Biology subjects

Gifford, S. M.

Publications and source records attributed to Gifford, S. M..

10 recordsLinked to original sources

bioRxiv2026

Transcriptional Markers of Organic Substrate Utilization in a Marine Bacterium

Marine dissolved organic carbon is a chemically complex substrate pool that fuels heterotrophic bacteria, yet it remains difficult to determine which compounds are used by specific microbes. Bacterial transcriptomes offer a potential biosensor of substrate availability, but the reliability of this approach in chemically mixed substrates remains uncertain. Here, we evaluated the reliability of this transcriptional sensor approach using the model marine bacterium Ruegeria pomeroyi DSS-3 grown on either glucose or a defined mixture containing glycerol, benzoate, succinate, leucine, dimethylsulfoniopropionate, and trimethylamine N-oxide. Genome-wide transcription differed strongly between treatments, with the mixed-substrate treatment enriched in genes associated with C1 metabolism, sulfur oxidation, benzoate degradation, and motility. Across substrates, the most diagnostic transcriptional responses occurred at pathway entry points and first committed reactions, including glucose transport and Entner-Doudoroff metabolism, trimethylamine N-oxide transport and catabolism, and early steps of aerobic benzoate oxidation. In contrast, downstream metabolic genes were less substrate-specific, likely because multiple pathways converged on shared central metabolic intermediates. Transporter transcription was also less consistently diagnostic than expected, although substrate-binding subunits often showed the strongest responses within transporter complexes. Comparisons with previous single-substrate studies indicated that some transcriptional markers, particularly benzoate oxidation genes, remained detectable in the substrate mixture, whereas glycerol and succinate responses were weakened or lost. These findings show that transcriptomics can provide useful insight into bacterial substrate use, but interpretation is most robust when focused on experimentally validated transporters and early pathway genes, and when evaluated in the context of pathway connectivity, cellular physiology, and substrate mixture complexity. IMPORTANCEMicrobes help control how carbon moves through the ocean, but it is often difficult to determine which organic compounds individual bacteria are using. This study tested whether gene expression in the marine bacterium Ruegeria pomeroyi can be used to identify the types of carbon compounds available in its environment. We found that some genes, especially those involved in initial steps of substrate use, provided clear signals of which compounds were present. However, these signals became harder to interpret when the bacterium was exposed to a mixture of compounds rather than a single substrate. We also found that some gene responses reflected broader physiological or behavioral changes rather than direct use of a specific compound. These results show both the promise and limits of using bacterial gene expression as a biosensor of marine carbon chemistry, and they help identify the kinds of genes most useful for interpreting complex environmental transcriptomic data.

microbiology↗

bioRxiv2026

Quantitative metagenomics reveals fine-scale population dynamics across bacteria, archaea, and microbial eukaryotes in an estuarine-coastal continuum

Microbial communities are foundational to marine ecosystem function, yet their diversity is often obscured by broad taxonomic groupings and relative-abundance surveys that mask the dynamics of individual populations. This limitation is especially important across estuarine-coastal gradients, where microbial standing stocks, environmental conditions, and community composition vary sharply over space and time. Here, we used quantitative, genome-resolved metagenomics to examine microbial population dynamics across a one-year estuary-to-ocean transect spanning the Neuse River Estuary, Pamlico Sound, and adjacent North Atlantic shelf waters. Internal standard normalization enabled absolute abundance estimates for single-copy genes and metagenome-assembled genomes (MAGs), allowing individual populations to be tracked as genome equivalents per liter. Bacterial standing stocks were higher in estuarine waters, and communities varied primarily with salinity and season. We recovered 415 MAGs, including 386 bacterial genomes that represented, on average, 52% of bacterial genome equivalents, along with archaeal and eukaryotic representatives. Many abundant MAGs lacked close reference genomes, demonstrating that numerically important coastal populations remain poorly characterized. Genome-resolved abundances revealed pronounced niche partitioning among closely related taxa, including seasonal and spatial turnover of Synechococcus, Cyanobium, and Vulcanococcus populations associated with distinct pigment-defined cytometric groups. Rhodobacteraceae MAGs also showed population-specific correlations with picoeukaryotic MAGs, including a winter offshore Planktomarina population that reached 18% of total bacterial genome equivalents during a Micromonas-associated bloom. By providing absolute population abundances, this study transformed coastal microbiome surveys into numerical frameworks for resolving microbial population structure, ecological interactions, and biogeochemical relevance across dynamic environmental gradients. IMPORTANCEEstuarine and coastal waters contain diverse microbial communities that help regulate food webs and the cycling of carbon and nutrients, but many of the individual microbial populations responsible for these processes remain poorly understood. In this study, we examined bacteria, archaea, and small algae across the Neuse River Estuary, Pamlico Sound, and nearby coastal ocean waters over one year. By measuring the absolute abundance of individual microbial genomes, rather than only their relative proportions, we showed that closely related populations can have very different seasonal and spatial patterns. This was especially clear for cyanobacteria related to Synechococcus and heterotrophic bacteria in the family Rhodobacteraceae, which showed distinct population dynamics and associations with small algae. These results demonstrate how quantitative genome-resolved measurements can reveal hidden microbial population structure and improve our understanding of how microorganisms shape coastal ecosystem function.

genomics↗

bioRxiv2024

Flagella are required to coordinately activate competition and host colonization factors in response to a mechanical signal

Bacteria employ antagonistic strategies to eliminate competitors of an ecological niche. Contact-dependent mechanisms, such as the type VI secretion system (T6SS), are prevalent in host-associated bacteria, yet we know relatively little about how T6SS+ strains make contact with competitors in highly viscous environments, such as host mucus. To better understand how cells respond to and contact one another in such environments, we performed a genome-wide transposon mutant screen of the T6SS-wielding beneficial bacterial symbiont, Vibrio fischeri, and identified two sets of genes that are conditionally required for killing. LPS/capsule and flagellar-associated genes do not affect T6SS directly and are therefore not required for interbacterial killing when cell contact is forced yet are necessary for killing in high-viscosity liquid (hydrogel) where cell-cell contact must be biologically mediated. Quantitative transcriptomics revealed that V. fischeri significantly increases expression of both T6SS genes and cell surface modification factors upon transition from low-to high-viscosity media. Consistent with coincubation and fluorescence microscopy data, flagella are not required for T6SS expression in hydrogel. However, flagella play a key role in responding to the physical environment by promoting expression of the surface modification genes identified in our screen, as well as additional functional pathways important for host colonization including uptake of host-relevant iron and carbon sources, and nitric oxide detoxification enzymes. Our findings suggest that flagella may act as a mechanosensor for V. fischeri to coordinately activate competitive strategies and host colonization factors, underscoring the significance of the physical environment in directing complex bacterial behaviors. SignificanceThe physical environment can have dramatic effects on bacterial behavior, but little is known about how mechanical signals impact antagonistic interactions. Symbiotic bacteria use molecular weapons to eliminate competitors for limited space within highly viscous host tissue and mucus. To better understand how the physical environment affects competition and adhesion within eukaryotic hosts, we used quantitative transcriptomics to reveal the flagella-dependent transcriptional response to bacterial transition from lower to a higher viscosity environment. This work revealed the T6SS interbacterial weapon is coordinately activated with host colonization factors, emphasizing the importance of integrating activation of interbacterial weapons into host colonization pathways to enhance a symbionts ability to successfully colonize the host while efficiently eliminating potential competitors from the host niche.

microbiology↗

bioRxiv2023

Collaborative metabolic curation of an emerging model marine bacterium, Alteromonas macleodii ATCC 27126

Inferring the metabolic capabilities of an organism from its genome is a challenging process, relying on computationally-derived or manually curated metabolic networks. Manual curation can correct mistakes in the draft network and add missing reactions based on the literature, but requires significant expertise and is often the bottleneck for high-quality metabolic reconstructions. Here, we present a synopsis of a community curation workshop for the emerging model marine bacterium Alteromonas macleodii ATCC 27126 and its genome database in BioCyc, focusing on pathways for utilizing organic carbon and nitrogen sources. Due to the scarcity of biochemical information or gene knock-outs, the curation process relied primarily on published growth phenotypes and bioinformatic analyses, including comparisons with related Alteromonas strains. We report full pathways for the utilization of the algal polysaccharides alginate and pectin in contrast to inconclusive evidence for one carbon metabolism and mixed acid fermentation, in accordance with the lack of growth on methanol and formate. Pathways for amino acid degradation are ubiquitous across Alteromonas macleodii strains, yet enzymes in the pathways for the degradation of threonine, tryptophan and tyrosine were not identified. Nucleotide degradation pathways are also partial in ATCC 27126. We postulate that demonstrated growth on nitrate as sole N source proceeds via a nitrate reductase pathway that is a hybrid of known pathways. Our evidence highlights the value of joint and interactive curation efforts, but also shows major knowledge gaps regarding Alteromonas metabolism. The manually-curated metabolic reconstruction is available as a "Tier-2" database on BioCyc. ImportanceMetabolic reconstructions are vital for the systemic understanding of an organisms ecology. Here, we report the outcome of a collaborative, interactive curation workshop to build a curated "metabolic encyclopedia" for Alteromonas macleodii ATCC 27126, a marine heterotrophic bacterium with widespread occurrence. Curating pathways for polysaccharide degradation, one-carbon metabolism, and others closed major knowledge gaps, and identified further avenues of research. Our study highlights how the combination of bioinformatic, genomic and physiological evidence can be harvested into a detailed metabolic model, but also identifies challenges if little experimental data is available for support. Overall, we show how an interactive get-together by a diverse group of scientists can advance the ecological understanding of emerging model bacteria, with relevance for the entire scientific community.

microbiology↗

bioRxiv2023

Microbial Respiration in Contrasting Ocean Provinces via High-Frequency Optical Assays

Microbial respiration plays a pivotal role in the marine carbon cycle, influencing the fraction of fixed carbon that undergoes remineralization versus export to depth. Despite its importance, methodological constraints have led to an inadequate understanding of this process, especially in low-activity oligotrophic and mesopelagic regions. Here, we quantify respiration rates as low as 0.2 {micro}mol O2 L-1 d-1 in contrasting ocean productivity provinces using optical oxygen sensors to identify size-fractionated respiration trends. At the low productivity North Pacific Ocean Station Papa, surface microbial respiration was relatively stable at 1.2 {micro}mol O2 L-1 d-1. Below the surface there was a decoupling of respiration and bacterial production potentially driven by phytodetritus remineralization. Size-fractionated rates showed cells <5 {micro}m contributed the most to Pacific respiration. At the North Atlantic Porcupine Abyssal Plain, the optode measurement frequency was drastically increased. Surface microbial respiration was higher (1.7 {micro}mol O2 L-1 d-1) and decreased by 3-fold below the euphotic zone. The Atlantic filtered fraction contributions to total respiration shifted with the phytoplankton bloom evolution. The high resolution optode method used in the Atlantic is consistent with coinciding in vivo 2-para-(iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride respiratory stain measurements and historical site estimates. We estimate that 58% of gross primary production was respired at the Pacific site and 34% at the Atlantic site, demonstrating that the Atlantic had a higher carbon export potential. This study highlights the dynamic nature of respiration across vertical, temporal, and size-fractionated factors, emphasizing the need for sensitive, high-throughput techniques to better understand ocean ecosystem metabolism.

microbiology↗

bioRxiv2023

Primary production dynamics during the decline phase of the North Atlantic annual spring bloom

The second field campaign of the NASA EXport Processes in the Ocean from RemoTe Sensing (EXPORTS) program was conducted in the late spring of 2021 within the vicinity of the Porcupine Abyssal Plain (49.0{degrees}N, 16.5{degrees}W) in the North Atlantic Ocean. Observations from EXPORTS support previous characterizations of this system as highly productive and organic matter rich, with the majority of primary production occurring in large cells ([≥] 5 {micro}m) such as diatoms that are primarily utilizing nitrate. Rates of total euphotic zone depth-integrated net primary production ranged from 36.4 to 146.6 mmol C m- 2 d-1, with an observational period average f-ratio of 0.74, indicating predominantly new production. Substantial variability in the contribution of small (<5 {micro}m) and large cells occurred over the observation period, coinciding with the end of the annual spring phytoplankton bloom. Physical changes associated with storms appear to have impacted the integrated production rates substantially, enhancing rates by [~]10%. These disturbances altered the balance between contributions of the different phytoplankton size fractions, thus highlighting the important role of mixed layer variability in nutrient entrainment into the upper water column and production dynamics. In diatoms, inputs of silicic acid related to deepening of the mixed layer increased silicic acid uptake rates yet concomitant increases in NPP in large cells was not observed. This campaign serves as the high productivity endmember within the EXPORTS program and as such, elucidates how nutrient concentrations and size class play key roles in both low and high productivity systems, but in differing ways.

ecology↗

bioRxiv2023

Quantitative analysis of food web dynamics in a low export ecosystem

Food webs trace the flow of organic matter and energy among producers and consumers; for pelagic marine food webs, network complexity directly influences the amount and form of carbon exported to the deep ocean via the biological pump. Here we present a synoptic view of mixed layer food web dynamics observed during the late summer 2018 EXport Processes in the Ocean from Remote Sensing (EXPORTS) field campaign in the subarctic Northeast Pacific at the long-running time-series site, Ocean Station Papa. Carbon biomass reservoirs of phytoplankton, microzooplankton, and bacterioplankton, were approximately equal while mesozooplankton biomass was 70% lower. Live organisms composed [~]40% of the total particulate organic carbon within the mixed layer: the remainder was attributed to detritus. Rates of carbon transfer among reservoirs indicated production and assimilation rates were well balanced by losses, leaving little organic carbon available for export. The slight positive net community production rate generated organic carbon that was exported from the system in the form of food web byproducts, such as large fecal pellets generated by mesozooplankton. This characteristically regenerative food web had relatively slow turnover times with small-magnitude transfers of carbon relative to standing stocks that occurred amidst a high background concentration of detrital particles and dissolved organic matter. The concurrent estimation of food web components and rates revealed that separated processes dominated the transfer of carbon within the food web compared to those that contributed to export. Plain Language SummaryThe biological carbon pump drives a downward flux of organic matter from the sunlit surface ocean to the vast ocean interior. Ecological interactions in the surface ocean directly affect the amount and type of carbon that is exported to the deep ocean. In this study, we present a synthesis of the late summer mixed layer food web in the Northeast Pacific that was extensively characterized during the 2018 EXport Processes in the Ocean from Remote Sensing (EXPORTS) field campaign. We found the majority of carbon was recycled within the mixed layer by microbes through multiple transfers between producers and consumers. Larger organisms, mesozooplankton and salps, only consumed a small amount of carbon but through the formation of sinking fecal pellets were the main mechanism of transporting carbon out of the system. The study highlights the need to concurrently study microbial and large organism dynamics to develop a predictive understanding of the fate of organic carbon in the oceans. Key PointsO_LIThe microbial loop dominated carbon flow in the late summer mixed layer food web of the North Pacific, most net production was respired leaving little carbon available for export. C_LIO_LIActive production and consumption of organic carbon occurred amid a high background of detrital particulate organic carbon (58% of total) with slow turnover time, 66 d. C_LIO_LIMesozooplankton which had relatively minor carbon consumption rates created the majority of export production due to efficient repackaging of consumed material. C_LI

ecology↗

bioRxiv2022

Synechococcus nitrogen gene loss in iron-limited ocean regions

Synechococcus are the most abundant cyanobacteria in high latitude regions and are responsible for an estimated 17% of annual marine primary productivity. Despite their biogeochemical importance, Synechococcus populations have been unevenly sampled across the ocean, with most studies focused on low-latitude strains. In particular, the near absence of Synechococcus genomes from high-latitude, High Nutrient Low Chlorophyll (HNLC) regions leaves a gap in our knowledge of picocyanobacterial adaptation to iron limitation and their influence on carbon, nitrogen, and iron cycles. We examined Synechococcus populations from the subarctic North Pacific, a well-characterized HNLC region, with quantitative metagenomics. Assembly with short and long reads produced two near complete Synechococcus metagenome-assembled genomes (MAGs). Quantitative metagenome-derived abundances of these populations matched well with flow cytometry counts, and the Synechococcus MAGs were estimated to comprise >99% of the Synechococcus at Station P. Whereas the Station P Synechococcus MAGs contained multiple genes for adaptation to iron limitation, both genomes lacked genes for uptake and assimilation of nitrate and nitrite, suggesting a dependence on ammonium, urea, and other forms of recycled nitrogen leading to reduced iron requirements. A global analysis of Synechococcus nitrate reductase abundance in the TARA Oceans dataset found nitrate assimilation genes are also lower in other HNLC regions. We propose nitrate and nitrite assimilation gene loss in Synechococcus represents an adaptation to severe iron limitation in high-latitude regions where ammonium availability is higher. Our findings have implications for models that quantify the contribution of cyanobacteria to primary production and subsequent carbon export. SignificanceThe cyanobacterium Synechococcus is a major contributor to ocean primary production and biogeochemistry. Here, we used quantitative metagenomics to assemble and enumerate two Synechococcus genomes from an iron-limited, High Nutrient Low Chlorophyll region. We show these genomes represent the majority of Synechococcus cells at the site and are the first known Synechococcus unable to assimilate either nitrate or nitrite. This gene loss is likely due to the high iron quota of these proteins and predominant availability of recycled forms of nitrogen. Synechococcus loss of nitrate assimilation affects their role in elemental cycles (e.g., carbon, nitrogen, and iron), limits their potential for carbon export, and enhances our understanding of Synechococcus evolution in response to nutrient limitation and competition.

ecology↗

bioRxiv2020

Transcriptional activity differentiates families of Marine Group II Euryarchaeota in the coastal ocean

Marine Group II Euryarchaeota (Candidatus Poseidoniales), abundant but yet-uncultivated members of marine microbial communities, are thought to be (photo)heterotrophs that metabolize dissolved organic matter (DOM) such as lipids and peptides. However, little is known about their transcriptional activity. We mapped reads from a metatranscriptomic time series collected at Sapelo Island (GA, USA) to metagenome-assembled genomes to determine the diversity of transcriptionally-active Ca. Poseidoniales. Summer metatranscriptomes had the highest abundance of Ca. Poseidoniales transcripts, mostly from the O1 and O3 genera within Ca. Thalassarchaeaceae (MGIIb). In contrast, transcripts from fall and winter samples were predominantly from Ca. Poseidoniaceae (MGIIa). Genes encoding proteorhodopsin, membrane-bound pyrophosphatase, peptidase/proteases, and part of the {beta}-oxidation pathway were highly transcribed across abundant genera. Highly transcribed genes specific to Ca. Thalassarchaeaceae included xanthine/uracil permease and receptors for amino acid transporters. Enrichment of Ca. Thalassarchaeaceae transcript reads related to protein/peptide, nucleic acid, and amino acid transport and metabolism, as well as transcript depletion during dark incubations, provided further evidence of heterotrophic metabolism. Quantitative PCR analysis of South Atlantic Bight samples indicated consistently abundant Ca. Poseidoniales in nearshore and inshore waters. Together, our data suggest Ca. Thalassarchaeaceae are important photoheterotrophs potentially linking DOM and nitrogen cycling in coastal waters.

microbiology↗

bioRxiv2018

Mosaic patterns of B-vitamin synthesis and utilization in a natural marine microbial community

Aquatic environments contain diverse microbial communities whose complex interactions mediate the cycling of major and trace nutrients such as vitamins. B-vitamins are essential coenzymes that many organisms cannot synthesize. Thus their exchange among de-novo synthesizers and auxotrophs is expected to play an important role in the microbial consortia and explain some of the temporal and spatial changes observed in diversity. In this study, we analyzed metatranscriptomes of a natural coastal microbial community, diel sampled-quarterly over one year to try to identify the potential major B-vitamin synthesizers and consumers. Our transcriptomic data show that the best-represented taxa dominated the expression of synthesis genes for some B-vitamins but lacked transcripts for others. For instance, Rhodobacterales dominated the expression of vitamin-B12 synthesis, but not of vitamin-B7, whose synthesis transcripts were mainly represented by Flavobacteria.In contrast, bacterial groups that constituted less than 4% of the community (e.g., Verrucomicrobia) accounted for most of the vitamin-B1 synthesis transcripts. Furthermore, ambient vitamin-B1 concentrations were higher in samples collected during the day, and were positively correlated with chlorophyll-a concentration. Our analysis supports the hypothesis that the mosaic of metabolic interdependencies through B-vitamin synthesis and exchange are key processes that contribute to shaping microbial communities in nature.

microbiology↗