Pulsed inputs of high molecular weight organic matter shift the mechanisms of substrate utilisation in marine bacterial communities.

Heterotrophic bacteria hydrolyze high molecular weight (HMW) organic matter extracellularly prior to uptake, resulting in diffusive loss of hydrolysis products. An alternative 'selfish' uptake mechanism that minimises this loss has recently been found to be common in the ocean. We investigated how HMW organic matter addition affects these two processing mechanisms in surface and bottom waters at three stations in the North Atlantic Ocean. A pulse of HMW organic matter increased cell numbers, as well as the rate and spectrum of extracellular enzymatic activities at both depths. The effects on selfish uptake were more differentiated: in Gulf Stream surface waters and productive surface waters south of Newfoundland, selfish uptake of structurally simple polysaccharides increased upon HMW organic matter addition. The number of selfish bacteria taking up structurally complex polysaccharides, however, was largely unchanged. In contrast, in the oligotrophic North Atlantic gyre, despite high external hydrolysis rates, the number of selfish bacteria was unchanged, irrespective of polysaccharide structure. In deep bottom waters (> 4000 m), structurally complex substrates were processed only by selfish bacteria. Mechanisms of substrate processing-and the extent to which hydrolysis products are released to the external environment-depend on substrate structural complexity and the resident bacterial community.

Distinct bacterial succession and functional response to alginate in the South, Equatorial, and North Pacific Ocean.

The availability of alginate, an abundant macroalgal polysaccharide, induces compositional and functional responses among marine microbes, but these dynamics have not been characterized across the Pacific Ocean. We investigated alginate-induced compositional and functional shifts (e.g., heterotrophic production, glucose turnover, hydrolytic enzyme activities) of microbial communities in the South Subtropical, Equatorial, and Polar Frontal North Pacific in mesocosms. We observed that shifts in response to alginate were site-specific. In the South Subtropical Pacific, prokaryotic cell counts, glucose turnover, and peptidase activities changed the most with alginate addition, along with the enrichment of the widest range of particle-associated taxa (161 amplicon sequence variants; ASVs) belonging to Alteromonadaceae, Rhodobacteraceae, Phormidiaceae, and Pseudoalteromonadaceae. Some of these taxa were detected at other sites but only enriched in the South Pacific. In the Equatorial Pacific, glucose turnover and heterotrophic prokaryotic production increased most rapidly; a single Alteromonas taxon dominated (60% of the community) but remained low (<2%) elsewhere. In the North Pacific, the particle-associated community response to alginate was gradual, with a more limited range of alginate-enriched taxa (82 ASVs). Thus, alginate-related ecological and biogeochemical shifts depend on a combination of factors that include the ability to utilize alginate, environmental conditions, and microbial interactions.

Distinct actors drive different mechanisms of biopolymer processing in polar marine coastal sediments.

Heterotrophic bacteria in the ocean initiate biopolymer degradation using extracellular enzymes that yield low molecular weight hydrolysis products in the environment, or by using a selfish uptake mechanism that retains the hydrolysate for the enzyme-producing cell. The mechanism used affects the availability of hydrolysis products to other bacteria, and thus also potentially the composition and activity of the community. In marine systems, these two mechanisms of substrate processing have been studied in the water column, but to date, have not been investigated in sediments. In surface sediments from an Arctic fjord of Svalbard, we investigated mechanisms of biopolymer hydrolysis using four polysaccharides and mucin, a glycoprotein. Extracellular hydrolysis of all biopolymers was rapid. Moreover, rapid degradation of mucin suggests that it may be a key substrate for benthic microbes. Although selfish uptake is common in ocean waters, only a small fraction (0.5%-2%) of microbes adhering to sediments used this mechanism. Selfish uptake was carried out primarily by Planctomycetota and Verrucomicrobiota. The overall dominance of extracellular hydrolysis in sediments, however, suggests that the bulk of biopolymer processing is carried out by a benthic community relying on the sharing of enzymatic capabilities and scavenging of public goods.

Strong seasonal differences of bacterial polysaccharide utilization in the North Sea over an annual cycle.

Marine heterotrophic bacteria contribute considerably to global carbon cycling, in part by utilizing phytoplankton-derived polysaccharides. The patterns and rates of two different polysaccharide utilization modes - extracellular hydrolysis and selfish uptake - have previously been found to change during spring phytoplankton bloom events. Here we investigated seasonal changes in bacterial utilization of three polysaccharides, laminarin, xylan and chondroitin sulfate. Strong seasonal differences were apparent in mode and speed of polysaccharide utilization, as well as in bacterial community compositions. Compared to the winter month of February, during the spring bloom in May, polysaccharide utilization was detected earlier in the incubations and a higher portion of all bacteria took up laminarin selfishly. Highest polysaccharide utilization was measured in June and September, mediated by bacterial communities that were significantly different from spring assemblages. Extensive selfish laminarin uptake, for example, was detectible within a few hours in June, while extracellular hydrolysis of chondroitin was dominant in September. In addition to the well-known Bacteroidota and Gammaproteobacteria clades, the numerically minor verrucomicrobial clade Pedosphaeraceae could be identified as a rapid laminarin utilizer. In summary, polysaccharide utilization proved highly variable over the seasons, both in mode and speed, and also by the bacterial clades involved.

Short-term changes in polysaccharide utilization mechanisms of marine bacterioplankton during a spring phytoplankton bloom.

Spring phytoplankton blooms in temperate environments contribute disproportionately to global marine productivity. Bloom-derived organic matter, much of it occurring as polysaccharides, fuels biogeochemical cycles driven by interacting autotrophic and heterotrophic communities. We tracked changes in the mode of polysaccharide utilization by heterotrophic bacteria during the course of a diatom-dominated bloom in the German Bight, North Sea. Polysaccharides can be taken up in a 'selfish' mode, where initial hydrolysis is coupled to transport into the periplasm, such that little to no low-molecular weight (LMW) products are externally released to the environment. Alternatively, polysaccharides hydrolyzed by cell-surface attached or free extracellular enzymes (external hydrolysis) yield LMW products available to the wider bacterioplankton community. In the early bloom phase, selfish activity was accompanied by low extracellular hydrolysis rates of a few polysaccharides. As the bloom progressed, selfish uptake increased markedly, and external hydrolysis rates increased, but only for a limited range of substrates. The late bloom phase was characterized by high external hydrolysis rates of a broad range of polysaccharides and reduced selfish uptake of polysaccharides, except for laminarin. Substrate utilization mode is related both to substrate structural complexity and to the bloom-stage dependent composition of the heterotrophic bacterial community.

Sequencing data discovery with MetaSeek.

SUMMARY: Sequencing data resources have increased exponentially in recent years, as has interest in large-scale meta-analyses of integrated next-generation sequencing datasets. However, curation of integrated datasets that match a user's particular research priorities is currently a time-intensive and imprecise task. MetaSeek is a sequencing data discovery tool that enables users to flexibly search and filter on any metadata field to quickly find the sequencing datasets that meet their needs. MetaSeek automatically scrapes metadata from all publicly available datasets in the Sequence Read Archive, cleans and parses messy, user-provided metadata into a structured, standard-compliant database and predicts missing fields where possible. MetaSeek provides a web-based graphical user interface and interactive visualization dashboard, as well as a programmatic API to rapidly search, filter, visualize, save, share and download matching sequencing metadata. AVAILABILITY AND IMPLEMENTATION: The MetaSeek online interface is available at https://www.metaseek.cloud/. The MetaSeek database can also be accessed via API to programmatically search, filter and download all metadata. MetaSeek source code, metadata scrapers and documents are available at https://github.com/MetaSeek-Sequencing-Data-Discovery/metaseek/.

Community structural differences shape microbial responses to high molecular weight organic matter.

The extent to which differences in microbial community structure result in variations in organic matter (OM) degradation is not well understood. Here, we tested the hypothesis that distinct marine microbial communities from North Atlantic surface and bottom waters would exhibit varying compositional succession and functional shifts in response to the same pool of complex high molecular weight (HMW-OM). We also hypothesized that microbial communities would produce a broader spectrum of enzymes upon exposure to HMW-OM, indicating a greater potential to degrade these compounds than reflected by initial enzymatic activities. Our results show that community succession in amended mesocosms was congruent with cell growth, increased bacterial production and most notably, with substantial shifts in enzymatic activities. In all amended mesocosms, closely related taxa that were initially rare became dominant at time frames during which a broader spectrum of active enzymes were detected compared to initial timepoints, indicating a similar response among different communities. However, succession on the whole-community level, and the rates, spectra and progression of enzymatic activities, reveal robust differences among distinct communities from discrete water masses. These results underscore the crucial role of rare bacterial taxa in ocean carbon cycling and the importance of bacterial community structure for HMW-OM degradation.

Structure and function of high Arctic pelagic, particle-associated and benthic bacterial communities.

Arctic marine microbes are affected by environmental changes that may ultimately influence their functions in carbon cycling. Here, we investigated in concert the structure and enzymatic activities of pelagic, particle-associated and benthic bacterial communities in the central Arctic Ocean, and used these data to evaluate microbial structure-function relationships. Our findings showed influences of hydrographic conditions and particle association on community composition, and sharp pelagic-benthic contrasts. In addition to community compositional differences, regional and depth-related patterns in enzymatic activities were observed. Peptide hydrolysis rates were highest in surface waters, especially at ice-free and first year ice-covered regions, and decreased with depth. While the range of hydrolysed polysaccharides showed varying geographic patterns, particles often showed a wider spectrum of polysaccharide hydrolase activities. Summed benthic peptidase rates differed across stations but showed similar proportions of individual enzyme activities. Analysing for potential linkages between structure and function after subtracting the effect of environmental conditions revealed no direct link, indicating functional redundancy to carry out peptide hydrolysis among pelagic microbes. Thus, while community composition and activities are influenced by environmental conditions, bacterial functional redundancy suggests that compositional shifts - in response to the changing Arctic - may have complex and less predictable functional consequences than previously anticipated. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Different utilization of alginate and other algal polysaccharides by marine Alteromonas macleodii ecotypes.

The marine bacterium Alteromonas macleodii is a copiotrophic r-strategist, but little is known about its potential to degrade polysaccharides. Here, we studied the degradation of alginate and other algal polysaccharides by A. macleodii strain 83-1 in comparison to other A. macleodii strains. Cell densities of strain 83-1 with alginate as sole carbon source were comparable to those with glucose, but the exponential phase was delayed. The genome of 83-1 was found to harbour an alginolytic system comprising five alginate lyases, whose expression was induced by alginate. The alginolytic system contains additional CAZymes, including two TonB-dependent receptors, and is part of a 24 kb genomic island unique to the A. macleodii 'surface clade' ecotype. In contrast, strains of the 'deep clade' ecotype contain only a single alginate lyase in a separate 7 kb island. This difference was reflected in an eightfold greater efficiency of surface clade strains to grow on alginate. Strain 83-1 furthermore hydrolysed laminarin, pullulan and xylan, and corresponding polysaccharide utilization loci were detected in the genome. Alteromonas macleodii alginate lyases were predominantly detected in Atlantic Ocean metagenomes. The demonstrated hydrolytic capacities are likely of ecological relevance and represent another level of adaptation among A. macleodii ecotypes.

Niche separation in bacterial communities and activities in porewater, loosely attached, and firmly attached fractions in permeable surface sediments.

Heterotrophic microbes are central to organic matter degradation and transformation in marine sediments. Currently, most investigations of benthic microbiomes do not differentiate between processes in the porewater and on the grains and, hence, only show a generalized picture of the community. This limits our understanding of the structure and functions of sediment microbiomes. To address this problem, we fractionated sandy surface sediment microbial communities from a coastal site in Isfjorden, Svalbard, into cells associated with the porewater, loosely attached to grains, and firmly attached to grains; we found dissimilar bacterial communities and metabolic activities in these fractions. Most (84%-89%) of the cells were firmly attached, and this fraction comprised more anaerobes, such as sulfate reducers, than the other fractions. The porewater and loosely attached fractions (3% and 8%-13% of cells, respectively) had more aerobic heterotrophs. These two fractions generally showed a higher frequency of dividing cells, polysaccharide (laminarin) hydrolysis rates, and per-cell O2 consumption than the firmly attached cells. Thus, the different fractions occupy distinct niches within surface sediments: the firmly attached fraction is potentially made of cells colonizing areas on the grain that are protected from abrasion, but might be more diffusion-limited for organic matter and electron acceptors. In contrast, the porewater and loosely attached fractions are less resource-limited and have faster growth. Their cell numbers are kept low possibly through abrasion and exposure to grazers. Differences in community composition and activity of these cell fractions point to their distinct roles and contributions to carbon cycling within surface sediments.

Selfish bacteria are active throughout the water column of the ocean.

Heterotrophic bacteria in the ocean invest carbon, nitrogen, and energy in extracellular enzymes to hydrolyze large substrates to smaller sizes suitable for uptake. Since hydrolysis products produced outside of a cell may be lost to diffusion, the return on this investment is uncertain. Selfish bacteria change the odds in their favor by binding, partially hydrolyzing, and transporting polysaccharides into the periplasmic space without loss of hydrolysis products. We expected selfish bacteria to be most common in the upper ocean, where phytoplankton produce abundant fresh organic matter, including complex polysaccharides. We, therefore, sampled water in the western North Atlantic Ocean at four depths from three stations differing in physiochemical conditions; these stations and depths also differed considerably in microbial community composition. To our surprise, we found that selfish bacteria are common throughout the water column of the ocean, including at depths greater than 5500 m. Selfish uptake as a strategy thus appears to be geographically-and phylogenetically-widespread. Since processing and uptake of polysaccharides require enzymes that are highly sensitive to substrate structure, the activities of these bacteria might not be reflected by measurements relying on uptake only of low molecular weight substrates. Moreover, even at the bottom of the ocean, the supply of structurally-intact polysaccharides, and therefore the return on enzymatic investment, must be sufficient to maintain these organisms.

Particles act as 'specialty centers' with expanded enzymatic function throughout the water column in the western North Atlantic.

Heterotrophic bacteria initiate the degradation of high molecular weight organic matter by producing an array of extracellular enzymes to hydrolyze complex organic matter into sizes that can be taken up into the cell. These bacterial communities differ spatially and temporally in composition, and potentially also in their enzymatic complements. Previous research has shown that particle-associated bacteria can be considerably more active than bacteria in the surrounding bulk water, but most prior studies of particle-associated bacteria have been focused on the upper ocean - there are few measurements of enzymatic activities of particle-associated bacteria in the mesopelagic and bathypelagic ocean, although the bacterial communities in the deep are dependent upon degradation of particulate organic matter to fuel their metabolism. We used a broad suite of substrates to compare the glucosidase, peptidase, and polysaccharide hydrolase activities of particle-associated and unfiltered seawater microbial communities in epipelagic, mesopelagic, and bathypelagic waters across 11 stations in the western North Atlantic. We concurrently determined bacterial community composition of unfiltered seawater and of samples collected via gravity filtration (>3 µm). Overall, particle-associated bacterial communities showed a broader spectrum of enzyme activities compared with unfiltered seawater communities. These differences in enzymatic activities were greater at offshore than at coastal locations, and increased with increasing depth in the ocean. The greater differences in enzymatic function measured on particles with depth coincided with increasing differences in particle-associated community composition, suggesting that particles act as 'specialty centers' that are essential for degradation of organic matter even at bathypelagic depths.

Linking lifestyle and foraging strategies of marine bacteria: selfish behaviour of particle-attached bacteria in the northern Adriatic Sea.

Microbe-mediated enzymatic hydrolysis of organic matter entails the production of hydrolysate, the recovery of which may be more or less efficient. The selfish uptake mechanism, recently discovered, allows microbes to hydrolyze polysaccharides and take up large oligomers, which are then degraded in the periplasmic space. By minimizing the hydrolysate loss, selfish behaviour may be profitable for free-living cells dwelling in a patchy substrate landscape. However, selfish uptake seems to be tailored to algal-derived polysaccharides, abundant in organic particles, suggesting that particle-attached microbes may use this strategy. We tracked selfish polysaccharides uptake in surface microbial communities of the northeastern Mediterranean Sea, linking the occurrence of this processing mode with microbial lifestyle. Additionally, we set up fluorescently labelled polysaccharides incubations supplying phytodetritus to investigate a 'pioneer' scenario for particle-attached microbes. Under both conditions, selfish behaviour was almost exclusively carried out by particle-attached microbes, suggesting that this mechanism may represent an advantage in the race for particle exploitation. Our findings shed light on the selfish potential of particle-attached microbes, suggesting multifaceted foraging strategies exerted by particle colonizers.

Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria.

Gut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as "medium grower" and "high grower." Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and "omics" approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem. Video abstract.

Thermophilic anaerobes in Arctic marine sediments induced to mineralize complex organic matter at high temperature.

Marine sediments harbour diverse populations of dormant thermophilic bacterial spores that become active in sediment incubation experiments at much higher than in situ temperature. This response was investigated in the presence of natural complex organic matter in sediments of two Arctic fjords, as well as with the addition of freeze-dried Spirulina or individual high-molecular-weight polysaccharides. During 50 degrees C incubation experiments, Arctic thermophiles catalysed extensive mineralization of the organic matter via extracellular enzymatic hydrolysis, fermentation and sulfate reduction. This high temperature-induced food chain mirrors sediment microbial processes occurring at cold in situ temperatures (near 0 degrees C), yet it is catalysed by a completely different set of microorganisms. Using sulfate reduction rates (SRR) as a proxy for organic matter mineralization showed that differences in organic matter reactivity determined the extent of the thermophilic response. Fjord sediments with higher in situ SRR also supported higher SRR at 50 degrees C. Amendment with Spirulina significantly increased volatile fatty acids production and SRR relative to unamended sediment in 50 degrees C incubations. Spirulina amendment also revealed temporally distinct sulfate reduction phases, consistent with 16S rRNA clone library detection of multiple thermophilic Desulfotomaculum spp. enriched at 50 degrees C. Incubations with four different fluorescently labelled polysaccharides at 4 degrees C and 50 degrees C showed that the thermophilic population in Arctic sediments produce a different suite of polymer-hydrolysing enzymes than those used in situ by the cold-adapted microbial community. Over time, dormant marine microorganisms like these are buried in marine sediments and might eventually encounter warmer conditions that favour their activation. Distinct enzymatic capacities for organic polymer degradation could allow specific heterotrophic populations like these to play a role in sustaining microbial metabolism in the deep, warm, marine biosphere.

Extensive Microbial Processing of Polysaccharides in the South Pacific Gyre via Selfish Uptake and Extracellular Hydrolysis.

Primary productivity occurs throughout the deep euphotic zone of the oligotrophic South Pacific Gyre (SPG), fueled largely by the regeneration of nutrients and thus recycling of organic matter. We investigated the heterotrophic capabilities of the SPG's bacterial communities by examining their ability to process polysaccharides, an important component of marine organic matter. We focused on the initial step of organic matter degradation by measuring the activities of extracellular enzymes that hydrolyze six different polysaccharides to smaller sizes. This process can occur by two distinct mechanisms: "selfish uptake," in which initial hydrolysis is coupled to transport of large polysaccharide fragments into the periplasmic space of bacteria, with little to no loss of hydrolysis products to the external environment, and "external hydrolysis," in which low molecular weight (LMW) hydrolysis products are produced in the external environment. Given the oligotrophic nature of the SPG, we did not expect high enzymatic activity; however, we found that all six polysaccharides were hydrolyzed externally and taken up selfishly in the central SPG, observations that may be linked to a comparatively high abundance of diatoms at the depth and location sampled (75 m). At the edge of the gyre and close to the center of the gyre, four of six polysaccharides were externally hydrolyzed, and a lower fraction of the bacterial community showed selfish uptake. One polysaccharide (fucoidan) was selfishly taken up without measurable external hydrolysis at two stations. Additional incubations of central gyre water from depths of 1,250 and 2,800 m with laminarin (an abundant polysaccharide in the ocean) led to extreme growth of opportunistic bacteria (Alteromonas), as tracked by cell counts and next generation sequencing of the bacterial communities. These Alteromonas appear to concurrently selfishly take up laminarin and release LMW hydrolysis products. Overall, extracellular enzyme activities in the SPG were similar to activities in non-oligotrophic regions, and a considerable fraction of the community was capable of selfish uptake at all three stations. A diverse set of bacteria responded to and are potentially important for the recycling of organic matter in the SPG.

Global ecotypes in the ubiquitous marine clade SAR86.

SAR86 is an abundant and ubiquitous heterotroph in the surface ocean that plays a central role in the function of marine ecosystems. We hypothesized that despite its ubiquity, different SAR86 subgroups may be endemic to specific ocean regions and functionally specialized for unique marine environments. However, the global biogeographical distributions of SAR86 genes, and the manner in which these distributions correlate with marine environments, have not been investigated. We quantified SAR86 gene content across globally distributed metagenomic samples and modeled these gene distributions as a function of 51 environmental variables. We identified five distinct clusters of genes within the SAR86 pangenome, each with a unique geographic distribution associated with specific environmental characteristics. Gene clusters are characterized by the strong taxonomic enrichment of distinct SAR86 genomes and partial assemblies, as well as differential enrichment of certain functional groups, suggesting differing functional and ecological roles of SAR86 ecotypes. We then leveraged our models and high-resolution, remote sensing-derived environmental data to predict the distributions of SAR86 gene clusters across the world's oceans, creating global maps of SAR86 ecotype distributions. Our results reveal that SAR86 exhibits previously unknown, complex biogeography, and provide a framework for exploring geographic distributions of genetic diversity from other microbial clades.

Selfish, sharing and scavenging bacteria in the Atlantic Ocean: a biogeographical study of bacterial substrate utilisation.

Identifying the roles played by individual heterotrophic bacteria in the degradation of high molecular weight (HMW) substrates is critical to understanding the constraints on carbon cycling in the ocean. At five sites in the Atlantic Ocean, we investigated the processing of organic matter by tracking changes in microbial community composition as HMW polysaccharides were enzymatically hydrolysed over time. During this investigation, we discovered that a considerable fraction of heterotrophic bacteria uses a newly-identified 'selfish' mode of substrate processing. We therefore additionally examined the balance of individual substrate utilisation mechanisms at different locations by linking individual microorganisms to distinct substrate utilisation mechanisms. Through FISH and uptake of fluorescently-labelled polysaccharides, 'selfish' organisms were identified as belonging to the Bacteroidetes, Planctomycetes and Gammaproteobacteria. 'Sharing' (extracellular enzyme producing) and 'scavenging' (non-enzyme producing) organisms predominantly belonged to the Alteromonadaceae and SAR11 clades, respectively. The extent to which individual mechanisms prevail depended on the initial population structure of the bacterial community at a given location and time, as well as the growth rate of specific bacteria. Furthermore, the same substrate was processed in different ways by different members of a pelagic microbial community, pointing to significant follow-on effects for carbon cycling.

Single cell fluorescence imaging of glycan uptake by intestinal bacteria.

Microbes in the intestines of mammals degrade dietary glycans for energy and growth. The pathways required for polysaccharide utilization are functionally diverse; moreover, they are unequally dispersed between bacterial genomes. Hence, assigning metabolic phenotypes to genotypes remains a challenge in microbiome research. Here we demonstrate that glycan uptake in gut bacteria can be visualized with fluorescent glycan conjugates (FGCs) using epifluorescence microscopy. Yeast α-mannan and rhamnogalacturonan-II, two structurally distinct glycans from the cell walls of yeast and plants, respectively, were fluorescently labeled and fed to Bacteroides thetaiotaomicron VPI-5482. Wild-type cells rapidly consumed the FGCs and became fluorescent; whereas, strains that had deleted pathways for glycan degradation and transport were non-fluorescent. Uptake of FGCs, therefore, is direct evidence of genetic function and provides a direct method to assess specific glycan metabolism in intestinal bacteria at the single cell level.

Polysaccharide hydrolysis in aggregates and free enzyme activity in aggregate-free seawater from the north-eastern Gulf of Mexico.

Marine snow aggregates represent hotspots of carbon remineralization in the ocean. Various aspects of bacterial dynamics have been investigated on marine snow. To date, extracellular enzymatic activities in aggregates have been measured using small substrate proxies that do not adequately reflect the complexity of biomacromolecules such as polysaccharides, proteins and lipids. To address this issue, we used six structurally distinct, fluorescently labelled polysaccharides to measure enzymatic hydrolysis on aggregates formed with a roller table and in aggregate-free (ambient) seawater from two near-coast sites, north-eastern Gulf of Mexico. A single polysaccharide was incubated in aggregates and ambient seawater. Changes in polysaccharide molecular weight were monitored over time to measure the course of enzymatic hydrolysis. All six polysaccharides were hydrolysed in aggregates, indicating a broad range of enzyme activities in aggregate-associated bacteria. Four substrates were also hydrolysed in ambient waters. Epifluorescence microscopy revealed that nearly all of the bacteria present in original waters were incorporated into aggregates. Therefore hydrolytic activities in ambient waters were presumably due to enzymes spatially disconnected from cells and aggregates. Our results show substantial enzymatic activity in cell/aggregate-free seawater, suggesting a significant role of free enzymes in hydrolytic activity in waters from the north-eastern Gulf of Mexico.