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ABSTRACT Microorganisms adapted to high hydrostatic pressures at depth in the oceans and within the subsurface of Earth’s crust represent a phylogenetically diverse community thriving under extreme pressure, temperature, and nutrient availability conditions. To better understand the microbial function, physiological responses, and metabolic strategies atin-situconditions requires high-pressure (HP) continuous culturing techniques that, although commonly used in bioengineering and biotechnology applications, remain relatively rare in the study of the Earth’s microbiomes. Here, we focus on recent developments in the design of HP chemostats, with particular emphasis on adaptations for delivery and sampling of dissolved gases. We present protocols for sterilization, inoculation, agitation, and sampling strategies that minimize cell lysis, applicable to a wide range of chemostat designs. 

ABSTRACT The abundant and metabolically versatile aquatic bacterial order,Rhodobacterales, influences marine biogeochemical cycles. We assessedRhodobacteralesmetagenome-assembled genome (MAG) abundance, estimated growth rates, and potential and expressed functions in the Chesapeake and Delaware Bays, two important US estuaries. Phylogenomics of draft and draft/closedRhodobacteralesgenomes from this study and others placed 46 nearly complete MAGs from these bays into 11 genera, many were not well characterized. Their abundances varied between the bays and were influenced by temperature, salinity, and silicate and phosphate concentrations.Rhodobacteralesgenera possessed unique and shared genes for transporters, photoheterotrophy, complex carbon degradation, nitrogen, and sulfur metabolism reflecting their seasonal differences in abundance and activity.Planktomarinagenomospecies were more ubiquitous than the more niche specialists, HIMB11, CPC320, LFER01, and MED-G52. Their estimated growth rates were correlated to various factors including phosphate and silicate concentrations, cell density, and light. Metatranscriptomic analysis of four abundant genomospecies commonly revealed that aerobic anoxygenic photoheterotrophy-associated transcripts were highly abundant at night. TheseRhodobacteralesalso differentially expressed genes for CO oxidation and nutrient transport and use between different environmental conditions. Phosphate concentrations and light penetration in the Chesapeake Bay likely contributed to higher estimated growth rates of HIMB11 and LFER01, respectively, in summer where they maintained higher ribosome concentrations and prevented physiological gene expression constraints by downregulating transporter genes compared to the Delaware Bay. Our study highlights the spatial and temporal shifts in estuarineRhodobacteraleswithin and between these bays reflected through their abundance, unique metabolisms, estimated growth rates, and activity changes. IMPORTANCEIn the complex web of global biogeochemical nutrient cycling, theRhodobacteralesemerge as key players, exerting a profound influence through their abundance and dynamic activity. While previous studies have primarily investigated these organisms within marine ecosystems, this study delves into their roles within estuarine environments using a combination of metagenomic and metatranscriptomic analyses. We uncovered a range ofRhodobacteralesgenera, from generalists to specialists, each exhibiting distinct abundance patterns and gene expression profiles. This diversity equips them with the capacity to thrive amidst the varying environmental conditions encountered within dynamic estuarine habitats. Crucially, our findings illuminate the adaptable nature of estuarineRhodobacterales, revealing their various energy production pathways and diverse resource management, especially during phytoplankton or algal blooms. Whether adopting a free-living or particle-attached existence, these organisms demonstrate remarkable flexibility in their metabolic strategies, underscoring their pivotal role in driving ecosystem dynamics within estuarine ecosystems. 

ABSTRACT The Winam Gulf in the Kenyan region of Lake Victoria experiences prolific, year-round cyanobacterial harmful algal blooms (cyanoHABs) which pose threats to human, livestock, and ecosystem health. To our knowledge, there is limited molecular research on the gulf’s cyanoHABs, and thus, the strategies employed for survival and proliferation by toxigenic cyanobacteria in this region remain largely unexplored. Here, we used metagenomics to analyze the Winam Gulf’s cyanobacterial composition, function, and biosynthetic potential.Dolichospermumwas the dominant bloom-forming cyanobacterium, co-occurring withMicrocystisat most sites.MicrocystisandPlanktothrixwere more abundant in shallow and turbid sites. Metagenome-assembled genomes (MAGs) ofDolichospermumharbored nitrogen fixation genes, suggesting diazotrophy as a potential mechanism supporting the proliferation ofDolichospermumin the nitrogen-limited gulf. Over 300 biosynthetic gene clusters (BGCs) putatively encoding the synthesis of toxins and other secondary metabolites were identified across the gulf, even at sites where there were no visible cyanoHAB events. Almost all BGCs identified had no known synthesis product, indicating a diverse and novel biosynthetic repertoire capable of synthesizing harmful or potentially therapeutic metabolites.MicrocystisMAGs containedmcygenes encoding the synthesis of hepatotoxic microcystins which are a concern for drinking water safety. These findings illustrate the spatial variation of bloom-forming cyanobacteria in the Winam Gulf and their available strategies to dominate different ecological niches. This study underscores the need for further use of genomic techniques to elucidate the dynamics and mitigate the potentially harmful effects of cyanoHABs and their associated toxins on human, environmental, and economic health. 

ABSTRACT Autotrophic bacteria are able to fix CO₂in a great diversity of habitats, even though this dissolved gas is relatively scarce at neutral pH and above. As many of these bacteria rely on CO₂fixation by ribulose 1,5-bisphospate carboxylase/oxygenase (RubisCO) for biomass generation, they must compensate for the catalytical constraints of this enzyme with CO₂-concentrating mechanisms (CCMs). CCMs consist of CO₂and HCO₃⁻transporters and carboxysomes. Carboxysomes encapsulate RubisCO and carbonic anhydrase (CA) within a protein shell and are essential for the operation of a CCM in autotrophicBacteriathat use the Calvin-Benson-Basham cycle. Members of the genusThiomicrospiralack genes homologous to those encoding previously described CA, and prior to this work, the mechanism of function for their carboxysomes was unclear. In this paper, we provide evidence that a member of the recently discovered iota family of carbonic anhydrase enzymes (ιCA) plays a role in CO₂fixation by carboxysomes from members ofThiomicrospiraand potentially otherBacteria. Carboxysome enrichments fromThiomicrospira pelophilaandThiomicrospira aerophilawere found to have CA activity and contain ιCA, which is encoded in their carboxysome loci. When the gene encoding ιCA was interrupted inT. pelophila, cells could no longer grow under low-CO₂conditions, and CA activity was no longer detectable in their carboxysomes. WhenT. pelophilaιCA was expressed in a strain ofEscherichia colilacking native CA activity, this strain recovered an ability to grow under low CO₂conditions, and CA activity was present in crude cell extracts prepared from this strain. IMPORTANCEHere, we provide evidence that iota carbonic anhydrase (ιCA) plays a role in CO₂fixation by some organisms with CO₂-concentrating mechanisms; this is the first time that ιCA has been detected in carboxysomes. While ιCA genes have been previously described in other members of bacteria, this is the first description of a physiological role for this type of carbonic anhydrase in this domain. Given its distribution in alkaliphilic autotrophic bacteria, ιCA may provide an advantage to organisms growing at high pH values and could be helpful for engineering autotrophic organisms to synthesize compounds of industrial interest under alkaline conditions. 

ABSTRACT Heterotrophic marine bacteria utilize and recycle dissolved organic matter (DOM), impacting biogeochemical cycles. It is currently unclear to what extent distinct DOM components can be used by different heterotrophic clades. Here, we ask how a natural microbial community from the Eastern Mediterranean Sea (EMS) responds to different molecular classes of DOM (peptides, amino acids, amino sugars, disaccharides, monosaccharides, and organic acids) comprising much of the biomass of living organisms. Bulk bacterial activity increased after 24 h for all treatments relative to the control, while glucose and ATP uptake decreased or remained unchanged. Moreover, while the per-cell uptake rate of glucose and ATP decreased, that of Leucin significantly increased for amino acids, reflecting their importance as common metabolic currencies in the marine environment.Pseudoalteromonadaceaedominated the peptides treatment, while differentVibrionaceaestrains became dominant in response to amino acids and amino sugars.Marinomonadaceaegrew well on organic acids, andAlteromonadaseaeon disaccharides. A comparison with a recent laboratory-based study reveals similar peptide preferences forPseudoalteromonadaceae, whileAlteromonadaceae, for example, grew well in the lab on many substrates but dominated in seawater samples only when disaccharides were added. We further demonstrate a potential correlation between the genetic capacity for degrading amino sugars and the dominance of specific clades in these treatments. These results highlight the diversity in DOM utilization among heterotrophic bacteria and complexities in the response of natural communities. IMPORTANCEA major goal of microbial ecology is to predict the dynamics of natural communities based on the identity of the organisms, their physiological traits, and their genomes. Our results show that several clades of heterotrophic bacteria each grow in response to one or more specific classes of organic matter. For some clades, but not others, growth in a complex community is similar to that of isolated strains in laboratory monoculture. Additionally, by measuring how the entire community responds to various classes of organic matter, we show that these results are ecologically relevant, and propose that some of these resources are utilized through common uptake pathways. Tracing the path between different resources to the specific microbes that utilize them, and identifying commonalities and differences between different natural communities and between them and lab cultures, is an important step toward understanding microbial community dynamics and predicting how communities will respond to perturbations. 

ABSTRACT Aerobic anoxygenic phototrophic (AAP) bacteria harvest light energy using bacteriochlorophyll-containing reaction centers to supplement their mostly heterotrophic metabolism. While their abundance and growth have been intensively studied in coastal environments, much less is known about their activity in oligotrophic open ocean regions. Therefore, we combinedin situsampling in the North Pacific Subtropical Gyre, north of O'ahu island, Hawaii, with two manipulation experiments. Infra-red epifluorescence microscopy documented that AAP bacteria represented approximately 2% of total bacteria in the euphotic zone with the maximum abundance in the upper 50 m. They conducted active photosynthetic electron transport with maximum rates up to 50 electrons per reaction center per second. Thein situdecline of bacteriochlorophyll concentration over the daylight period, an estimate of loss rates due to predation, indicated that the AAP bacteria in the upper 50 m of the water column turned over at rates of 0.75–0.90 d⁻¹. This corresponded well with the specific growth rate determined in dilution experiments where AAP bacteria grew at a rate 1.05 ± 0.09 d⁻¹. An amendment of inorganic nitrogen to obtain N:P = 32 resulted in a more than 10 times increase in AAP abundance over 6 days. The presented data document that AAP bacteria are an active part of the bacterioplankton community in the oligotrophic North Pacific Subtropical Gyre and that their growth was mostly controlled by nitrogen availability and grazing pressure.IMPORTANCEMarine bacteria represent a complex assembly of species with different physiology, metabolism, and substrate preferences. We focus on a specific functional group of marine bacteria called aerobic anoxygenic phototrophs. These photoheterotrophic organisms require organic carbon substrates for growth, but they can also supplement their metabolic needs with light energy captured by bacteriochlorophyll. These bacteria have been intensively studied in coastal regions, but rather less is known about their distribution, growth, and mortality in the oligotrophic open ocean. Therefore, we conducted a suite of measurements in the North Pacific Subtropical Gyre to determine the distribution of these organisms in the water column and their growth and mortality rates. A nutrient amendment experiment showed that aerobic anoxygenic phototrophs were limited by inorganic nitrogen. Despite this, they grew more rapidly than average heterotrophic bacteria, but their growth was balanced by intense grazing pressure. 

ABSTRACT Marine oxygen-deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs is still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase (nod) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribednodgenes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria,rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribenod, but at lower relative abundance than UBA11136 in the upper ODZ. Thenod-transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here namedptd) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs.IMPORTANCEIn marine oxygen-deficient zones (ODZs), microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the world’s largest ODZ, but the identity of the microbes transforming nitric oxide remains unknown. Here, we show that highly transcribed nitric oxide dismutase (nod) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribenodat lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria. 

ABSTRACT Global climate change impacts marine ecosystems through rising surface temperatures, ocean acidification, and deoxygenation. While the response of the coral holobiont to the first two effects has been relatively well studied, less is known about the response of the coral microbiome to deoxygenation. In this study, we investigated the response of the microbiome to hypoxia in two coral species that differ in their tolerance to hypoxia. We conductedin situoxygen manipulations on a coral reef in Bahía Almirante on the Caribbean coast of Panama, which has previously experienced documented episodes of hypoxia. Naïve coral colonies (previously unexposed to hypoxia) ofSiderastrea sidereaandAgaricia lamarckiwere transplanted to a reef and either enclosed in chambers that created hypoxic conditions or left at ambient oxygen levels. We collected samples of surface mucus and tissue after 48 hours of exposure and characterized the microbiome by sequencing 16S rRNA genes. We found that the microbiomes of the two coral species were distinct from one another and remained so after exhibiting similar shifts in microbiome composition in response to hypoxia. There was an increase in both abundance and number of taxa of anaerobic microbes after exposure to hypoxia. Some of these taxa may play beneficial roles in the coral holobiont by detoxifying the surrounding environment during hypoxic stress or may represent opportunists exploiting host stress. This work describes the first characterization of the coral microbiome under hypoxia and is an initial step toward identifying potential beneficial bacteria for corals facing this environmental stressor. IMPORTANCEMarine hypoxia is a threat for corals but has remained understudied in tropical regions where coral reefs are abundant. Though microbial symbioses can alleviate the effects of ecological stress, we do not yet understand the taxonomic or functional response of the coral microbiome to hypoxia. In this study, we experimentally lowered oxygen levels aroundSiderastrea sidereaandAgaricia lamarckicoloniesin situto observe changes in the coral microbiome in response to deoxygenation. Our results show that hypoxia triggers a stochastic change of the microbiome overall, with some bacterial families changing deterministically after just 48 hours of exposure. These families represent an increase in anaerobic and opportunistic taxa in the microbiomes of both coral species. Thus, marine deoxygenation destabilizes the coral microbiome and increases bacterial opportunism. This work provides novel and fundamental knowledge of the microbial response in coral during hypoxia and may provide insight into holobiont function during stress. 

ABSTRACT Extracellular vesicles are small (approximately 50 to 250 nm in diameter), membrane-bound structures that are released by cells into their surrounding environment. Heterogeneous populations of vesicles are abundant in the global oceans, and they likely play a number of ecological roles in these microbially dominated ecosystems. Here, we examine how vesicle production and size vary among different strains of cultivated marine microbes as well as explore the degree to which this is influenced by key environmental variables. We show that both vesicle production rates and vesicle sizes significantly differ among cultures of marine Proteobacteria, Cyanobacteria, and Bacteroidetes. Further, these properties vary within individual strains as a function of differences in environmental conditions, such as nutrients, temperature, and light irradiance. Thus, both community composition and the local abiotic environment are expected to modulate the production and standing stock of vesicles in the oceans. Examining samples from the oligotrophic North Pacific Gyre, we show depth-dependent changes in the abundance of vesicle-like particles in the upper water column in a manner that is broadly consistent with culture observations: the highest vesicle abundances are found near the surface, where the light irradiances and the temperatures are the greatest, and they then decrease with depth. This work represents the beginnings of a quantitative framework for describing extracellular vesicle dynamics in the oceans, which is essential as we begin to incorporate vesicles into our ecological and biogeochemical understanding of marine ecosystems. IMPORTANCE Bacteria release extracellular vesicles that contain a wide variety of cellular compounds, including lipids, proteins, nucleic acids, and small molecules, into their surrounding environment. These structures are found in diverse microbial habitats, including the oceans, where their distributions vary throughout the water column and likely affect their functional impacts within microbial ecosystems. Using a quantitative analysis of marine microbial cultures, we show that bacterial vesicle production in the oceans is shaped by a combination of biotic and abiotic factors. Different marine taxa release vesicles at rates that vary across an order of magnitude, and vesicle production changes dynamically as a function of environmental conditions. These findings represent a step forward in our understanding of bacterial extracellular vesicle production dynamics and provide a basis for the quantitative exploration of the factors that shape vesicle dynamics in natural ecosystems.