Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.
Microbial Community Response to Polysaccharide Amendment in Anoxic Hydrothermal Sediments of the Guaymas Basin
Organic-rich, hydrothermal sediments of the Guaymas Basin are inhabited by diverse microbial communities including many uncultured lineages with unknown metabolic potential. Here we investigated the short-term effect of polysaccharide amendment on a sediment microbial community to identify taxa involved in the initial stage of macromolecule degradation. We incubated anoxic sediment with cellulose, chitin, laminarin, and starch and analyzed the total and active microbial communities using bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Our results show a response of an initially minor but diverse population of Clostridia particularly after amendment with the lower molecular weight polymers starch and laminarin. Thus, Clostridia may readily become key contributors to the heterotrophic community in Guaymas Basin sediments when substrate availability and temperature range permit their metabolic activity and growth, which expands our appreciation of the potential diversity and niche differentiation of heterotrophs in hydrothermally influenced sediments. BONCAT-FACS, although challenging in its application to complex samples, detected metabolic responses prior to growth and thus can provide complementary insight into a microbial community’s metabolic potential and succession pattern. As a primary application of BONCAT-FACS on a diverse deep-sea sediment community, our study highlights important considerations and demonstrates inherent limitations associated with this experimental approach.
more »
« less
- Award ID(s):
- 1817428
- NSF-PAR ID:
- 10378941
- Date Published:
- Journal Name:
- Frontiers in Microbiology
- Volume:
- 12
- ISSN:
- 1664-302X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
Brazelton, William J. (Ed.)The flanking regions of Guaymas Basin, a young marginal rift basin located in the Gulf of California, are covered with thick sediment layers that are hydrothermally altered due to magmatic intrusions. To explore environmental controls on microbial community structure in this complex environment, we analyzed site- and depth-related patterns of microbial community composition (bacteria, archaea, and fungi) in hydrothermally influenced sediments with different thermal conditions, geochemical regimes, and extent of microbial mats. We compared communities in hot hydrothermal sediments (75-100°C at ~40 cm depth) covered by orange-pigmented Beggiatoaceae mats in the Cathedral Hill area, temperate sediments (25-30°C at ~40 cm depth) covered by yellow sulfur precipitates and filamentous sulfur oxidizers at the Aceto Balsamico location, hot sediments (>115°C at ~40 cm depth) with orange-pigmented mats surrounded by yellow and white mats at the Marker 14 location, and background, non-hydrothermal sediments (3.8°C at ~45 cm depth) overlain with ambient seawater. Whereas bacterial and archaeal communities are clearly structured by site-specific in-situ thermal gradients and geochemical conditions, fungal communities are generally structured by sediment depth. Unexpectedly, chytrid sequence biosignatures are ubiquitous in surficial sediments whereas deeper sediments contain diverse yeasts and filamentous fungi. In correlation analyses across different sites and sediment depths, fungal phylotypes correlate to each other to a much greater degree than Bacteria and Archaea do to each other or to fungi, further substantiating that site-specific in-situ thermal gradients and geochemical conditions that control bacteria and archaea do not extend to fungi.more » « less
-
The surficial hydrothermal sediments of Guaymas Basin harbor complex microbial communities where oxidative and reductive nitrogen, sulfur, and carbon-cycling populations and processes overlap and coexist. Here, we resolve microbial community profiles in hydrothermal sediment cores of Guaymas Basin on a scale of 2 millimeters, using Denaturing Gradient Gel Electrophoresis (DGGE) to visualize the rapid downcore changes among dominant bacteria and archaea. DGGE analysis of bacterial 16S rRNA gene amplicons identified free-living and syntrophic deltaproteobacterial sulfate-reducing bacteria, fermentative Cytophagales, members of the Chloroflexi (Thermoflexia), Aminicenantes, and uncultured sediment clades. The DGGE pattern indicates a gradually changing downcore community structure where small changes on a 2-millimeter scale accumulate to significantly changing populations within the top 4 cm sediment layer. Functional gene DGGE analyses identified anaerobic methane-oxidizing archaea (ANME) based on methyl-coenzyme M reductase genes, and members of the Betaproteobacteria and Thaumarchaeota based on bacterial and archaeal ammonia monooxygenase genes, respectively. The co-existence and overlapping habitat range of aerobic, nitrifying, sulfate-reducing and fermentative bacteria and archaea, including thermophiles, in the surficial sediments is consistent with dynamic redox and thermal gradients that sustain highly complex microbial communities in the hydrothermal sediments of Guaymas Basin.more » « less
-
null (Ed.)International Ocean Discovery Program (IODP) Expedition 385 drilled organic-rich sediments with sill intrusions on the flanking regions and in the northern axial graben in Guaymas Basin, a young marginal rift basin in the Gulf of California. Guaymas Basin is characterized by a widely distributed, intense heat flow and widespread off-axis magmatism expressed by a dense network of sill intrusions across the flanking regions, which is in contrast to classical mid-ocean ridge spreading centers. The numerous off-axis sills provide multiple transient heat sources that mobilize buried sedimentary carbon, in part as methane and other hydrocarbons, and drive hydrothermal circulation. The resulting thermal and geochemical gradients shape abundance, composition, and activity of the deep subsurface biosphere of the basin. Drill sites extend over the flanking regions of Guaymas Basin, covering a distance of ~81 km from the from the northwest to the southeast. Adjacent Sites U1545 and U1546 recovered the oldest and thickest sediment successions (to ~540 meters below seafloor [mbsf]; equivalent to the core depth below seafloor, Method A [CSF-A] scale), one with a thin sill (a few meters in thickness) near the drilled bottom (Site U1545), and one with a massive, deeply buried sill (~356–430 mbsf) that chemically and physically affects the surrounding sediments (Site U1546). Sites U1547 and U1548, located in the central part of the northern Guaymas Basin segment, were drilled to investigate a 600 m wide circular mound (bathymetric high) and its periphery. The dome-like structure is outlined by a ring of active vent sites called Ringvent. It is underlain by a remarkably thick sill at shallow depth (Site U1547). Hydrothermal gradients steepen at the Ringvent periphery (Holes U1548A–U1548C), which in turn shifts the zones of authigenic carbonate precipitation and of highest microbial cell abundance toward shallower depths. The Ringvent sill was drilled several times and yielded remarkably diverse igneous rock textures, sediment–sill interfaces, and hydrothermal alteration, reflected by various secondary minerals in veins and vesicles. Thus, the Ringvent sill became the target of an integrated sampling and interdisciplinary research effort that included geological, geochemical, and microbiological specialties. The thermal, lithologic, geochemical, and microbiological contrasts between the two deep northwestern sites (U1545 and U1546) and the Ringvent sites (U1547 and U1548) form the scientific centerpiece of the expedition. These observations are supplemented by results from sites that represent attenuated cold seepage conditions in the central basin (Site U1549), complex and disturbed sediments overlying sills in the northern axial trough (Site U1550), terrigenous sedimentation events on the southeastern flanking regions (Site U1551), and hydrate occurrence in shallow sediments proximal to the Sonora margin (Site U1552). The scientific outcomes of Expedition 385 will (1) revise long-held assumptions about the role of sill emplacement in subsurface carbon mobilization versus carbon retention, (2) comprehensively examine the subsurface biosphere of Guaymas Basin and its responses and adaptations to hydrothermal conditions, (3) redefine hydrothermal controls of authigenic mineral formation in sediments, and (4) yield new insights into many geochemical and geophysical aspects of both architecture and sill–sediment interaction in a nascent spreading center. The generally high quality and high degree of completeness of the shipboard datasets present opportunities for interdisciplinary and multidisciplinary collaborations during shore-based studies. In comparison to Deep Sea Drilling Project Leg 64 to Guaymas Basin in 1979, sophisticated drilling strategies (for example, the advanced piston corer [APC] and half-length APC systems) and numerous analytical innovations have greatly improved sample recovery and scientific yield, particularly in the areas of organic geochemistry and microbiology. For example, microbial genomics did not exist 40 y ago. However, these technical refinements do not change the fact that Expedition 385 will in many respects build on the foundations laid by Leg 64 for understanding Guaymas Basin, regardless of whether adjustments are required in the near future.more » « less
-
more » « less
The Guaymas Basin in the Gulf of California is characterized by active seafloor spreading, the rapid deposition of organic-rich sediments, steep geothermal gradients, and abundant methane of mixed thermogenic and microbial origin. Subsurface sediment samples from eight drilling sites with distinct geochemical and thermal profiles were selected for DNA extraction and PCR amplification to explore the diversity of methane-cycling archaea in the Guaymas Basin subsurface. We performed PCR amplifications with general (mcrIRD), and ANME-1 specific primers that target the alpha (α) subunit of methyl coenzyme M reductase (mcrA). Diverse ANME-1 lineages associated with anaerobic methane oxidation were detected in seven out of the eight drilling sites, preferentially around the methane-sulfate interface, and in several cases, showed preferences for specific sampling sites. Phylogenetically, most ANME-1 sequences from the Guaymas Basin subsurface were related to marine mud volcanoes, seep sites, and the shallow marine subsurface. The most frequently recovered methanogenic phylotypes were closely affiliated with the hyperthermophilic Methanocaldococcaceae, and found at the hydrothermally influenced Ringvent site. The coolest drilling site, in the northern axial trough of Guaymas Basin, yielded the greatest diversity in methanogen lineages. Our survey indicates the potential for extensive microbial methane cycling within subsurface sediments of Guaymas Basin.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmicb.2021.763971
