes and, notably, from undefined microbial communities for polysaccharide quantification. This advancement addresses limitations associated with traditional chemical hydrolysis methods.

The activity of bacterial transcription factors (TFs) is typically modulated through direct interactions with small molecules. However, these input signals remain unknown for most TFs, even in well-studied model bacteria. Identifying these signals typically requires tedious experiments for each TF. Here, we develop a systematic workflow for the identification of TF input signals in bacteria based on metabolomics and transcriptomics data. We inferred the activity of 173 TFs from published transcriptomics data and determined the abundance of 279 metabolites across 40 matched experimental conditions in Escherichia coli . By correlating TF activities with metabolites abundances, we successfully identified previously known TF-metabolite interactions and predicted novel TF effector metabolites for 41 TFs. To validate our predictions, we conducted in vitro assays and confirmed a predicted effector metabolite for LeuO. As a result, we established a network of 80 regulatory interactions between 71 metabolites and 41 E. coli TFs. This network includes 76 novel interactions that encompass a diverse range of chemical classes and regulatory patterns, bringing us closer to a comprehensive TF regulatory network in E. coli . ### Competing Interest Statement The authors have declared no competing interest.

Microorganisms primarily utilize nutrients to generate biomass and replicate. When a single nutrient source is available, the produced biomass typically increases linearly with the initial amount of that nutrient. This linear trend can be accurately predicted by “black box models”, which conceptualize growth as a single chemical reaction, treating nutrients as substrates and biomass as a product. However, natural environments usually present multiple nutrient sources, prompting us to extend the black box framework to incorporate catabolism, anabolism, and biosynthesis of biomass precursors. This modification allows for the quantification of co-utilization effects among multiple nutrients on microbial biomass production. The extended model differentiates between different types of nutrients: non-degradable nutrients, which can only serve as a biomass precursor, and degradable nutrients, which can also be used as an energy source. We experimentally demonstrated using Escherichia coli that, in contrast to initial model predictions, different nutrients affect each other’s utilization in a mutually dependent manner; i.e., for some combinations, the produced biomass was no longer proportional to the initial amounts of nutrients present. To account for these mutual effects within a black box framework, we phenomenologically introduced an interaction between the metabolic processes involved in utilizing the nutrient sources. This phenomenological model qualitatively captures the experimental observations and, unexpectedly, predicts that the total produced biomass is influenced not only by the combination of nutrient sources but also by their relative initial amounts – a prediction we subsequently validated experimentally. Moreover, the model identifies which metabolic processes – catabolism, anabolism, or precursor biosynthesis—is affected in each specific nutrient combination, offering insights into microbial metabolic coordination.

Recent physical binding screens suggest that protein-metabolite interactions are more extensive than previously recognized. To elucidate the functional relevance of these interactions, we developed a mass spectrometry-based screening method for higher throughput in vitro enzyme assays. By systematically quantifying the effects of 79 metabolites on the activity of 20 central Escherichia coli enzymes, we not only assess functional relevance but also gauge the depth of the current understanding of regulatory interactions within one of the best-characterized networks. Our identification of 50 inhibitors and 14 activators not only expands the range of known input signals but also uncovers novel regulatory logic. For instance, we observed that AMP inhibits malic enzyme to safeguard the cyclic operation of the tricarboxylic acid cycle, and erythrose-4-phosphate inhibits 6-phosphogluconate dehydrogenase to redirect flux from the pentose phosphate pathway into the Entner-Doudoroff pathway. Discrepancies between our standardized assays and existing database entries suggest that many previously reported interactions might occur only under specific, often nonphysiological conditions. Our dataset represents a systematically determined functional protein-metabolite interaction network, establishing a baseline for allosteric regulation in central metabolism. These results enhance our understanding of the regulatory logic governing metabolic processes and underscore its significance in cellular adaptation and growth.

A hallmark of the main secreted antibody immunoglobulin A (IgA) is its mutational load that accumulates throughout life. Although this is mainly interpreted in terms of continuing microbial induction, we show that dietary composition during early life can promote IgA induction, its repertoire, and mutational diversification independently of microbial exposure. Using germ-free and colonized mice fed different diets formulated with proprietary grain-based processing or from purified chemicals with different principal macronutrient calorie sources, we found that dietary lipopolysaccharide contamination led to Toll-like receptor (TLR) 4 signaling and promoted germinal center activity in the intestinal immune compartment. The effects of lipopolysaccharide on mucosal immune induction were phenocopied only when presented within colloidal liposomes rather than in dispersed solution. These findings indicate that dietary composition and its formulation can leave a durable impression on the resultant IgA repertoire.

The carbohydrates that fuel gut colonization by S. Typhimurium are not fully known. To investigate this, we designed a quality-controlled mutant pool to probe the metabolic capabilities of this enteric pathogen. Using neutral genetic barcodes, we tested 35 metabolic mutants across five different mouse models with varying microbiome complexities, allowing us to differentiate between context-dependent and context-independent nutrient sources. Results showed that S. Typhimurium uses D-mannose, D-fructose and likely D-glucose as context-independent carbohydrates across all five mouse models. The utilization of D-galactose, N-acetylglucosamine and hexuronates, on the other hand, was context-dependent. Furthermore, we showed that D-fructose is important in strain-to-strain competition between Salmonella serovars. Complementary experiments confirmed that D-glucose, D-fructose, and D-galactose are excellent niches for S. Typhimurium to exploit during colonization. Quantitative measurements revealed sufficient amounts of carbohydrates, such as D-glucose or D-galactose, in the murine cecum to drive S. Typhimurium colonization. Understanding these key substrates and their context-dependent or -independent use by enteric pathogens will inform the future design of probiotics and therapeutics to prevent diarrheal infections such as non-typhoidal salmonellosis.