ABSTRACT Anoxic subsurface sediments contain communities of heterotrophic microorganisms that metabolize organic carbon at extraordinarily low rates. In order to assess the mechanisms by which subsurface microorganisms access detrital sedimentary organic matter, we measured kinetics of a range of extracellular peptidases in anoxic sediments of the White Oak River Estuary, NC. Nine distinct peptidase substrates were enzymatically hydrolyzed at all depths. Potential peptidase activities ( V max ) decreased with increasing sediment depth, although V max expressed on a per-cell basis was approximately the same at all depths. Half-saturation constants ( K m ) decreased with depth, indicating peptidases that functioned more efficiently at low substrate concentrations. Potential activities of extracellular peptidases acting on molecules that are enriched in degraded organic matter ( d -phenylalanine and l -ornithine) increased relative to enzymes that act on l -phenylalanine, further suggesting microbial community adaptation to access degraded organic matter. Nineteen classes of predicted, exported peptidases were identified in genomic data from the same site, of which genes for class C25 (gingipain-like) peptidases represented more than 40% at each depth. Methionine aminopeptidases, zinc carboxypeptidases, and class S24-like peptidases, which are involved in single-stranded-DNA repair, were also abundant. These results suggest a subsurface heterotrophic microbial community that primarily accesses low-quality detrital organic matter via a diverse suite of well-adapted extracellular enzymes. IMPORTANCE Burial of organic carbon in marine and estuarine sediments represents a long-term sink for atmospheric carbon dioxide. Globally, ∼40% of organic carbon burial occurs in anoxic estuaries and deltaic systems. However, the ultimate controls on the amount of organic matter that is buried in sediments, versus oxidized into CO 2 , are poorly constrained. In this study, we used a combination of enzyme assays and metagenomic analysis to identify how subsurface microbial communities catalyze the first step of proteinaceous organic carbon degradation. Our results show that microbial communities in deeper sediments are adapted to access molecules characteristic of degraded organic matter, suggesting that those heterotrophs are adapted to life in the subsurface.
more »
« less
On Single-Cell Enzyme Assays in Marine Microbial Ecology and Biogeochemistry
Extracellular enzyme activity is a well-established parameter for evaluating microbial biogeochemical roles in marine ecosystems. The presence and activity of extracellular enzymes in seawater provide insights into the quality and quantity of organic matter being processed by the present microorganisms. A key challenge in our understanding of these processes is to decode the extracellular enzyme repertoire and activities of natural communities at the single-cell level. Current measurements are carried out on bulk or size-fractionated samples capturing activities of mixed populations. This approach – even with size-fractionation – cannot be used to trace enzymes back to their producers, nor distinguish the active microbial members, leading to a disconnect between measured activities and the producer cells. By targeting extracellular enzymes and resolving their activities at the single-cell level, we can investigate underlying phenotypic heterogeneity among clonal or closely related organisms, characterize enzyme kinetics under varying environmental conditions, and resolve spatio-temporal distribution of individual enzyme producers within natural communities. In this perspective piece, we discuss state-of-the-art technologies in the fields of microfluidic droplets and functional screening of prokaryotic cells for measuring enzyme activity in marine seawater samples, one cell at a time. We further elaborate on how this single-cell approach can be used to address research questions that cannot be answered with current methods, as pertinent to the enzymatic degradation of organic matter by marine microorganisms.
more »
« less
- PAR ID:
- 10338553
- Date Published:
- Journal Name:
- Frontiers in Marine Science
- Volume:
- 9
- ISSN:
- 2296-7745
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
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.more » « less
-
Yin, Yanbin (Ed.)more » « less
Microbial communities in terrestrial geothermal systems often contain chemolithoautotrophs with well-characterized distributions and metabolic capabilities. However, the extent to which organic matter produced by these chemolithoautotrophs supports heterotrophs remains largely unknown. Here we compared the abundance and activity of peptidases and carbohydrate active enzymes (CAZymes) that are predicted to be extracellular identified in metagenomic assemblies from 63 springs in the Central American and the Andean convergent margin (Argentinian backarc of the Central Volcanic Zone), as well as the plume-influenced spreading center in Iceland. All assemblies contain two orders of magnitude more peptidases than CAZymes, suggesting that the microorganisms more often use proteins for their carbon and/or nitrogen acquisition instead of complex sugars. The CAZy families in highest abundance are GH23 and CBM50, and the most abundant peptidase families are M23 and C26, all four of which degrade peptidoglycan found in bacterial cells. This implies that the heterotrophic community relies on autochthonous dead cell biomass, rather than allochthonous plant matter, for organic material. Enzymes involved in the degradation of cyanobacterial- and algal-derived compounds are in lower abundance at every site, with volcanic sites having more enzymes degrading cyanobacterial compounds and non-volcanic sites having more enzymes degrading algal compounds. Activity assays showed that many of these enzyme classes are active in these samples. High temperature sites (> 80°C) had similar extracellular carbon-degrading enzymes regardless of their province, suggesting a less well-developed population of secondary consumers at these sites, possibly connected with the limited extent of the subsurface biosphere in these high temperature sites. We conclude that in < 80°C springs, chemolithoautotrophic production supports heterotrophs capable of degrading a wide range of organic compounds that do not vary by geological province, even though the taxonomic and respiratory repertoire of chemolithoautotrophs and heterotrophs differ greatly across these regions.
-
null (Ed.)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.more » « less
-
more » « less
Abstract As tall shrubs increase in extent and abundance in response to a changing climate, they have the potential to substantially alter Arctic and boreal ecosystem nutrient cycling and carbon (C) balance. Siberian alder (
Alnus viridis ssp.fruticosa ), a nitrogen (N) fixing shrub, is among the species responding to climate warming in both the Arctic and boreal forests. By relieving N limitation of microbial activity, alder‐fixed N has the potential to increase decomposition of labile soil C. Simultaneously, it may also decrease decomposition of recalcitrant soil C by downregulating microbial N mining. The microbial response to N additions is influenced by differences in the soil organic matter (SOM) chemistry and could ultimately determine whether alder N additions result in a net sink or source of C to the atmosphere. We measured the activities of three extracellular enzymes in bulk organic soils under and away from alder canopies in stands differing in SOM chemistry in both the arctic and boreal forest regions of Alaska, USA. In the Arctic, samples taken from under alder had higher activities of both recalcitrant and labile C‐degrading enzymes than samples taken away, regardless of SOM chemistry. In the boreal forest, enzyme activities did not differ with alder proximity nor stand SOM chemistry, possibly due to long legacies of alder N inputs in these stands. As arctic and boreal forest ecosystems experience shifts in the distribution and abundance of this N‐fixing shrub, alders' influence on soil decomposition could have significant consequences for high latitude soil C budgets.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmars.2022.846656
