Conservation of Energetic Pathways for Electroautotrophy in the Uncultivated Candidate Order Tenderiales.

Electromicrobiology can be used to understand extracellular electron uptake in previously undescribed chemolithotrophs. Enrichment and characterization of the uncultivated electroautotroph "Candidatus Tenderia electrophaga" using electromicrobiology led to the designation of the order Tenderiales. Representative Tenderiales 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 Tenderiales and nearest-neighbor orders important for extracellular electron uptake based on a previously proposed pathway from "Ca. Tenderia electrophaga." The Tenderiales contained a conserved cluster we designated uetABCDEFGHIJ, which encodes proteins containing features that would enable transport of extracellular electrons to cytoplasmic membrane-bound energy-transducing complexes such as two conserved cytochrome cbb3 oxidases. For example, UetJ is predicted to be an extracellular undecaheme c-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 Tenderiales 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 Tenderiales and other known neutrophilic iron oxidizers were revealed, including very few Cyc2 genes in the Tenderiales. Our results reveal a possible conserved pathway for extracellular electron uptake and suggest that the Tenderiales have an ecological role in coupling metal or mineral redox chemistry and the carbon cycle in marine and brackish sediments. IMPORTANCE Chemolithotrophic bacteria capable of extracellular electron uptake to drive energy metabolism and CO2 fixation are known as electroautotrophs. The recently described order Tenderiales contains the uncultivated electroautotroph "Ca. Tenderia electrophaga." The "Ca. 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 Tenderiales spp. recovered by metagenome-assembled genomes. This conservation extends to near neighbors of the Tenderiales 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 Tenderiales, and the geographic locations from which metagenome-assembled genomes were recovered offer clues to their natural ecological niche.

Genomic insights into diverse bacterial taxa that degrade extracellular DNA in marine sediments.

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. 13C-DNA added to sediment microcosms was largely degraded within 10 d and mineralized to 13CO2. SIP probing of DNA revealed diverse 'Candidatus Izemoplasma', Lutibacter, Shewanella and Fusibacteraceae incorporated DNA-derived 13C-carbon. NanoSIMS confirmed incorporation of 13C into individual bacterial cells of Fusibacteraceae sorted from microcosms. Genomes of the 13C-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.

Anaerobic bacterial degradation of protein and lipid macromolecules in subarctic marine sediment.

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 13C-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 13C-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.

Proposal to reclassify the proteobacterial classes Deltaproteobacteria and Oligoflexia, and the phylum Thermodesulfobacteria into four phyla reflecting major functional capabilities.

The class Deltaproteobacteria comprises an ecologically and metabolically diverse group of bacteria best known for dissimilatory sulphate reduction and predatory behaviour. Although this lineage is the fourth described class of the phylum Proteobacteria, it rarely affiliates with other proteobacterial classes and is frequently not recovered as a monophyletic unit in phylogenetic analyses. Indeed, one branch of the class Deltaproteobacteria encompassing Bdellovibrio-like predators was recently reclassified into a separate proteobacterial class, the Oligoflexia. Here we systematically explore the phylogeny of taxa currently assigned to these classes using 120 conserved single-copy marker genes as well as rRNA genes. The overwhelming majority of markers reject the inclusion of the classes Deltaproteobacteria and Oligoflexia in the phylum Proteobacteria. Instead, the great majority of currently recognized members of the class Deltaproteobacteria are better classified into four novel phylum-level lineages. We propose the names Desulfobacterota phyl. nov. and Myxococcota phyl. nov. for two of these phyla, based on the oldest validly published names in each lineage, and retain the placeholder name SAR324 for the third phylum pending formal description of type material. Members of the class Oligoflexia represent a separate phylum for which we propose the name Bdellovibrionota phyl. nov. based on priority in the literature and general recognition of the genus Bdellovibrio. Desulfobacterota phyl. nov. includes the taxa previously classified in the phylum Thermodesulfobacteria, and these reclassifications imply that the ability of sulphate reduction was vertically inherited in the Thermodesulfobacteria rather than laterally acquired as previously inferred. Our analysis also indicates the independent acquisition of predatory behaviour in the phyla Myxococcota and Bdellovibrionota, which is consistent with their distinct modes of action. This work represents a stable reclassification of one of the most taxonomically challenging areas of the bacterial tree and provides a robust framework for future ecological and systematic studies.

Glacial Runoff Promotes Deep Burial of Sulfur Cycling-Associated Microorganisms in Marine Sediments.

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 (dsrB) 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 Desulfobacteraceae members, correlated positively with SRRs, indicating their active contributions to sulfur-cycling processes. In contrast, other surface community members, such as Desulfatiglans, Atribacteria, and Chloroflexi, 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.

A Bioinformatics Guide to Plant Microbiome Analysis.

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.

Diversity decoupled from sulfur isotope fractionation in a sulfate-reducing microbial community.

The extent of fractionation of sulfur isotopes by sulfate-reducing microbes is dictated by genomic and environmental factors. A greater understanding of species-specific fractionations may better inform interpretation of sulfur isotopes preserved in the rock record. To examine whether gene diversity influences net isotopic fractionation in situ, we assessed environmental chemistry, sulfate reduction rates, diversity of putative sulfur-metabolizing organisms by 16S rRNA and dissimilatory sulfite reductase (dsrB) gene amplicon sequencing, and net fractionation of sulfur isotopes along a sediment transect of a hypersaline Arctic spring. In situ sulfate reduction rates yielded minimum cell-specific sulfate reduction rates < 0.3 × 10-15 moles cell-1  day-1 . Neither 16S rRNA nor dsrB diversity indices correlated with relatively constant (38‰-45‰) net isotope fractionation (ε34 Ssulfide-sulfate ). Measured ε34 S values could be reproduced in a mechanistic fractionation model if 1%-2% of the microbial community (10%-60% of Deltaproteobacteria) were engaged in sulfate respiration, indicating heterogeneous respiratory activity within sulfate-reducing populations. This model indicated enzymatic kinetic diversity of Apr was more likely to correlate with sulfur fractionation than DsrB. We propose that, above a threshold Shannon diversity value of 0.8 for dsrB, the influence of the specific composition of the microbial community responsible for generating an isotope signal is overprinted by the control exerted by environmental variables on microbial physiology.

Proteomic Response of Three Marine Ammonia-Oxidizing Archaea to Hydrogen Peroxide and Their Metabolic Interactions with a Heterotrophic Alphaproteobacterium.

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 (H2O2)-detoxifying enzyme catalase, and some AOA have been shown to grow poorly under conditions of exposure to H2O2 However, differences in the degrees of H2O2 sensitivity of different AOA strains, the physiological status of AOA cells exposed to H2O2, and their molecular response to H2O2 remain poorly characterized. Further, AOA might rely on heterotrophic bacteria to detoxify H2O2, 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 Nitrosopumilus strains grown in the presence and absence of catalase and in coculture with the heterotrophic alphaproteobacterium Oceanicaulis alexandrii We observed that most proteins detected at a higher relative abundance in H2O2-exposed Nitrosopumilus 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 H2O2 into the cells. Using RNA-stable isotope probing, we confirmed that O. alexandrii cells growing in coculture with the Nitrosopumilus strains assimilated Nitrosopumilus-derived organic carbon, suggesting that AOA could recruit H2O2-detoxifying bacteria through the release of labile organic matter. Our results contribute new insights into the response of AOA to H2O2 and highlight the potential ecological importance of their interactions with heterotrophic free-living bacteria in marine environments.IMPORTANCE Ammonia-oxidizing archaea (AOA) are the most abundant chemolithoautotrophic microorganisms in the oxygenated water column of the global ocean. Although H2O2 appears to be a universal by-product of aerobic metabolism, genes encoding the hydrogen peroxide (H2O2)-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 H2O2, which may contribute to niche differentiation between these organisms. Furthermore, our results suggest that marine AOA rely on H2O2 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.

Long-Term Transcriptional Activity at Zero Growth of a Cosmopolitan Rare Biosphere Member.

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 "Candidatus 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 in situ-like conditions for 50 days by Desulfosporosinus-targeted qPCR and metatranscriptomics. The Desulfosporosinus population stayed at a constant low abundance under all incubation conditions, averaging 1.2 × 106 16S rRNA gene copies per cm³ soil. In contrast, transcriptional activity of "Ca. 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.IMPORTANCE 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.

Visualisation of the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans in co-cultures with micro-algae by CARD-FISH.

Some studies have described the isolation and 16S rRNA gene sequence-based identification of hydrocarbon-degrading bacteria living associated with marine eukaryotic phytoplankton, and thus far the direct visual observation of these bacteria on micro-algal cell surfaces ('phycosphere') has not yet been reported. Here, we developed two new 16S rRNA-targeted oligonucleotide probes, PCY223 and ALGAR209, to respectively detect and enumerate the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH). To enhance the hybridization specificity with the ALGAR209 probe, a competitor probe was developed. These probes were tested and optimized using pure cultures, and then used in enrichment experiments with laboratory cultures of micro-algae exposed to phenanthrene, and with coastal water enriched with crude oil. Microscopic analysis revealed these bacteria are found in culture with the micro-algal cells, some of which were found attached to algal cells, and whose abundance increased after phenanthrene or crude oil enrichment. These new probes are a valuable tool for identifying and studying the ecology of P. algicola and A. aromaticivorans in laboratory and field samples of micro-algae, as well as opening new fields of research that could harness their ability to enhance the bioremediation of contaminated sites.

Bacterial interactions during sequential degradation of cyanobacterial necromass in a sulfidic arctic marine sediment.

Seafloor microorganisms impact global carbon cycling by mineralizing vast quantities of organic matter (OM) from pelagic primary production, which is predicted to increase in the Arctic because of diminishing sea ice cover. We studied microbial interspecies-carbon-flow during anaerobic OM degradation in arctic marine sediment using stable isotope probing. We supplemented sediment incubations with 13 C-labeled cyanobacterial necromass (spirulina), mimicking fresh OM input, or acetate, an important OM degradation intermediate and monitored sulfate reduction rates and concentrations of volatile fatty acids (VFAs) during substrate degradation. Sequential 16S rRNA gene and transcript amplicon sequencing and fluorescence in situ hybridization combined with Raman microspectroscopy revealed that only few bacterial species were the main degraders of 13 C-spirulina necromass. Psychrilyobacter, Psychromonas, Marinifilum, Colwellia, Marinilabiaceae and Clostridiales species were likely involved in the primary hydrolysis and fermentation of spirulina. VFAs, mainly acetate, produced from spirulina degradation were mineralized by sulfate-reducing bacteria and an Arcobacter species. Cellular activity of Desulfobacteraceae and Desulfobulbaceae species during acetoclastic sulfate reduction was largely decoupled from relative 16S rRNA gene abundance shifts. Our findings provide new insights into the identities and physiological constraints that determine the population dynamics of key microorganisms during complex OM degradation in arctic marine sediments.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Peatland Acidobacteria with a dissimilatory sulfur metabolism.

Sulfur-cycling microorganisms impact organic matter decomposition in wetlands and consequently greenhouse gas emissions from these globally relevant environments. However, their identities and physiological properties are largely unknown. By applying a functional metagenomics approach to an acidic peatland, we recovered draft genomes of seven novel Acidobacteria species with the potential for dissimilatory sulfite (dsrAB, dsrC, dsrD, dsrN, dsrT, dsrMKJOP) or sulfate respiration (sat, aprBA, qmoABC plus dsr genes). Surprisingly, the genomes also encoded DsrL, which so far was only found in sulfur-oxidizing microorganisms. Metatranscriptome analysis demonstrated expression of acidobacterial sulfur-metabolism genes in native peat soil and their upregulation in diverse anoxic microcosms. This indicated an active sulfate respiration pathway, which, however, might also operate in reverse for dissimilatory sulfur oxidation or disproportionation as proposed for the sulfur-oxidizing Desulfurivibrio alkaliphilus. Acidobacteria that only harbored genes for sulfite reduction additionally encoded enzymes that liberate sulfite from organosulfonates, which suggested organic sulfur compounds as complementary energy sources. Further metabolic potentials included polysaccharide hydrolysis and sugar utilization, aerobic respiration, several fermentative capabilities, and hydrogen oxidation. Our findings extend both, the known physiological and genetic properties of Acidobacteria and the known taxonomic diversity of microorganisms with a DsrAB-based sulfur metabolism, and highlight new fundamental niches for facultative anaerobic Acidobacteria in wetlands based on exploitation of inorganic and organic sulfur molecules for energy conservation.

Bottled aqua incognita: microbiota assembly and dissolved organic matter diversity in natural mineral waters.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Corrigendum: A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes.

[This corrects the article on p. 731 in vol. 6, PMID: 26236305.].

Diversity analysis of sulfite- and sulfate-reducing microorganisms by multiplex dsrA and dsrB amplicon sequencing using new primers and mock community-optimized bioinformatics.

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.

A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes.

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.