To decipher the influence of depth stratification and surface provincialism on the dark ocean prokaryotic community composition, we sampled the major deep-water masses in the eastern North Atlantic covering three biogeographic provinces. Their diversity was evaluated using ordination and canonical analysis of 454 pyrotag sequences. Variance partitioning suggested that 16% of the variation in the bacterial community composition was based on depth stratification while 9% of the variation was due to geographic location. General linear mixed effect models showed that the community of the subsurface waters was connected to the dark ocean prokaryotic communities in different biogeographic provinces. Cluster analysis indicated that some prokaryotic taxa are specific to distinct regions in bathypelagic water masses. Taken together, our data suggest that the dark ocean prokaryotic community composition of the eastern North Atlantic is primed by the formation and the horizontal transport of water masses.

The deazaflavin cofactor F420 enhances the persistence of mycobacteria during hypoxia, oxidative stress, and antibiotic treatment. However, the identities and functions of the mycobacterial enzymes that utilize F420 under these conditions have yet to be resolved. In this work, we used sequence similarity networks to analyze the distribution of the largest F420-dependent protein family in mycobacteria. We show that these enzymes are part of a larger split β-barrel enzyme superfamily (flavin/deazaflavin oxidoreductases, FDORs) that include previously characterized pyridoxamine/pyridoxine-5'-phosphate oxidases and heme oxygenases. We show that these proteins variously utilize F420, flavin mononucleotide, flavin adenine dinucleotide, and heme cofactors. Functional annotation using phylogenetic, structural, and spectroscopic methods revealed their involvement in heme degradation, biliverdin reduction, fatty acid modification, and quinone reduction. Four novel crystal structures show that plasticity in substrate binding pockets and modifications to cofactor binding motifs enabled FDORs to carry out a variety of functions. This systematic classification and analysis provides a framework for further functional analysis of the roles of FDORs in mycobacterial pathogenesis and persistence.

Anthropogenically induced changes in precipitation are projected to generate increased river runoff to semi-enclosed seas, increasing loads of terrestrial dissolved organic matter and decreasing salinity. To determine how bacterial community structure and functioning adjust to such changes, we designed microcosm transplant experiments with Baltic Proper (salinity 7.2) and Bothnian Sea (salinity 3.6) water. Baltic Proper bacteria generally reached higher abundances than Bothnian Sea bacteria in both Baltic Proper and Bothnian Sea water, indicating higher adaptability. Moreover, Baltic Proper bacteria growing in Bothnian Sea water consistently showed highest bacterial production and beta-glucosidase activity. These metabolic responses were accompanied by basin-specific changes in bacterial community structure. For example, Baltic Proper Pseudomonas and Limnobacter populations increased markedly in relative abundance in Bothnian Sea water, indicating a replacement effect. In contrast, Roseobacter and Rheinheimera populations were stable or increased in abundance when challenged by either of the waters, indicating an adjustment effect. Transplants to Bothnian Sea water triggered the initial emergence of particular Burkholderiaceae populations, and transplants to Baltic Proper water triggered Alteromonadaceae populations. Notably, in the subsequent re-transplant experiment, a priming effect resulted in further increases to dominance of these populations. Correlated changes in community composition and metabolic activity were observed only in the transplant experiment and only at relatively high phylogenetic resolution. This suggested an importance of successional progression for interpreting relationships between bacterial community composition and functioning. We infer that priming effects on bacterial community structure by natural episodic events or climate change induced forcing could translate into long-term changes in bacterial ecosystem process rates.

Taurine, an amino acid-like compound, acts as an osmostress protectant in many marine metazoans and algae and is released via various processes into the oceanic dissolved organic matter pool. Taurine transporters are widespread among members of the marine prokaryotic community, tentatively indicating that taurine might be an important substrate for prokaryotes in the ocean. In this study, we determined prokaryotic taurine assimilation and respiration throughout the water column along two transects in the North Atlantic off the Iberian Peninsula. Taurine assimilation efficiency decreased from the epipelagic waters from 55 ± 14% to 27 ± 20% in the bathypelagic layers (means of both transects). Members of the ubiquitous alphaproteobacterial SAR11 clade accounted for a large fraction of cells taking up taurine, especially in surface waters. Archaea (Thaumarchaeota + Euryarchaeota) were also able to take up taurine in the upper water column, but to a lower extent than Bacteria. The contribution of taurine assimilation to the heterotrophic prokaryotic carbon biomass production ranged from 21% in the epipelagic layer to 16% in the bathypelagic layer. Hence, we conclude that dissolved free taurine is a significant carbon and energy source for prokaryotes throughout the oceanic water column being utilized with similar efficiencies as dissolved free amino acids.

Even though the uptake and assimilation of organic compounds by phytoplankton has been long recognized, very little is still known about its potential ecological role in natural marine communities and whether it varies depending on the light regimes the algae experience. We combined measurements of size-fractionated assimilation of trace additions of (3)H-leucine and (35)S-dimethylsulfoniopropionate (DMSP) with microautoradiography to assess the extent and relevance of osmoheterotrophy in summer phytoplankton assemblages from Arctic and Antarctic waters, and the role of solar radiation on it was further investigated by exposing samples to different radiation spectra. Significant assimilation of both substrates occurred in the size fraction containing most phytoplankton (>5 µm), sunlight exposure generally increasing (35)S-DMSP-sulfur assimilation and decreasing (3)H-leucine assimilation. Microautoradiography revealed that the capacity to take up both organic substrates seemed widespread among different polar algal phyla, particularly in pennate and centric diatoms, and photosynthetic dinoflagellates. Image analysis of the microautoradiograms showed for the first time interspecific variability in the uptakes of (35)S-DMSP and (3)H-leucine by phytoplankton depending on the solar spectrum. Overall, these results suggest that the role of polar phytoplankton in the utilization of labile dissolved organic matter may be significant under certain conditions and further confirm the relevance of solar radiation in regulating heterotrophy in the pelagic ocean.

Aerobic methanotrophic bacteria have evolved a specialist lifestyle dependent on consumption of methane and other short-chain carbon compounds. However, their apparent substrate specialism runs contrary to the high relative abundance of these microorganisms in dynamic environments, where the availability of methane and oxygen fluctuates. In this work, we provide in situ and ex situ evidence that verrucomicrobial methanotrophs are mixotrophs. Verrucomicrobia-dominated soil communities from an acidic geothermal field in Rotokawa, New Zealand rapidly oxidised methane and hydrogen simultaneously. We isolated and characterised a verrucomicrobial strain from these soils, Methylacidiphilum sp. RTK17.1, and showed that it constitutively oxidises molecular hydrogen. Genomic analysis confirmed that this strain encoded two [NiFe]-hydrogenases (group 1d and 3b), and biochemical assays revealed that it used hydrogen as an electron donor for aerobic respiration and carbon fixation. While the strain could grow heterotrophically on methane or autotrophically on hydrogen, it grew optimally by combining these metabolic strategies. Hydrogen oxidation was particularly important for adaptation to methane and oxygen limitation. Complementary to recent findings of hydrogenotrophic growth by Methylacidiphilum fumariolicum SolV, our findings illustrate that verrucomicrobial methanotrophs have evolved to simultaneously utilise hydrogen and methane from geothermal sources to meet energy and carbon demands where nutrient flux is dynamic. This mixotrophic lifestyle is likely to have facilitated expansion of the niche space occupied by these microorganisms, allowing them to become dominant in geothermally influenced surface soils. Genes encoding putative oxygen-tolerant uptake [NiFe]-hydrogenases were identified in all publicly available methanotroph genomes, suggesting hydrogen oxidation is a general metabolic strategy in this guild.

Zooplankton and microbes play a key role in the ocean's biological cycles by releasing and consuming copious amounts of particulate and dissolved organic matter. Additionally, zooplankton provide a complex microhabitat rich in organic and inorganic nutrients in which bacteria thrive. In this study, we assessed the phylogenetic composition and metabolic potential of microbial communities associated with crustacean zooplankton species collected in the North Atlantic. Using Illumina sequencing of the 16S rRNA gene, we found significant differences between the microbial communities associated with zooplankton and those inhabiting the surrounding seawater. Metagenomic analysis of the zooplankton-associated microbial community revealed a highly specialized bacterial community able to exploit zooplankton as microhabitat and thus, mediating biogeochemical processes generally underrepresented in the open ocean. The zooplankton-associated bacterial community is able to colonize the zooplankton's internal and external surfaces using a large set of adhesion mechanisms and to metabolize complex organic compounds released or exuded by the zooplankton such as chitin, taurine and other complex molecules. Moreover, the high number of genes involved in iron and phosphorus metabolisms in the zooplankton-associated microbiome suggests that this zooplankton-associated bacterial community mediates specific biogeochemical processes (through the proliferation of specific taxa) that are generally underrepresented in the ambient waters.

Mesophilic ammonia-oxidizing Archaea (AOA) are abundant in a diverse range of marine environments, including the deep ocean, as revealed by the quantification of the archaeal amoA gene encoding the alpha-subunit of the ammonia monooxygenase. Using two different amoA primer sets, two distinct ecotypes of marine Crenarchaeota Group I (MCGI) were detected in the waters of the tropical Atlantic and the coastal Arctic. The HAC-AOA ecotype (high ammonia concentration AOA) was ≈ 8000 times and 15 times more abundant in the coastal Arctic and the top 300 m layer of the open equatorial Atlantic, respectively, than the LAC-AOA (low ammonia concentration AOA) ecotype. In contrast, the LAC-AOA ecotype dominated the lower meso- and bathypelagic waters of the tropical Atlantic (≈ 50 times more abundant than the HAC-AOA) where ammonia concentrations are well below the detection limit using conventional spectrophotometric or fluorometric methods. Cluster analysis of the sequences from the clone libraries obtained by the two amoA primer sets revealed two phylogenetically distinct clusters. Taken together, our results suggest the presence of two ecotypes of archaeal ammonia oxidizers corresponding to the medium (1.24 µM on average in the coastal Arctic) and low ammonia concentration (< 0.01 µM) in the shallow and the deep waters respectively.

Messenger RNA can provide valuable insights into the variability of metabolic processes of microorganisms. However, due to uncertainties that include the stability of RNA, its application for activity profiling of environmental samples is questionable. We explored different factors affecting the decay rate of transcripts of three marine bacterial isolates using qPCR and determined mRNA half-life time of specific bacterial taxa and of functional genes by metatranscriptomics of a coastal environmental prokaryotic community. The half-life time of transcripts from 11 genes from bacterial isolates ranged from 1 to 46 min. About 80% of the analysed transcripts exhibited half-live times shorter than 10 min. Significant differences were found in the half-life time between mRNA and rRNA. The half-life time of mRNA obtained from a coastal metatranscriptome ranged from 9 to 400 min. The shortest half-life times of the metatranscriptome corresponded to transcripts from the same clusters of orthologous groups (COGs) in all bacterial classes. The prevalence of short mRNA half-life time in genes related to defence mechanisms and motility indicate a tight connection of RNA decay rate to environmental stressors. The short half-life time of RNA and its high variability needs to be considered when assessing metatranscriptomes especially in environmental samples.

Ammonia-oxidizing archaea (AOA) constitute a considerable fraction of microbial biomass in the global ocean, comprising 20%-40% of the ocean's prokaryotic plankton. However, it remains enigmatic to what extent these chemolithoautotrophic archaea release dissolved organic carbon (DOC). A combination of targeted and untargeted metabolomics was used to characterize the exometabolomes of three model AOA strains of the Nitrosopumilus genus. Our results indicate that marine AOA exude a suite of organic compounds with potentially varying reactivities, dominated by nitrogen-containing compounds. A significant fraction of the released dissolved organic matter (DOM) consists of labile compounds, which typically limit prokaryotic heterotrophic activity in open ocean waters, including amino acids, thymidine and B vitamins. Amino acid release rates corresponded with ammonia oxidation activity and the three Nitrosopumilus strains predominantly released hydrophobic amino acids, potentially as a result of passive diffusion. Despite the low contribution of DOC released by AOA (~0.08%-1.05%) to the heterotrophic prokaryotic carbon demand, the release of physiologically relevant metabolites could be crucial for microbes that are auxotrophic for some of these compounds, including members of the globally abundant and ubiquitous SAR11 clade.

Increasing urban populations have led to the growth of informal settlements, with contaminated environments linked to poor human health through a range of interlinked pathways. Here, we describe the design and methods for the Revitalising Informal Settlements and their Environments (RISE) study, a transdisciplinary randomised trial evaluating impacts of an intervention to upgrade urban informal settlements in two Asia-Pacific countries.

Throughout coastal Antarctica, ice shelves separate oceanic waters from sunlight by hundreds of meters of ice. Historical studies have detected activity of nitrifying microorganisms in oceanic cavities below permanent ice shelves. However, little is known about the microbial composition and pathways that mediate these activities. In this study, we profiled the microbial communities beneath the Ross Ice Shelf using a multi-omics approach. Overall, beneath-shelf microorganisms are of comparable abundance and diversity, though distinct composition, relative to those in the open meso- and bathypelagic ocean. Production of new organic carbon is likely driven by aerobic lithoautotrophic archaea and bacteria that can use ammonium, nitrite, and sulfur compounds as electron donors. Also enriched were aerobic organoheterotrophic bacteria capable of degrading complex organic carbon substrates, likely derived from in situ fixed carbon and potentially refractory organic matter laterally advected by the below-shelf waters. Altogether, these findings uncover a taxonomically distinct microbial community potentially adapted to a highly oligotrophic marine environment and suggest that ocean cavity waters are primarily chemosynthetically-driven systems.

Ergosterol has traditionally been used as a proxy to estimate fungal biomass as it is almost exclusively found in fungal lipid membranes. Ergosterol determination has been mostly used for fungal samples from terrestrial, freshwater, salt marsh- and mangrove-dominated environments or to describe fungal degradation of plant matter. In the open ocean, however, the expected concentrations of ergosterol are orders of magnitude lower than in terrestrial or macrophyte-dominated coastal systems. Consequently, the fungal biomass in the open ocean remains largely unknown. Recent evidence based on microscopy and -omics techniques suggests, however, that fungi contribute substantially to the microbial biomass in the oceanic water column, highlighting the need to accurately determine fungal biomass in the open ocean. We performed ergosterol extractions of an oceanic fungal isolate (Rhodotorula sphaerocarpa) with biomass concentrations varying over nine orders of magnitude. While after the initial chloroform-methanol extraction ~87% of the ergosterol was recovered, a second extraction recovered an additional ~10%. Testing this extraction method on samples collected from the open Atlantic Ocean, we successfully determined ergosterol concentrations as low as 0.12 pM. Thus, this highly sensitive method is well suited for measuring fungal biomass from open ocean waters, including deep-sea environments.

The ocean-atmosphere exchange of CO2 largely depends on the balance between marine microbial photosynthesis and respiration. Despite vast taxonomic and metabolic diversity among marine planktonic bacteria and archaea (prokaryoplankton)1-3, their respiration usually is measured in bulk and treated as a 'black box' in global biogeochemical models4; this limits the mechanistic understanding of the global carbon cycle. Here, using a technology for integrated phenotype analyses and genomic sequencing of individual microbial cells, we show that cell-specific respiration rates differ by more than 1,000× among prokaryoplankton genera. The majority of respiration was found to be performed by minority members of prokaryoplankton (including the Roseobacter cluster), whereas cells of the most prevalent lineages (including Pelagibacter and SAR86) had extremely low respiration rates. The decoupling of respiration rates from abundance among lineages, elevated counts of proteorhodopsin transcripts in Pelagibacter and SAR86 cells and elevated respiration of SAR86 at night indicate that proteorhodopsin-based phototrophy3,5-7 probably constitutes an important source of energy to prokaryoplankton and may increase growth efficiency. These findings suggest that the dependence of prokaryoplankton on respiration and remineralization of phytoplankton-derived organic carbon into CO2 for its energy demands and growth may be lower than commonly assumed and variable among lineages.

Although terrestrial and aquatic fungi are well-known decomposers of organic matter, the role of marine fungi remains largely unknown. Recent studies based on omics suggest that marine fungi potentially play a major role in elemental cycles. However, there is very limited information on the diversity of extracellular enzymatic activities performed by pelagic fungi in the ocean and how these might be affected by community composition and/or critical environmental parameters such as temperature. In order to obtain information on the potential metabolic activity of marine fungi, extracellular enzymatic activities (EEA) were investigated. Five marine fungal species belonging to the most abundant pelagic phyla (Ascomycota and Basidiomycota) were grown at 5 °C and 20 °C, and fluorogenic enzymatic assays were performed using six substrate analogues for the hydrolysis of carbohydrates (β-glucosidase, β-xylosidase, and N-acetyl-β-D-glucosaminidase), amino acids (leucine aminopeptidase), and of organic phosphorus (alkaline phosphatase) and sulfur compounds (sulfatase). Remarkably, all fungal strains were capable of hydrolyzing all the offered substrates. However, the hydrolysis rate (Vmax) and half-saturation constant (Km) varied among the fungal strains depending on the enzyme type. Temperature had a strong impact on the EEAs, resulting in Q10 values of up to 6.1 and was species and substrate dependent. The observed impact of temperature on fungal EEA suggests that warming of the global ocean might alter the contribution of pelagic fungi in marine biogeochemical cycles.

Molecular hydrogen (H2) is an abundant and readily accessible energy source in marine systems, but it remains unknown whether marine microbial communities consume this gas. Here we use a suite of approaches to show that marine bacteria consume H2 to support growth. Genes for H2-uptake hydrogenases are prevalent in global ocean metagenomes, highly expressed in metatranscriptomes and found across eight bacterial phyla. Capacity for H2 oxidation increases with depth and decreases with oxygen concentration, suggesting that H2 is important in environments with low primary production. Biogeochemical measurements of tropical, temperate and subantarctic waters, and axenic cultures show that marine microbes consume H2 supplied at environmentally relevant concentrations, yielding enough cell-specific power to support growth in bacteria with low energy requirements. Conversely, our results indicate that oxidation of carbon monoxide (CO) primarily supports survival. Altogether, H2 is a notable energy source for marine bacteria and may influence oceanic ecology and biogeochemistry.

Heterotrophic prokaryotes express extracellular hydrolytic enzymes to cleave large organic molecules before taking up the hydrolyzed products. According to foraging theory, extracellular enzymes should be cell associated in dilute systems such as deep sea habitats, but secreted into the surrounding medium in diffusion-limited systems. However, extracellular enzymes in the deep sea are found mainly dissolved in ambient water rather than cell associated. In order to resolve this paradox, we conducted a global survey of peptidases and carbohydrate-active enzymes (CAZymes), two key enzyme groups initiating organic matter assimilation, in an integrated metagenomics, metatranscriptomics, and metaproteomics approach. The abundance, percentage, and diversity of genes encoding secretory processes, i.e., dissolved enzymes, consistently increased from epipelagic to bathypelagic waters, indicating that organic matter cleavage, and hence prokaryotic metabolism, is mediated mainly by particle-associated prokaryotes releasing their extracellular enzymes into diffusion-limited particles in the bathypelagic realm.

Studies of unculturable microbes often combine methods, such as 16S rRNA sequencing, metagenomics, and metaproteomics. To apply these techniques to the microbial community inhabiting the surfaces of marine macrophytes, it is advisable to perform a selective DNA and protein isolation prior to the analysis to avoid biases due to the host material being present in high quantities. Two protocols for DNA and protein isolation were adapted for selective extractions of DNA and proteins from epiphytic communities inhabiting the surfaces of two marine macrophytes, the seagrass Cymodocea nodosa and the macroalga Caulerpa cylindracea. Protocols showed an almost complete removal of the epiphytic community regardless of the sampling season, station, settlement, or host species. The obtained DNA was suitable for metagenomic and 16S rRNA sequencing, while isolated proteins could be identified by mass spectrometry. Low presence of host DNA and proteins in the samples indicated a high specificity of the protocols. The procedures are based on universally available laboratory chemicals making the protocols widely applicable. Taken together, the adapted protocols ensure an almost complete removal of the macrophyte epiphytic community. The procedures are selective for microbes inhabiting macrophyte surfaces and provide DNA and proteins applicable in 16S rRNA sequencing, metagenomics, and metaproteomics.

Carbon monoxide (CO) gas is infamous for its acute toxicity. This toxicity predominantly stems from its tendency to form carbonyl complexes with transition metals, thus inhibiting the heme-prosthetic groups of proteins, including respiratory terminal oxidases. While CO has been proposed as an antibacterial agent, the evidence supporting its toxicity toward bacteria is equivocal, and its cellular targets remain poorly defined. In this work, we investigate the physiological response of mycobacteria to CO. We show that Mycobacterium smegmatis is highly resistant to the toxic effects of CO, exhibiting only minor inhibition of growth when cultured in its presence. We profiled the proteome of M. smegmatis during growth in CO, identifying strong induction of cytochrome bd oxidase and members of the dos regulon, but relatively few other changes. We show that the activity of cytochrome bd oxidase is resistant to CO, whereas cytochrome bcc-aa 3 oxidase is strongly inhibited by this gas. Consistent with these findings, growth analysis shows that M. smegmatis lacking cytochrome bd oxidase displays a significant growth defect in the presence of CO, while induction of the dos regulon appears to be unimportant for adaptation to CO. Altogether, our findings indicate that M. smegmatis has considerable resistance to CO and benefits from respiratory flexibility to withstand its inhibitory effects.IMPORTANCE Carbon monoxide has an infamous reputation as a toxic gas, and it has been suggested that it has potential as an antibacterial agent. Despite this, how bacteria resist its toxic effects is not well understood. In this study, we investigated how CO influences growth, proteome, and aerobic respiration of wild-type and mutant strains of Mycobacterium smegmatis We show that this bacterium produces the CO-resistant cytochrome bd oxidase to tolerate poisoning of its CO-sensitive complex IV homolog. Further, we show that aside from this remodeling of its respiratory chain, M. smegmatis makes few other functional changes to its proteome, suggesting it has a high level of inherent resistance to CO.

Bacteria have evolved sophisticated uptake machineries in order to obtain the nutrients required for growth. Gram-negative plant pathogens of the genus Pectobacterium obtain iron from the protein ferredoxin, which is produced by their plant hosts. This iron-piracy is mediated by the ferredoxin uptake system (Fus), a gene cluster encoding proteins that transport ferredoxin into the bacterial cell and process it proteolytically. In this work we show that gene clusters related to the Fus are widespread in bacterial species. Through structural and biochemical characterisation of the distantly related Fus homologues YddB and PqqL from Escherichia coli, we show that these proteins are analogous to components of the Fus from Pectobacterium. The membrane protein YddB shares common structural features with the outer membrane ferredoxin transporter FusA, including a large extracellular substrate binding site. PqqL is an active protease with an analogous periplasmic localisation and iron-dependent expression to the ferredoxin processing protease FusC. Structural analysis demonstrates that PqqL and FusC share specific features that distinguish them from other members of the M16 protease family. Taken together, these data provide evidence that protease associated import systems analogous to the Fus are widespread in Gram-negative bacteria.