Heterotrophic Carbon Cycling Research Articles

Lytic archaeal viruses infect abundant primary producers in Earth\u2019s crust

The continental subsurface houses a major portion of life’s abundance and diversity, yet little is known about viruses infecting microbes that reside there. Here, we use a combination of metagenomics and virus-targeted direct-geneFISH (virusFISH) to show that highly abundant carbon-fixing organisms of the uncultivated genus Candidatus Altiarchaeum are frequent targets of previously unrecognized viruses in the deep subsurface. Analysis of CRISPR spacer matches display resistances of Ca. Altiarchaea against eight predicted viral clades, which show genomic relatedness across continents but little similarity to previously identified viruses. Based on metagenomic information, we tag and image a putatively viral genome rich in protospacers using fluorescence microscopy. VirusFISH reveals a lytic lifestyle of the respective virus and challenges previous predictions that lysogeny prevails as the dominant viral lifestyle in the subsurface. CRISPR development over time and imaging of 18 samples from one subsurface ecosystem suggest a sophisticated interplay of viral diversification and adapting CRISPR-mediated resistances of Ca. Altiarchaeum. We conclude that infections of primary producers with lytic viruses followed by cell lysis potentially jump-start heterotrophic carbon cycling in these subsurface ecosystems.

Molecular Basis for Substrate Recognition and Catalysis by a Marine Bacterial Laminarinase.

Laminarin is an abundant algal polysaccharide that serves as carbon storage and fuel to meet the nutrition demands of heterotrophic microbes. Laminarin depolymerization catalyzed by microbial extracellular enzymes initiates remineralization, a key process in ocean biogeochemical cycles. Here, we described a glycoside hydrolase 16 (GH16) family laminarinase from a marine alga-associated Flavobacterium at the biochemical and structural levels. We found that the endolytic enzyme cleaved laminarin with a preference for β-1,3-glycoside linkages and showed transglycosylation activity across a broad range of acceptors. We also solved and compared high-resolution crystal structures of laminarinase in the apo form and in complex with β-1,3-tetrasaccharides, revealing an expanded catalytic cleft formed following substrate binding. Moreover, structure and mutagenesis studies identified multiple specific contacts between the enzyme and glucosyl residues essential for the substrate specificity for β-1,3-glucan. These results provide novel insights into the structural requirements for substrate binding and catalysis of GH16 family laminarinase, enriching our understanding of bacterial utilization of algal laminarin.IMPORTANCE Heterotrophic bacterial communities are key players in marine biogeochemical cycling due to their ability to remineralize organic carbon. Processing of complex organic matter requires heterotrophic bacteria to produce extracellular enzymes with precise specificity to depolymerize substrates to sizes sufficiently small for uptake. Thus, extracellular enzymatic hydrolysis initiates microbe-driven heterotrophic carbon cycling. In this study, based on biochemical and structural analyses, we revealed the depolymerization mechanism of β-1,3-glucan, a carbon reserve in algae, by laminarinase from an alga-associated marine Flavobacterium The findings provide new insights into the substrate recognition and catalysis of bacterial laminarinase and promote a better understanding of how extracellular enzymes are involved in organic matter cycling.

Improved Measurement of Extracellular Enzymatic Activities in Subsurface Sediments Using Competitive Desorption Treatment

Extracellular enzymatic activities initiate microbially-driven heterotrophic carbon cycling in subsurface sediments. While measurement of hydrolytic activities in sediments is fundamental to our understanding of carbon cycling, these measurements are often technically difficult due to sorption of organic substrates to the sediment matrix. Most methods that measure hydrolysis of organic substrates in sediments rely on recovery of a fluorophore or fluorescently-labeled target substrate from a sediment incubation. The tendency for substrates to sorb to sediments results in lower recovery of an added substrate, and can result in data that are unusable or difficult to interpret. We developed a treatment using competitive desorption of a fluorescently-labeled, high molecular weight organic substrate that improves recovery of the labeled substrate from sediment subsamples. Competitive desorption treatment improved recovery of the fluorescent substrate by a median of 66%, expanded the range of sediments for which activity measurements could be made, and was effective in sediments from a broad range of geochemical contexts. More reliable measurements of hydrolytic activities in sediments will yield usable and more easily interpretable data from a wider range of sedimentary environments, enabling better understanding of microbially-catalyzed carbon cycling in subsurface environments.

Patterns of Microbially Driven Carbon Cycling in the Ocean: Links between Extracellular Enzymes and Microbial Communities

Heterotrophic microbial communities play a central role in the marine carbon cycle. They are active in nearly all known environments, from the surface to the deep ocean, in the sediments, and from the equator to the Poles. In order to process complex organic matter, these communities produce extracellular enzymes of the correct structural specificity to hydrolyze substrates to sizes sufficiently small for uptake. Extracellular enzymatic hydrolysis thus initiates heterotrophic carbon cycling. Our knowledge of the enzymatic capabilities of microbial communities in the ocean is still underdeveloped. Recent studies, however, suggest that there may be large-scale patterns of enzymatic function in the ocean, patterns of community function that may be connected to emerging patterns of microbial community composition. Here I review some of these large-scale contrasts in microbial enzyme activities, between high-latitude and temperate surface ocean waters, contrasts between inshore and offshore waters, changes with depth gradients in the ocean, and contrasts between the water column and underlying sediments. These contrasting patterns are set in the context of recent studies of microbial communities and patterns of microbial biogeography. Focusing on microbial community function as well as composition and potential should yield clearer understanding of the factors driving carbon cycling in the ocean.

Dynamics of extracellular enzyme activities in seawater under changed atmospheric pCO2: a mesocosm investigation

As part of the PeECE II mesocosm project, we investigated the effects of pCO2 levels on the initial step of heterotrophic carbon cycling in the surface ocean. The activities of microbial extracellular enzymes hydrolyzing 4 polysaccharides were measured during the development of a natural phytoplankton bloom under pCO2 conditions representing glacial (190 µatm) and future (750 µatm) atmospheric pCO2. We observed that (1) chondroitin hydrolysis was variable throughout the pre-, early- and late-bloom phases, (2) fucoidanase activity was measurable only in the glacial mesocosm as the bloom developed, (3) laminarinase activity was low and constant, and (4) xylanase activity declined as the bloom progressed. Concurrent measurements of microbial community composition, using denaturing-gradient gel electrophoresis (DGGE), showed that the 2 mesocosms diverged temporally, and from one another, especially in the late-bloom phase. Enzyme activities correlated with bloom phase and pCO2, suggesting functional as well as compositional changes in microbial communities in the different pCO2 environments. These changes, however, may be a response to temporal changes in the development of phytoplankton communities that differed with the pCO2 environment. We hypothesize that the phytoplankton communities produced dissolved organic carbon (DOC) differing in composition, a hypothesis supported by changing amino acid composition of the DOC, and that enzyme activities responded to changes in substrates. Enzyme activities observed under different pCO2 conditions likely reflect both genetic and population-level responses to changes occurring among multiple components of the microbial loop.

Carbon isotope fractionation in phospholipid fatty acid biomarkers of bacteria and fungi native to an acid mine drainage lake

This study identifies isotope signatures associated with autotrophic and heterotrophic microbial communities that may provide a means to determine carbon cycling relationships in situ for acid mine drainage (AMD) sites. Stable carbon isotope ratios (δ13C) of carbon sources, bulk cells, and membrane phospholipids (PLFA) were measured for autotrophic and heterotrophic microbial enrichment cultures from a mine tailings impoundment in northern Ontario, Canada, and for pure strains of the sulfur oxidizing bacteria Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. The autotrophic enrichments had indistinguishable PLFA distributions from the pure cultures, and the PLFA cyc-C19:0 was determined to be a unique biomarker in this system for these sulfur oxidizing bacteria. The PLFA distributions produced by the heterotrophic enrichments were distinct from the autotrophic distributions and the C18:2 PLFA was identified as a biomarker for these heterotrophic enrichments. Genetic analysis (16S, 18S rRNA) of the heterotrophic cultures indicated that these communities were primarily composed of Acremonium fungi.Stable carbon isotope analysis revealed that bulk cellular material in all autotrophic cultures was depleted in δ13C by 5.6–10.9‰ relative to their atmospheric CO2 derived carbon source, suggesting that inorganic carbon fixation in these cultures is carbon limited. Individual PLFA from these autotrophs were further depleted by 8.2–14.6‰ compared to the bulk cell δ13C, which are among the largest biosynthetic isotope fractionation factors between bulk cell and PLFA reported in the literature. In contrast, the heterotrophic bulk cells were not significantly fractionated in δ13C relative to their carbon source and heterotrophic PLFA ranged from 3‰ enriched to 4‰ depleted relative to the isotopic composition of their total biomass. These distinct PLFA biomarkers and isotopic fractionations associated with autotrophic and heterotrophic activity in this laboratory study provide potential biomarkers for delineating autotrophic and heterotrophic carbon cycling in AMD environments.

Linkages between bacterioplankton community composition, heterotrophic carbon cycling and environmental conditions in a highly dynamic coastal ecosystem

We used mesocosm experiments to study the bacterioplankton community in a highly dynamic coastal ecosystem during four contrasting periods of the seasonal cycle: winter mixing, spring phytoplankton bloom, summer stratification and autumn upwelling. A correlation approach was used in order to measure the degree of coupling between the dynamics of major bacterial groups, heterotrophic carbon cycling and environmental factors. We used catalysed reporter deposition-fluorescence in situ hybridization to follow changes in the relative abundance of the most abundant groups of bacteria (Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes). Bacterial carbon flux-related variables included bacterial standing stock, bacterial production and microbial respiration. The environmental factors included both, biotic variables such as chlorophyll-a concentration, primary production, phytoplankton extracellular release, and abiotic variables such as the concentration of dissolved inorganic and organic nutrients. Rapid shifts in the dominant bacterial groups occurred associated to environmental changes and bacterial bulk functions. An alternation between Alphaproteobacteria and Bacteroidetes was observed associated to different phytoplankton growth phases. The dominance of the group Bacteroidetes was related to high bacterial biomass and production. We found a significant, non-spurious, linkage between the relative abundances of major bacterial groups and bacterial carbon cycling. Our results suggest that bacteria belonging to these major groups could actually share a function in planktonic ecosystems.

Toxicity assessment by community analysis

Toxicity assessments are conducted to determine the potential for environmental effects of commercial chemicals. Assessments made with single species testing are commonly used in environmental risk assessment. Community-level analyses can be used to test the predictions of the single-species' tests and determine the potential for indirect effects. Mesocosms, such as the P & G Experimental Stream Facility, are used to develop an experimental ecosystem in which this community-level analysis can be performed. Microbial communities, as base of the photoautotrophic carbon input and heterotrophic carbon cycling within streams, are key to the ecotoxicological assessment and understanding of these effects. These communities are evaluated for changes in biomass, activity, and community structure over an 8-week exposure to an added test chemical. These studies are not evaluated based on a ‘most sensitive species’ or key endpoint approach. Rather, an ecosystem-level evaluation is made using a decision tree that sequentially evaluates: (1) statistical differences compared to a control stream; (2) ecological consistency of complementary endpoints; (3) dose response of effect; and (4) temporal pattern of effect (transient or progressive). The goal of this analysis is to reduce the results of these complex studies down to a single No-Observed-Effect-Concentration (NOEC) for the ecosystem tested. Results from experiments with two anionic surfactants illustrate how analytical evaluation of microbial communities is used in the derivation of ecosystem-level NOECs when both direct and indirect effects are observed.

Bacterial biomass in warm-core Gulf Stream ring 82-B: mesoscale distributions, temporal changes and production

The distribution of bacterioplankton biomass and productivity in warm-core Gulf Stream ring 82-B generally corresponded to the physical and dynamical structure of the ring. Mean cell volumes were uniform for 4 months, but were larger by a factor of 2–3 in the high velocity (frontal) region (HVR) near the ring edge. As a result of this gradient and higher abundances, water column biomass and production were highest in the front, which appeared to be a local maximum in those properties. In this regard bacterioplankton contrasted strongly to phytoplankton, which exhibited strong local maxima at the center of the ring in June. In April when the water column inside the ring was isothermal to 450 m, bacterial biomass and production were low and uniform to 250 and 50 m, respectively. Bacterioplankton responded dramatically to the vernal restratification of the ring. In June when the surface layer was characterized by a strong pycnocline at 10–40 m, bacterial biomass and production often had strong subsurface maxima, and were 3 and 5 times greater than in April, respectively. Abundance exceeded 1.5 × 10 9 cells l −1 at ring center and exceeded 3 × 10 9 l −1 in the HVR. Turnover rates for the euphotic zone bacterioplankton as a whole were 0.24 d −1 in April, 0.56 d −1 in June, and 0.27 d −1 in August at ring center. Bacterial production averaged 12% of hourly primary production (range 1–32%), suggesting that bacteria control a significant and sometimes large portion of the carbon cycling in the euphotic zone. These data suggest that warm-core rings are sites of enhanced variability of bacterioplankton properties in the open sea. Furthermore, the data strongly support recent work showing that frontal zones are sites of locally enhanced bacterial biomass and production. In the ring system as a whole, the euphotic zone bacterioplankton biomass and production were comparable to and occasionally greater than the biomass and production of the >64 μm zooplankton, especially in the HVR. Bacterioplankton appear to carry out a major fraction of the heterotrophic carbon cycling in oceanic frontal regions.