Salmonella outbreaks are often linked to contaminated cantaloupes, and bacterial multiple antibiotic resistance is frequently observed. Research is needed to understand the growth and survival of Salmonella on cantaloupe surfaces, and the mechanisms of antibiotic resistance in that environment. This study investigated the growth, survival, and capacity for horizontal gene transfer of antibiotic resistance genes in Salmonella on the surface of cantaloupe. Bovine-derived Salmonella isolates were inoculated onto the surface of cantaloupe and incubated at 35°C under 65-75% humidity for 5 days. Each isolate was co-inoculated with donor E. coli CSH56 harboring helper plasmid RP4-8 and mobilizable plasmid pSC27, which carries kanamycin, neomycin, and ampicillin resistance genes. One hundred microliters of the Salmonella suspension (1 × 10[8] CFU/mL) and 100 µL of the E. coli suspension (1 × 10[7] CFU/mL) was applied to each cantaloupe (1:10 ratio). Salmonella survival and growth were monitored on Hektoen agar, transconjugants were enumerated on Hektoen agar with kanamycin, and donor E. coli were quantified on MacConkey agar. All Salmonella isolates survived and exhibited growth within the 5-day period, with increases ranging from 0.94 log10 CFU/mL to 3.43 log10 CFU/mL. Notably, all Salmonella isolates possess the capability to accept and integrate sequences derived from the pSC27 plasmid from E. coli into their chromosome, both in vitro and on cantaloupe. After conjugation, the count of Salmonella mutants carrying the plasmid was 2.79 log10 CFU/mL in vitro. The survival, growth, and horizontal gene transfer of Salmonella on the surface of cantaloupe pose significant risks to public health.IMPORTANCESalmonella, a leading cause of foodborne diseases, poses a global health threat, with multidrug resistance emerging as a severe problem. Cantaloupes have been implicated in salmonellosis outbreaks in multiple countries over the last three decades. We hypothesize that acquisition of antibiotic resistance genes from other bacteria may occur on the surface of the fruit. This study established that Salmonella can survive, proliferate, and acquire antibiotic resistance genes on cantaloupe surfaces. This observation underscores the potential risks on the growth and transfer of genes between pathogens such as Salmonella and E. coli in foods. This impact goes beyond disease production and contributes to the rapid escalation of antimicrobial resistance and transmission of virulence genes in foods and the environment, which is a significant threat to public health.

Dietary supplement formulations that contain co-biotics or compounds that modulate biological processes in both the host and microbiome to confer a health benefit are an emerging strategy to fine-tune both host physiology and gut microbiome function. Here, we evaluated three novel formulations containing co-biotic compounds (DM-02, a multivitamin; AM-02, formulated for energy and focus; and PM-02, formulated to promote sleep) for their effects on human gut microbiome composition and function. The three formulations and untreated control (Unt-Ctrl) were subjected to upper gastrointestinal digestion, after which the digesta were exposed to 24-h simulated colonic fermentation in the validated ex vivo systemic intestinal fermentation research (SIFR) technology (n = 6 healthy adults). Outcomes included pH, short-chain fatty acid (SCFA) and gas production, ultra-deep metagenomic profiling, and untargeted metabolomics of post-colonic supernatants, each compared to Unt-Ctrl. All three formulations significantly reduced pH (3%-4%) and increased total SCFAs (9%-11%) and acetate (9%-12%), with AM-02 and PM-02 also increasing butyrate by 20% and propionate by 7%-8%, without increasing gas production. Each formulation significantly enriched specific SCFA- and B-vitamin-producing taxa. AM-02 significantly increased the abundance of two pyruvate fermentation to acetate/lactate pathways. PM-02 significantly increased the abundance of two tryptophan biosynthesis pathways, accompanied by an increase in available tryptophan and the abundance of tryptophan-producing microbes. All three formulations increased the availability of microbiome-derived metabolites, indicating microbiome functional modulation by the treatments. These findings support clinical evaluation of these novel formulations as a strategy to enhance microbiome composition and function.

Drosophila suzukii (spotted-wing drosophila) is an invasive pest that infests ripe, market-ready fruit, posing a significant threat to fruit production. To develop alternatives to conventional chemical control, environmentally derived fungi (EF) were evaluated for their potential to modulate D. suzukii behavior and viability. Pichia occidentalis, isolated from Drosophila melanogaster oral and fecal secretions, exhibited species-specific repellency in D. suzukii. In D. suzukii, exposure to P. occidentalis reduced survival, most pronounced in males, and significantly inhibited female oviposition. In contrast, D. melanogaster showed no reduction in survival and increased oviposition in the presence of the fungus. Solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME GC-MS) identified volatile metabolites produced by P. occidentalis. Subsequent cafeteria-style assays of individual volatiles revealed that several of these compounds elicited aversive responses in D. suzukii while remaining attractive to D. melanogaster, highlighting species-specific olfactory effects. Isoamyl butanoate, 2-phenyl-1-propanal, and benzyl alcohol demonstrated consistent repellent activity with superior efficacy compared to established D. suzukii repellents (2-pentylfuran, 1-octen-3-ol, and geosmin). Collectively, these findings demonstrate that EF metabolites can differentially influence closely related species and may be leveraged as targeted, behavior-based tools for managing D. suzukii.IMPORTANCEDrosophila suzukii is a major invasive pest that damages fresh fruit by laying eggs in ripening crops, leading to significant agricultural losses. Current control strategies rely heavily on chemical insecticides, which provide incomplete control and can harm non-target organisms. Here, we show that volatile metabolites produced by the environmental yeast Pichia occidentalis differentially influence closely related Drosophila species, decreasing oviposition and longevity in D. suzukii while stimulating oviposition in D. melanogaster. This species-specific divergence highlights the potential of microbial volatiles as precision tools for pest management, further supporting a behavior-based approach that selectively disrupts pest reproduction while minimizing ecological impact.

Phage steering is an emerging antimicrobial strategy that exploits the evolutionary consequences of phage predation to sensitize bacterial populations to existing antimicrobials. While phage steering toward hypersensitivity to antibiotics and immune effectors has been well documented, little is known about whether a similar mechanism can enhance bacterial sensitivity to commercial disinfectants. Here, we show that SF1, a lytic siphovirus infecting Salmonella enterica, requires an intact O-antigen layer for adsorption and lytic replication, and that in-frame deletion mutants with O-antigen or deeper LPS defects had increased susceptibility to several commercial disinfectants. Critically, SF1 enhanced the activity of all disinfectants tested in combination assays and, moreover, exhibited mathematical synergy with benzalkonium chloride, hydrogen peroxide, and triclosan, compounds to which SF1-selected LPS mutants were hypersensitive. These findings are consistent with the hypothesis that SF1 functions not only as a bactericidal agent, but as an evolutionary steering tool that potentiates sanitizer activity through selection for sanitizer-hypersensitive phenotypes. Broadly, these results support phage steering as a promising strategy for deploying bacteriophages as adjuvants to commercial disinfectants.IMPORTANCECommercial disinfectants are essential for controlling pathogens throughout the food production and processing chain, but their effectiveness is increasingly compromised by bacterial tolerance and persistence. Although bacteriophages are being investigated as direct bactericidal agents, their utility as adjuvants to commercial sanitizers remains underexplored. In this study, we demonstrate that Salmonella phage SF1 selects for LPS-deficient mutants that exhibit increased susceptibility to several commercial disinfectants. Importantly, we further show that SF1 synergizes with sanitizers to which phage-selected mutants are hypersensitive, supporting a steering-mediated mechanism whereby phage predation enhances sanitizer activity. To our knowledge, this is the first report demonstrating phage steering as a strategy for deploying bacteriophages as synergizing adjuvants to commercial disinfectants. These findings highlight the potential value of exploiting evolutionary trade-offs to improve pathogen control in food processing environments.

The coordination of intracellular carbon and nitrogen levels is essential for optimal bacterial growth and, in rhizobia, for survival in the soil, root colonization, and symbiotic interactions with their host plants. Here, we show that the phosphotransferase system PTS[Ntr], a global regulator of carbon and nitrogen metabolism, modulates the tricarboxylic acid (TCA) cycle activity and the accumulation of three major carbon storage polymers: glycogen, polyhydroxybutyrate, and exopolysaccharide in Rhizobium leguminosarum. The unphosphorylated form of the effector protein ManX is sufficient for the full activation of the TCA cycle dehydrogenase enzymes. Accordingly, loss of manX reduced dehydrogenase activity and redirected overflow carbon into storage polymers, phenocopying wild-type cells grown under nitrogen starvation. We further demonstrate that carbon metabolism in R. leguminosarum is also tightly regulated by the HPr kinase (HprK) protein. In an hprK mutant, loss of NPr Ser48 phosphorylation favors phosphotransfer through the histidyl arm and is predicted to increase phosphorylation of downstream PTS[Ntr] components. When grown on glucose, the hprK mutant accumulates elevated intracellular pyrimidine levels and exhibits increased malate dehydrogenase and TCA cycle activity, consistent with a misregulated shift toward gluconeogenesis. This mutant also overproduces exopolysaccharide, an effect potentially mediated by crosstalk between phosphorylated PtsN and the ChvI/ChvG two-component regulatory system. Together, these data suggest that HprK exerts pleiotropic control over processes regulated by PTS[Ntr], influencing central metabolism while repressing exopolysaccharide production, likely by promoting the unphosphorylated state of PtsN.IMPORTANCEBacteria must continuously balance growth, nutrient availability, and storage to ensure fitness and survival in fluctuating environments. However, how regulatory networks integrate central metabolism with carbon storage remains poorly understood. This study reinforces the role of the PTS[Ntr] system as a central coordinator of carbon routing and polymer accumulation. By revealing how ManX and HprK-dependent phosphorylation influence central metabolism and the production of major carbon storage polymers in Rhizobium leguminosarum, we show that disruption of PTS[Ntr] signaling reprograms carbon allocation, uncoupling growth from carbon storage and mimicking nitrogen starvation even under nitrogen-replete conditions. These findings highlight a regulatory link between carbon-nitrogen signaling and bacterial storage strategies, with implications for cellular survival and plant symbiosis. More broadly, our work illustrates how global metabolic regulators shape bacterial physiological states, providing new insights into mechanisms that support microbial adaptation in complex environments.

Listeria monocytogenes integrates into multi-species biofilms and persists in food processing facilities. Its persistence depends on the composition of the microbial communities, but those species that accommodate L. monocytogenes, or not, remain to be identified. Moreover, the decision-making on biofilm vs pellicle formation in microbial communities remains poorly understood. It was the aim of this study to identify communities that accommodate Listeria, or not, and to observe biofilm vs pellicle formation in multi-species biofilms. We quantified L. monocytogenes in six reconstituted communities, each consisting of 5-19 strains. A 15-member community was simplified into five species to quantify L. monocytogenes in all 31 combinations of these five strains. Janthinobacterium tructae favored pellicle formation, while Pseudomonas shahriarae convinced others to join a surface-attached biofilm. In the six microbial communities, cell counts of L. monocytogenes were lowest when Carnobacterium maltaromaticum was also present. The antagonistic effect of C. maltaromaticum was confirmed when analyzing all combinations of five strains. The presence of C. maltaromaticum reduced cell counts of L. monocytogenes by about 1 log CFU/cm[2]. Serratia liquefaciens also antagonized L. monocytogenes, but this was observed (P < 0.05) only in the absence of C. maltaromaticum. Genome analyses of the community identified close to 100 different quorum-sensing systems, including peptides that are also bactericidal against L. monocytogenes. We conclude that the language used to describe human social interactions is also suited to describe microbial interactions in multi-species biofilm communities, but unraveling mechanisms of communication among biofilm-embedded bacteria remains subject to future studies.

Biogeochemical cycles exert significant influences on the fate and behavior of antibiotics in estuarine and coastal environments; however, ferrous sulfide (FeS)-mediated enrichment and dissemination of antibiotic resistance genes (ARGs) remain poorly understood. Here, we conducted microcosm experiments and field investigations to examine the relationships of FeS amendment, microbial communities, functional potentials in relation to elemental cycling, and ARG abundance. The positive effects of FeS on ARG abundance were aerobic dependent, which were accompanied by a significant enrichment of Proteobacteria, as the crucial hosts of ARGs. Proteobacteria were associated with systematic metabolic pathways for carbon fixation (Calvin-Benson-Bassham [CBB] cycle), N2 fixation, and FeS oxidation. In addition, field samples from China estuarine and coastal wetlands further showed that CBB cycle, N2 fixation, and sulfur oxidation pathways were significantly correlated with the relative abundance of ARGs. These findings collectively indicate that FeS oxidation facilitates enrichment and dissemination of ARGs through selective enrichment of Proteobacteria and consequent enhancement of metabolic synergies between sulfur, iron, carbon, and nitrogen cycles. However, FeS-mediated oxidation may also degrade microbial DNA and suppress ARG proliferation, indicating that the net outcome of ARG enrichment depends on oxygen conditions and microbial community filtering. The cascade processes of environmental regulation-community restructuring-metabolic synergy are of great significance to enhance ARG enrichment and dissemination. Therefore, redox fluctuations can enhance elemental cycling and further promote significant enrichment and dissemination of ARGs. These findings highlight the importance of redox dynamics in ARG monitoring and risk assessment in estuarine and coastal wetlands.IMPORTANCEEstuarine and coastal wetlands are important ecosystems for antibiotic resistance gene (ARG) enrichments. This study investigated FeS oxidation-mediated biogeochemical cycling and ARG dissemination. FeS oxidation increased enrichment and dissemination of the ARGs in estuarine and coastal sediments. The FeS oxidation reshaped microbial community structure and selectively enriched the critical Proteobacteria, which played the crucial hosts of the ARGs. The enrichment and dissemination of ARGs in the presence of FeS oxidation were due mainly to the enhanced metabolic coupling of sulfur oxidation, nitrogen fixation, and carbon fixation. CBB cycle, N2 fixation, and sulfur oxidation pathways were also significantly correlated with ARG abundance along the latitudinal gradient. Therefore, enhanced elemental metabolic pathways under FeS oxidation exacerbated enrichment and dissemination of ARGs, highlighting the coupling mechanisms of biogeochemical cycle and ARGs in estuarine and coastal wetlands.

Shewanella oneidensis is one of the most extensively studied electrochemically active bacteria because of its unparalleled respiratory versatility, which is largely built on diverse cytochromes (cyts) c. It has been firmly established that Fur, RyhB, and Hfq are critical for cyt c production through regulation of iron homeostasis, but how they interplay remains largely unknown. In this study, we show that S. oneidensis RyhB carries novel features, such as unusual length and functional exchangeability with its Escherichia coli counterpart but not its Vibrio cholerae counterpart. The RyhB loss suppresses the signature phenotypes caused by the Fur loss, including impaired growth and cyt c production in S. oneidensis. Despite this, neither the direct regulation of expression of most cyt c genes nor the suppression of disrupted iron homeostasis resulting from Fur loss appears to be a primary mechanism underlying RyhB's influence on cyt c content. Rather, this effect is achieved by modulating heme levels; RyhB mediates the expression of the key genes in heme biosynthesis and the entire heme uptake/degradation system. Overall, our work highlights the significance of RyhB-mediated post-transcriptional regulation in the physiology of S. oneidensis, unraveling unexpected mechanisms by which RyhB affects cyt c biosynthesis.

In vitro viability assays are essential for studying the survival of Borrelia burgdorferi after exposure to stressful conditions. In contrast to the cumbersome and tedious manual microscopic counting method, fluorescence-based assays are more rapid and convenient and thus can be used in a high-throughput format. In the SYBR Green I/propidium iodide (PI) assay, spirochetes are labeled with different fluorescent dyes, and fluorescence ratios are then compared to calculate the ratio between live and dead spirochetes. Stained spirochetes are also manually counted under a fluorescence microscope or analyzed via flow cytometry to obtain the live/dead ratio. In this study, we compared these methods to determine the most accurate technique for quantifying spirochete viability. Our results showed that, compared with the SYBR Green I/PI assay that carries inherent caveats, both the fluorescent microscopic counting method and the flow cytometry method provide more reliable and accurate results for B. burgdorferi viability assays, and flow cytometry offers significant advantages in speed and reduced labor compared to manual microscopic counting. Leveraging the flow cytometry-based viability assay, we investigated the role of gene bbi29, encoding a putative virulence-associated lipoprotein, in B. burgdorferi stress response. The contribution of bbi29 to B. burgdorferi mammalian infection was also examined using a murine model of Lyme disease.

Microbial cultivation remains essential for understanding the physiology, ecology, and biotechnological potential of environmental microbes, yet conventional plate-based methods (CPM) recover only a minute fraction of the environmental microbiome. Polar regions, particularly Arctic soils, represent unique reservoirs of "microbial dark matter" that remain challenging to cultivate, owing to oligotrophic conditions, low temperatures, and freeze-thaw cycles that impose severe physiological constraints on microbial growth. Here, we report the first systematic application of microfluidic droplet technology (MDT) to Arctic active-layer soil microbiota and benchmark its performance against CPM using identical starting cell numbers, R2A medium, and incubation at 15°C. MDT achieved 6.5- to 8.1-fold higher recovery rates than CPM and improved isolation throughput by >180-fold. Near-full-length 16S rRNA gene sequencing (PacBio) revealed that MDT recovered significantly higher taxonomic richness across all taxonomic levels, with 256 genera detected in the high-cell-input group (DropAS_H) versus 211 in the corresponding plate group (PlateAS_H). Notably, MDT yielded a more even community distribution, significantly reducing the dominance of fast-growing copiotrophs, such as Pseudomonas and Flavobacterium. Moreover, approximately 50% of the sequences from MDT were affiliated with potential novel species (<98.65% identity to type strains), and 27% with potential novel genera (<95% identity). Strain verification by Sanger sequencing confirmed 12 of 17 isolates as candidate novel species, among which one strain represented a potential novel genus within Devosiaceae. This study demonstrates that MDT is a powerful platform for accessing the uncultured majority of polar soil microbiota and establishes a pipeline for high-throughput isolation of novel cold-adapted bacteria.IMPORTANCEArctic soils harbor a vast reservoir of microbial diversity that remains largely inaccessible due to the extreme oligotrophic conditions and low temperatures characteristic of polar environments, leading to slow growth and extended lag phases in most microbes. Conventional plate-based methods (CPM) inherently favor fast-growing copiotrophs while suppressing rare or slow-growing lineages. Here, we demonstrate that microfluidic droplet technology (MDT) overcomes these fundamental constraints, representing its first systematic application to polar microbiology. By physically isolating individual cells into microliter-scale bioreactors, MDT provides independent microenvironments that allow slow-growing and oligotrophic taxa to proliferate without being outcompeted by fast-growing copiotrophs in bulk cultures. The water-in-oil emulsion format further enables extended low-temperature incubation without evaporative loss or airborne fungal contamination, issues that frequently compromise long-term plate-based cultivation of Arctic samples. Relative to CPM, MDT increased recovery rates by over 6-fold and isolation throughput by 180-fold, while markedly enhancing both taxonomic richness and evenness. Exclusively recovered by MDT, the oligotrophic genus Caulobacter and numerous cold-adapted genera underscore that MDT accesses physiologically distinct fractions of the cryospheric microbiome. Furthermore, the integration of near-full-length 16S rRNA gene sequencing with MDT cultivation assessment provided substantially improved phylogenetic resolution for species-level identification and novel taxon delineation. Collectively, these findings establish MDT as a transformative platform for cryospheric culturomics, accelerating the construction of comprehensive polar strain collections essential for understanding cold-adaptation mechanisms and exploiting the biotechnological potential of Earth's frozen microbiomes.

Francisella tularensis is a zoonotic pathogen transmitted to humans through contact with infected animals, arthropod vectors, or contaminated environmental sources. Although replication occurs primarily within eukaryotic host cells, persistence outside a host is thought to be critical for long-term maintenance and transmission in natural ecosystems. To better understand persistence in aquatic habitats outside of mammalian hosts, we studied virulent strains of F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B) incubated in a planktonic state (free living in water) under various axenic aquatic conditions. After 1 year in cold, nutrient-poor treatments, the type B strain (FSC200) caused disease in mice and remained culturable on laboratory media, whereas the type A strain (Schu S4) caused disease but with delayed onset and became non-responsive to laboratory growth conditions. In 1.5-year persistence experiments, type B still caused disease in mice, but with delayed onset, whereas type A no longer caused disease. The rate of bacterial decline depended on subspecies, temperature, and the initial inoculum size. We further explored the persistence of type A and type B populations in natural pond water, which corroborated findings from axenic conditions and revealed that both strains perpetuated disease from a non-replicative inactive persistent state. This study provides timeframes for the persistence of virulent planktonic F. tularensis subspecies tularensis and holarctica populations in various aquatic conditions, contributing to increased understanding of host-independent survival as a component of the perpetuation of tularemia.

Sulfate reduction is estimated to account for half of the organic carbon respiration in anoxic, organic-rich ocean sediments. Sulfate-reducing microorganisms (SRMs) typically oxidize simple carbon compounds, such as primary alcohols, or small fatty acids. However, much of the organic input to the seafloor is complex, driving interactions between terminal respirers, such as SRMs and methanogens, and primary degraders that initiate the breakdown of complex organic carbon. To explore how these interactions shape sediment microbiomes, we conducted multi-month microcosm experiments using serially diluted sediments in deep 96-well plates. Sediments from a former deep-sea whalefall site were amended with either the insoluble polymer chitin or its soluble monomer N-acetylglucosamine. The high replication in these experiments allowed us to evaluate the effects of complex versus labile carbon on degradation activity, microbial diversity, community structure, and functional redundancy over time. Geochemical analysis, combined with 16S rRNA gene sequencing and metagenomics, revealed that chitin addition preserved higher microbial diversity and increased predicted interactions among microorganisms. Despite anaerobic chitin degradation producing N-acetylglucosamine, microbial communities enriched by these substrates showed low nestedness over the 7-month experiment. Complex carbon inputs fostered unique microbial assemblages and functional interactions, including the emergence of diverse methanogenic lineages at medium to high dilutions-less apparent in monomer treatments. In microoxic sediments, methanogens and other rare biosphere members co-exist with SRM and dynamically respond to complex carbon inputs. Our results highlight the contribution of carbon complexity and recalcitrance in stimulating metabolically diverse community members in sediments, driving the assembly of functional microbial networks.

YggS (COG0325 family) was widely conserved across the three domains of life and had been implicated in pyridoxal 5'-phosphate (PLP) homeostasis. However, transcriptional regulators that directly controlled yggS had remained unknown. Here, using Alcaligenes faecalis JQ135, PhpR, a previously unrecognized MocR-family regulator, was identified as a transcriptional regulator that directly controlled yggS. Deletion of yggS in strain JQ135 impaired cell growth and caused intracellular PLP imbalance. PhpR (PLP homeostasis-related protein regulator, phpR) directly repressed yggS transcription by binding a conserved non-palindromic promoter motif in a PLP-dependent manner. Comparative genomic analyses further revealed that the adjacent yggS-phpR gene pair was divergently transcribed and highly conserved, but restricted to Alcaligenes species. Together, these findings established the first direct transcriptional regulatory mechanism for a YggS-family protein and revealed a PLP-responsive regulatory circuit linking cofactor homeostasis in bacteria.

Metagenomic sequencing is increasingly applied to wastewater to characterize the diversity, dynamics, and relative abundance of human and animal viruses. Among these sequencing approaches are those that enrich viral nucleic acids from the wastewater matrix, aiming to increase the viral read fraction for analysis. However, the feasibility of scaling targeted viral sequencing to diverse sewersheds across large geographic scales is currently unknown. In this study, we apply hybrid capture metagenomic sequencing to nearly 450 weekly wastewater samples collected during the respiratory virus season in the United States and evaluate sequencing performance for generating public health-relevant data. Analysis of data from 15 wastewater treatment plants demonstrates that our approach enabled efficient capture of pathogens of interest, achieving a median viral read fraction over 19%. Importantly, relative abundance estimates of common pathogens correlated with direct quantification of viral targets using reverse transcription digital droplet PCR. Together, our results demonstrate that hybrid capture sequencing of wastewater is a viable tool to monitor both common and rare pathogens across geographically diverse sewersheds.IMPORTANCEWastewater testing is commonly used to identify and quantify human pathogens at a community scale. However, the most commonly used approaches rely on targeted, PCR-based methods that are highly specific to a single virus. Metagenomic sequencing provides an opportunity to detect and quantify the relative abundance of a wide range of viruses that are important for human health from wastewater, and hybrid capture approaches work by first enriching samples for these extremely rare targets to increase sensitivity. We demonstrate that hybrid capture metagenomic sequencing successfully enriches wastewater samples from diverse locations across the United States and that data derived from sequences are associated with detections of 11 key viruses using standard RT-ddPCR methods. This work suggests that scaling hybrid capture metagenomics for viruses in wastewater is a feasible way to generate data critical for public health to support infectious disease outbreak response.

Inorganic carbon use in cellular metabolism is ubiquitous in all living organisms. The assimilation of inorganic carbon into biomass via heterotrophic inorganic carbon fixation (HICF) is estimated to contribute significantly to global carbon fixation, yet the controls on this process in strictly heterotrophic microorganisms remain poorly understood. To identify potential drivers of HICF, we cultivated well-characterized heterotrophic Gammaproteobacteria (Pseudomonas putida and Pseudoalteromonas carrageenovora) at two temperatures (optimal vs colder) and resource treatments (high vs low organic carbon), all of which were amended with NaH[13]CO3. We performed bulk and single-cell stable carbon isotope probing (SIP) analysis to detect and quantify HICF in each condition, and assessed other measures of metabolic state, including C/N ratio, natural abundance nitrogen isotope ratios, and cell size. We observed HICF in all treatments, with the percent of biomass originating from [13]C-dissolved inorganic carbon (DIC) for P. putida (0.006% to 0.029%) and P. carrageenovora (0.18% to 1.1%) on the lower end of estimates for the contribution of anaplerotic fixation to heterotrophic biomass (1% to 8%). HICF was generally highest during exponential phase, at the optimal growth temperature, and under low carbon conditions, and these trends were also observed at the single-cell level. Overall, temperature and resource availability appear to influence HICF rates. These findings suggest that HICF may be more relevant under environmental conditions where DIC and nitrogen are not limiting, and the flux of digestible organic carbon is limited or intermittent. Environmental applications of [13]C-DIC SIP in such systems may record heterotrophic contributions to community carbon fixation.IMPORTANCEHeterotrophic microorganisms are abundant across ecosystems and play a central role in environmental carbon cycling by storing, transforming, and decomposing organic matter. However, heterotrophs can also incorporate inorganic carbon into biomass, but this is rarely considered in models of the carbon cycle. Our experiments on heterotrophic bacteria from soil (Pseudomonas putida) and marine environments (Pseudoalteromonas carrageenovora) show that inorganic carbon uptake is impacted by both resource availability and temperature and may be undetectable without stable isotope probing. By integrating single-cell techniques, our study measures variation in inorganic carbon uptake among individual cells across growth phases and conditions. Our study highlights heterotrophic inorganic carbon fixation as a variable source of [13]C-dissolved inorganic carbon incorporation into microbial biomass, underscoring the importance of considering this process in the design and interpretation of environmental stable isotope probing studies.