Microorganisms in marine sediments play major roles in marine biogeochemical cycles by mineralizing substantial quantities of organic matter from decaying cells. Proteins and lipids are abundant ...components of necromass, yet the taxonomic identities of microorganisms that actively degrade them remain poorly resolved. Here, we revealed identities, trophic interactions, and genomic features of bacteria that degraded
C-labeled proteins and lipids in cold anoxic microcosms containing sulfidic subarctic marine sediment. Supplemented proteins and lipids were rapidly fermented to various volatile fatty acids within 5 days. DNA-stable isotope probing (SIP) suggested Psychrilyobacter atlanticus was an important primary degrader of proteins, and Psychromonas members were important primary degraders of both proteins and lipids. Closely related Psychromonas populations, as represented by distinct 16S rRNA gene variants, differentially utilized either proteins or lipids. DNA-SIP also showed
C-labeling of various Deltaproteobacteria within 10 days, indicating trophic transfer of carbon to putative sulfate-reducers. Metagenome-assembled genomes revealed the primary hydrolyzers encoded secreted peptidases or lipases, and enzymes for catabolism of protein or lipid degradation products. Psychromonas species are prevalent in diverse marine sediments, suggesting they are important players in organic carbon processing in situ. Together, this study provides new insights into the identities, functions, and genomes of bacteria that actively degrade abundant necromass macromolecules in the seafloor.
Microbial diversity in the environment is mainly concealed within the rare biosphere (all species with <0.1% relative abundance). While dormancy explains a low-abundance state very well, the ...mechanisms leading to rare but active microorganisms remain elusive. We used environmental systems biology to genomically and transcriptionally characterize "
Desulfosporosinus infrequens," a low-abundance sulfate-reducing microorganism cosmopolitan to freshwater wetlands, where it contributes to cryptic sulfur cycling. We obtained its near-complete genome by metagenomics of acidic peat soil. In addition, we analyzed anoxic peat soil incubated under
-like conditions for 50 days by
-targeted qPCR and metatranscriptomics. The
population stayed at a constant low abundance under all incubation conditions, averaging 1.2 × 10
16S rRNA gene copies per cm³ soil. In contrast, transcriptional activity of "
Desulfosporosinus infrequens" increased at day 36 by 56- to 188-fold when minor amendments of acetate, propionate, lactate, or butyrate were provided with sulfate, compared to the no-substrate-control. Overall transcriptional activity was driven by expression of genes encoding ribosomal proteins, energy metabolism, and stress response but not by expression of genes encoding cell growth-associated processes. Since our results did not support growth of these highly active microorganisms in terms of biomass increase or cell division, they had to invest their sole energy for maintenance, most likely counterbalancing acidic pH conditions. This finding explains how a rare biosphere member can contribute to a biogeochemically relevant process while remaining in a zero-growth state over a period of 50 days.
The microbial rare biosphere represents the largest pool of biodiversity on Earth and constitutes, in sum of all its members, a considerable part of a habitat's biomass. Dormancy or starvation is typically used to explain the persistence of low-abundance microorganisms in the environment. We show that a low-abundance microorganism can be highly transcriptionally active while remaining in a zero-growth state for at least 7 weeks. Our results provide evidence that this zero growth at a high cellular activity state is driven by maintenance requirements. We show that this is true for a microbial keystone species, in particular a cosmopolitan but permanently low-abundance sulfate-reducing microorganism in wetlands that is involved in counterbalancing greenhouse gas emissions. In summary, our results provide an important step forward in understanding time-resolved activities of rare biosphere members relevant for ecosystem functions.
High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a ...highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology.
Summary
Genes encoding dissimilatory sulfite reductase (DsrAB) are commonly used as diagnostic markers in ecological studies of sulfite‐ and sulfate‐reducing microorganisms. Here, we developed new ...high‐coverage primer sets for generation of reductive bacterial‐type dsrA and dsrB polymerase chain reaction (PCR) products for highly parallel amplicon sequencing and a bioinformatics workflow for processing and taxonomic classification of short dsrA and dsrB reads. We employed two diverse mock communities that consisted of 45 or 90 known dsrAB sequences derived from environmental clones to precisely evaluate the performance of individual steps of our amplicon sequencing approach on the Illumina MiSeq platform. Although PCR cycle number, gene‐specific primer mismatches and stringent filtering for high‐quality sequences had notable effects on the observed dsrA and dsrB community structures, recovery of most mock community sequences was generally proportional to their relative input abundances. Successful dsrA and dsrB diversity analysis in selected environmental samples further proved that the multiplex amplicon sequencing approach is adequate for monitoring spatial distribution and temporal abundance dynamics of dsrAB‐containing microorganisms. Although tested for reductive bacterial‐type dsrAB, this method is readily applicable for oxidative‐type dsrAB of sulfur‐oxidizing bacteria and also provides guidance for processing short amplicon reads of other functional genes.
Recent evidence for intimate relationship of plants with their microbiota shows that plants host individual and diverse microbial communities that are essential for their survival. Understanding ...their relatedness using genome-based and high-throughput techniques remains a hot topic in microbiome research. Molecular analysis of the plant holobiont necessitates the application of specific sampling and preparatory steps that also consider sources of unwanted information, such as soil, co-amplified plant organelles, human DNA, and other contaminations. Here, we review state-of-the-art and present practical guidelines regarding experimental and computational aspects to be considered in molecular plant–microbiome studies. We discuss sequencing and “omics” techniques with a focus on the requirements needed to adapt these methods to individual research approaches. The choice of primers and sequence databases is of utmost importance for amplicon sequencing, while the assembly and binning of shotgun metagenomic sequences is crucial to obtain quality data. We discuss specific bioinformatic workflows to overcome the limitation of genome database resources and for covering large eukaryotic genomes such as fungi. In transcriptomics, it is necessary to account for the separation of host mRNA or dual-RNAseq data. Metaproteomics approaches provide a snapshot of the protein abundances within a plant tissue which requires the knowledge of complete and well-annotated plant genomes, as well as microbial genomes. Metabolomics offers a powerful tool to detect and quantify small molecules and molecular changes at the plant–bacteria interface if the necessary requirements with regard to (secondary) metabolite databases are considered. We highlight data integration and complementarity which should help to widen our understanding of the interactions among individual players of the plant holobiont in the future.
Ammonia-oxidizing archaea (AOA) play an important role in the nitrogen cycle and account for a considerable fraction of the prokaryotic plankton in the ocean. Most AOA lack the hydrogen peroxide (H
O
...)-detoxifying enzyme catalase, and some AOA have been shown to grow poorly under conditions of exposure to H
O
However, differences in the degrees of H
O
sensitivity of different AOA strains, the physiological status of AOA cells exposed to H
O
, and their molecular response to H
O
remain poorly characterized. Further, AOA might rely on heterotrophic bacteria to detoxify H
O
, and yet the extent and variety of costs and benefits involved in these interactions remain unclear. Here, we used a proteomics approach to compare the protein profiles of three
strains grown in the presence and absence of catalase and in coculture with the heterotrophic alphaproteobacterium
We observed that most proteins detected at a higher relative abundance in H
O
-exposed
cells had no known function in oxidative stress defense. Instead, these proteins were putatively involved in the remodeling of the extracellular matrix, which we hypothesize to be a strategy limiting the influx of H
O
into the cells. Using RNA-stable isotope probing, we confirmed that
cells growing in coculture with the
strains assimilated
-derived organic carbon, suggesting that AOA could recruit H
O
-detoxifying bacteria through the release of labile organic matter. Our results contribute new insights into the response of AOA to H
O
and highlight the potential ecological importance of their interactions with heterotrophic free-living bacteria in marine environments.
Ammonia-oxidizing archaea (AOA) are the most abundant chemolithoautotrophic microorganisms in the oxygenated water column of the global ocean. Although H
O
appears to be a universal by-product of aerobic metabolism, genes encoding the hydrogen peroxide (H
O
)-detoxifying enzyme catalase are largely absent in genomes of marine AOA. Here, we provide evidence that closely related marine AOA have different degrees of sensitivity to H
O
, which may contribute to niche differentiation between these organisms. Furthermore, our results suggest that marine AOA rely on H
O
detoxification during periods of high metabolic activity and release organic compounds, thereby potentially attracting heterotrophic prokaryotes that provide this missing function. In summary, this report provides insights into the metabolic interactions between AOA and heterotrophic bacteria in marine environments and suggests that AOA play an important role in the biogeochemical carbon cycle by making organic carbon available for heterotrophic microorganisms.
Extracellular DNA is a major macromolecule in global element cycles, and is a particularly crucial phosphorus, nitrogen and carbon source for microorganisms in the seafloor. Nevertheless, the ...identities, ecophysiology and genetic features of DNA-foraging microorganisms in marine sediments are largely unknown. Here, we combined microcosm experiments, DNA stable isotope probing (SIP), single-cell SIP using nano-scale secondary isotope mass spectrometry (NanoSIMS) and genome-centric metagenomics to study microbial catabolism of DNA and its subcomponents in marine sediments.
C-DNA added to sediment microcosms was largely degraded within 10 d and mineralized to
CO
. SIP probing of DNA revealed diverse 'Candidatus Izemoplasma', Lutibacter, Shewanella and Fusibacteraceae incorporated DNA-derived
C-carbon. NanoSIMS confirmed incorporation of
C into individual bacterial cells of Fusibacteraceae sorted from microcosms. Genomes of the
C-labelled taxa all encoded enzymatic repertoires for catabolism of DNA or subcomponents of DNA. Comparative genomics indicated that diverse 'Candidatus Izemoplasmatales' (former Tenericutes) are exceptional because they encode multiple (up to five) predicted extracellular nucleases and are probably specialized DNA-degraders. Analyses of additional sediment metagenomes revealed extracellular nuclease genes are prevalent among Bacteroidota at diverse sites. Together, our results reveal the identities and functional properties of microorganisms that may contribute to the key ecosystem function of degrading and recycling DNA in the seabed.
Marine fjords with active glacier outlets are hot spots for organic matter burial in the sediments and subsequent microbial mineralization. Here, we investigated controls on microbial community ...assembly in sub-arctic glacier-influenced (GI) and non-glacier-influenced (NGI) marine sediments in the Godthåbsfjord region, south-western Greenland. We used a correlative approach integrating 16S rRNA gene and dissimilatory sulfite reductase (
) amplicon sequence data over six meters of depth with biogeochemistry, sulfur-cycling activities, and sediment ages. GI sediments were characterized by comparably high sedimentation rates and had "young" sediment ages of <500 years even at 6 m sediment depth. In contrast, NGI stations reached ages of approximately 10,000 years at these depths. Sediment age-depth relationships, sulfate reduction rates (SRR), and C/N ratios were strongly correlated with differences in microbial community composition between GI and NGI sediments, indicating that age and diagenetic state were key drivers of microbial community assembly in subsurface sediments. Similar bacterial and archaeal communities were present in the surface sediments of all stations, whereas only in GI sediments were many surface taxa also abundant through the whole sediment core. The relative abundance of these taxa, including diverse
members, correlated positively with SRRs, indicating their active contributions to sulfur-cycling processes. In contrast, other surface community members, such as
,
, and
, survived the slow sediment burial at NGI stations and dominated in the deepest sediment layers. These taxa are typical for the energy-limited marine deep biosphere and their relative abundances correlated positively with sediment age. In conclusion, our data suggests that high rates of sediment accumulation caused by glacier runoff and associated changes in biogeochemistry, promote persistence of sulfur-cycling activity and burial of a larger fraction of the surface microbial community into the deep subsurface.
Non-carbonated natural mineral waters contain microorganisms that regularly grow after bottling despite low concentrations of dissolved organic matter (DOM). Yet, the compositions of bottled water ...microbiota and organic substrates that fuel microbial activity, and how both change after bottling, are still largely unknown.
We performed a multifaceted analysis of microbiota and DOM diversity in 12 natural mineral waters from six European countries. 16S rRNA gene-based analyses showed that less than 10 species-level operational taxonomic units (OTUs) dominated the bacterial communities in the water phase and associated with the bottle wall after a short phase of post-bottling growth. Members of the betaproteobacterial genera Curvibacter, Aquabacterium, and Polaromonas (Comamonadaceae) grew in most waters and represent ubiquitous, mesophilic, heterotrophic aerobes in bottled waters. Ultrahigh-resolution mass spectrometry of DOM in bottled waters and their corresponding source waters identified thousands of molecular formulae characteristic of mostly refractory, soil-derived DOM.
The bottle environment, including source water physicochemistry, selected for growth of a similar low-diversity microbiota across various bottled waters. Relative abundance changes of hundreds of multi-carbon molecules were related to growth of less than ten abundant OTUs. We thus speculate that individual bacteria cope with oligotrophic conditions by simultaneously consuming diverse DOM molecules.
Electromicrobiology can be used to understand extracellular electron uptake in previously undescribed chemolithotrophs. Enrichment and characterization of the uncultivated electroautotroph "
Tenderia ...electrophaga" using electromicrobiology led to the designation of the order
Representative
metagenome-assembled genomes (MAGs) have been identified in a number of environmental surveys, yet a comprehensive characterization of conserved genes for extracellular electron uptake has thus far not been conducted. Using comparative genomics, we identified conserved orthologous genes within the
and nearest-neighbor orders important for extracellular electron uptake based on a previously proposed pathway from "
Tenderia electrophaga." The
contained a conserved cluster we designated
, which encodes proteins containing features that would enable transport of extracellular electrons to cytoplasmic membrane-bound energy-transducing complexes such as two conserved cytochrome
oxidases. For example, UetJ is predicted to be an extracellular undecaheme
-type cytochrome that forms a heme wire. We also identified clusters of genes predicted to facilitate assembly and maturation of electron transport proteins, as well as cellular attachment to surfaces. Autotrophy among the
is supported by the presence of carbon fixation and stress response pathways that could allow cellular growth by extracellular electron uptake. Key differences between the
and other known neutrophilic iron oxidizers were revealed, including very few Cyc2 genes in the
. Our results reveal a possible conserved pathway for extracellular electron uptake and suggest that the
have an ecological role in coupling metal or mineral redox chemistry and the carbon cycle in marine and brackish sediments.
Chemolithotrophic bacteria capable of extracellular electron uptake to drive energy metabolism and CO
fixation are known as electroautotrophs. The recently described order
contains the uncultivated electroautotroph "
Tenderia electrophaga." The "
Tenderia electrophaga" genome contains genes proposed to make up a previously undescribed extracellular electron uptake pathway. Here, we use comparative genomics to show that this pathway is well conserved among
spp. recovered by metagenome-assembled genomes. This conservation extends to near neighbors of the
but not to other well-studied chemolithotrophs, including iron and sulfur oxidizers, indicating that these genes may be useful markers of growth using insoluble extracellular electron donors. Our findings suggest that extracellular electron uptake and electroautotrophy may be pervasive among the
, and the geographic locations from which metagenome-assembled genomes were recovered offer clues to their natural ecological niche.
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