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

A mechanistic microbial underpinning for the size-reactivity continuum of dissolved organic carbon degradation

The reservoir of dissolved organic carbon (DOC) in the ocean is modified by multiple input and removal processes. Incubation experiments as well as measurements of oceanic DOC have demonstrated that the high molecular weight (HMW) fraction of DOC typically has a younger radiocarbon age and is more reactive biologically than the low molecular weight (LMW) fraction of DOC. These observations have been summarized as a ‘size-reactivity continuum’ of DOC reactivity, but mechanistic explanations for these observations have been lacking. Here we describe how our recent discovery of ‘selfish’ HMW organic matter uptake among bacteria in surface ocean waters may help explain the rapid removal of HMW DOC. ‘Selfish’ substrate uptake by bacteria encompasses rapid binding and partial hydrolysis of intact polysaccharides on the outer membrane of bacteria, seamlessly followed by the transport of large oligosaccharide fragments into the periplasm with little to no loss of LMW hydrolysis products. ‘Selfish’ bacteria therefore process HMW substrates in a manner distinct from bacteria that carry out extracellular hydrolysis that yields LMW hydrolysis products in the environment. Recognition of the presence and prevalence of selfish bacteria in the ocean has profound implications for carbon flow – the source and quantity of LMW substrates made available to non-extracellular-enzyme producing bacteria – as well as for efforts to model and measure bacterial interactions during organic matter degradation. This discovery also highlights the importance of targeted substrate binding and uptake as key (often understudied) factors in geochemical investigations of microbially driven carbon cycling in the ocean. We conclude with some speculative thoughts about the factors that may determine the prevalence of selfish substrate uptake in the environment

Approaches to Investigate Selective Dietary Polysaccharide Utilization by Human Gut Microbiota at a Functional Level

The human diet is temporally and spatially dynamic, and influenced by culture, regional food systems, socioeconomics, and consumer preference. Such factors result in enormous structural diversity of ingested glycans that are refractory to digestion by human enzymes. To convert these glycans into metabolizable nutrients and energy, humans rely upon the catalytic potential encoded within the gut microbiome, a rich collective of microorganisms residing in the gastrointestinal tract. The development of high-throughput sequencing methods has enabled microbial communities to be studied with more coverage and depth, and as a result, cataloging the taxonomic structure of the gut microbiome has become routine. Efforts to unravel the microbial processes governing glycan digestion by the gut microbiome, however, are still in their infancy and will benefit by retooling our approaches to study glycan structure at high resolution and adopting next-generation functional methods. Also, new bioinformatic tools specialized for annotating carbohydrate-active enzymes and predicting their functions with high accuracy will be required for deciphering the catalytic potential of sequence datasets. Furthermore, physiological approaches to enable genotype-phenotype assignments within the gut microbiome, such as fluorescent polysaccharides, has enabled rapid identification of carbohydrate interactions at the single cell level. In this review, we summarize the current state-of-knowledge of these methods and discuss how their continued development will advance our understanding of gut microbiome function

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

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

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

Implementing a public web based GIS service for feedback of surveillance data on communicable diseases in Sweden

BACKGROUND: Surveillance data allow for analysis, providing public health officials and policy-makers with a basis for long-term priorities and timely information on possible outbreaks for rapid response (data for action). In this article we describe the considerations and technology behind a newly introduced public web tool in Sweden for easy retrieval of county and national surveillance data on communicable diseases. METHODS: The web service was designed to automatically present updated surveillance statistics of some 50 statutory notifiable diseases notified to the Swedish Institute for Infectious Disease Control (SMI). The surveillance data is based on clinical notifications from the physician having treated the patient and laboratory notifications, merged into cases using a unique personal identification number issued to all Swedish residents. The web service use notification data from 1997 onwards, stored in a relational database at the SMI. RESULTS: The web service presents surveillance data to the user in various ways; tabulated data containing yearly and monthly disease data per county, age and sex distribution, interactive maps illustrating the total number of cases and the incidence per county and time period, graphs showing the total number of cases per week and graphs illustrating trends in the disease data. The system design encompasses the database (storing the data), the web server (holding the web service) and an in-the-middle computer (to ensure good security standards). CONCLUSIONS: The web service has provided the health community, the media, and the public with easy access to both timely and detailed surveillance data presented in various forms. Since it was introduced in May 2003, the system has been accessed more than 1,000,000 times, by more than 10,000 different viewers (over 12.600 unique IP-numbers)

Metabolism of a hybrid algal galactan by members of the human gut microbiome

Native porphyran is a hybrid of porphryan and agarose. As a common element of edible seaweed, this algal galactan is a frequent component of the human diet. Bacterial members of the human gut microbiota have acquired polysaccharide utilization loci (PULs) that enable the metabolism of porphyran or agarose. However, the molecular mechanisms that underlie the deconstruction and use of native porphyran remains incompletely defined. Here, we have studied two human gut bacteria, porphyranolytic Bacteroides plebeius and agarolytic Bacteroidesuniformis, that target native porphyran. This reveals an exo-based cycle of porphyran depolymerization that incorporates a keystone sulfatase. In both PULs this cycle also works together with a PUL-encoded agarose depolymerizing machinery to synergistically reduce native porphyran to monosaccharides. This provides a framework for understanding the deconstruction of a hybrid algal galactan, and insight into the competitive and/or syntrophic relationship of gut microbiota members that target rare nutrients

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

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