The Biogeochemistry of Marine Polysaccharides: Sources, Inventories, and Bacterial Drivers of the Carbohydrate Cycle.

Polysaccharides are major components of macroalgal and phytoplankton biomass and constitute a large fraction of the organic matter produced and degraded in the ocean. Until recently, however, our knowledge of marine polysaccharides was limited due to their great structural complexity, the correspondingly complicated enzymatic machinery used by microbial communities to degrade them, and a lack of readily applied means to isolate andcharacterize polysaccharides in detail. Advances in carbohydrate chemistry, bioinformatics, molecular ecology, and microbiology have led to new insights into the structures of polysaccharides, the means by which they are degraded by bacteria, and the ecology of polysaccharide production and decomposition. Here, we survey current knowledge, discuss recent advances, and present a new conceptual model linking polysaccharide structural complexity and abundance to microbially driven mechanisms of polysaccharide processing. We conclude by highlighting specific future research foci that will shed light on this central but poorly characterized component of the marine carbon cycle.

Verrucomicrobia are candidates for polysaccharide-degrading bacterioplankton in an arctic fjord of Svalbard.

In Arctic marine bacterial communities, members of the phylum Verrucomicrobia are consistently detected, although not typically abundant, in 16S rRNA gene clone libraries and pyrotag surveys of the marine water column and in sediments. In an Arctic fjord (Smeerenburgfjord) of Svalbard, members of the Verrucomicrobia, together with Flavobacteria and smaller proportions of Alpha- and Gammaproteobacteria, constituted the most frequently detected bacterioplankton community members in 16S rRNA gene-based clone library analyses of the water column. Parallel measurements in the water column of the activities of six endo-acting polysaccharide hydrolases showed that chondroitin sulfate, laminarin, and xylan hydrolysis accounted for most of the activity. Several Verrucomicrobia water column phylotypes were affiliated with previously sequenced, glycoside hydrolase-rich genomes of individual Verrucomicrobia cells that bound fluorescently labeled laminarin and xylan and therefore constituted candidates for laminarin and xylan hydrolysis. In sediments, the bacterial community was dominated by different lineages of Verrucomicrobia, Bacteroidetes, and Proteobacteria but also included members of multiple phylum-level lineages not observed in the water column. This community hydrolyzed laminarin, xylan, chondroitin sulfate, and three additional polysaccharide substrates at high rates. Comparisons with data from the same fjord in the previous summer showed that the bacterial community in Smeerenburgfjord changed in composition, most conspicuously in the changing detection frequency of Verrucomicrobia in the water column. Nonetheless, in both years the community hydrolyzed the same polysaccharide substrates.

Microbial community composition and function in permanently cold seawater and sediments from an arctic fjord of svalbard.

Heterotrophic microbial communities in seawater and sediments metabolize much of the organic carbon produced in the ocean. Although carbon cycling and preservation depend critically on the capabilities of these microbial communities, their compositions and capabilities have seldom been examined simultaneously at the same site. To compare the abilities of seawater and sedimentary microbial communities to initiate organic matter degradation, we measured the extracellular enzymatic hydrolysis rates of 10 substrates (polysaccharides and algal extracts) in surface seawater and bottom water as well as in surface and anoxic sediments of an Arctic fjord. Patterns of enzyme activities differed between seawater and sediments, not just quantitatively, in accordance with higher cell numbers in sediments, but also in their more diversified enzyme spectrum. Sedimentary microbial communities hydrolyzed all of the fluorescently labeled polysaccharide and algal extracts, in most cases at higher rates in subsurface than surface sediments. In seawater, in contrast, only 5 of the 7 polysaccharides and 2 of the 3 algal extracts were hydrolyzed, and hydrolysis rates in surface and deepwater were virtually identical. To compare bacterial communities, 16S rRNA gene clone libraries were constructed from the same seawater and sediment samples; they diverged strongly in composition. Thus, the broader enzymatic capabilities of the sedimentary microbial communities may result from the compositional differences between seawater and sedimentary microbial communities, rather than from gene expression differences among compositionally similar communities. The greater number of phylum- and subphylum-level lineages and operational taxonomic units in sediments than in seawater samples may reflect the necessity of a wider range of enzymatic capabilities and strategies to access organic matter that has already been degraded during passage through the water column. When transformations of marine organic matter are considered, differences in community composition and their different abilities to access organic matter should be taken into account.

Fluorescent derivatization of polysaccharides and carbohydrate-containing biopolymers for measurement of enzyme activities in complex media.

Fluorescence derivatization provides a means of tracing the dynamics of polysaccharides even in the presence of high concentrations of other organic compounds or salts. A method of labeling polysaccharides with fluoresceinamine was extended to polysaccharides of a wide range of chemical composition, and alternative means of preparation were established for polysaccharides not initially amenable to column chromatography. The polysaccharides were activated with cyanogen bromide, coupled to fluoresceinamine, and separated from unreacted fluorophore via gel filtration chromatography or dialysis. Since the resulting derivatized polysaccharides proved to be stable to further physical and chemical manipulation, methods were also developed for re-activation and labeling with a second fluorophore, as well as for tethering the labeled polysaccharides to agarose beads. As an example of the application of this approach, five distinct fluorescently-labeled polysaccharides (pullulan, laminarin, xylan, chondroitin sulfate, and alginic acid) were used to investigate the activities and structural specificities of extracellular enzymes produced in situ by marine microbial communities, providing a means of measuring specifically the activities of endo-acting extracellular enzymes and avoiding use of low molecular mass substrate proxies. These labeled polysaccharides could be used to explore the dynamics of polysaccharides in other types of complex media, as well as to investigate the activities and specificities of endo-acting enzymes in other systems.

Extracellular enzyme activity in anaerobic bacterial cultures: evidence of pullulanase activity among mesophilic marine bacteria.

The extracellular enzymatic activity of a mixed culture of anaerobic marine bacteria enriched on pullulan [alpha(1,6)-linked maltotriose units] was directly assessed with a combination of gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (NMR). Hydrolysis products of pullulan were separated by GPC into three fractions with molecular weights of > or = 10,000, approximately 5,000, and < or = 1,200. NMR spectra of these fractions demonstrated that pullulan was rapidly and specifically hydrolyzed at alpha(1,6) linkages by pullulanase enzymes, most likely type II pullulanase. Although isolated pullulanase enzymes have been shown to hydrolyze pullulan completely to maltotriose (S. H. Brown, H. R. Costantino, and R. M. Kelly, Appl. Environ. Microbiol. 56:1985-1991, 1990; M. Klingeberg, H. Hippe, and G. Antranikian, FEMS Microbiol. Lett. 69:145-152, 1990; R. Koch, P. Zablowski, A. Spreinat, and G. Antranikian, FEMS Microbiol. Lett. 71:21-26, 1990), the smallest carbohydrate detected in the bacterial cultures consisted of two maltotriose units linked through one alpha(1,6) linkage. Either the final hydrolysis step was closely linked to substrate uptake, or specialized porins similar to maltoporin might permit direct transport of large oligosaccharides into the bacterial cell. This is the first report of pullulanase activity among mesophilic marine bacteria. The combination of GPC and NMR could easily be used to assess other types of extracellular enzyme activity in bacterial cultures.