High‐throughput sequencing of the 16S rRNA gene on the Illumina platform is commonly used to assess microbial diversity in environmental samples. The MiniSeq, Illumina's latest benchtop sequencer, ...enables more cost‐efficient DNA sequencing relative to larger Illumina sequencing platforms (e.g., MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high‐throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed additional sequencing primers that enabled application of a dual‐index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples in four different sequencing runs as a quality control benchmark. We were able to recapture a realistic richness of the mock community in all sequencing runs, and identify meaningful differences in alpha and beta diversity in the environmental samples. Furthermore, rarefaction analysis indicated diversity in many environmental samples was close to saturation. These results show that the MiniSeq can produce similar quantities of high‐quality V4 reads compared to the MiSeq, yet is a cost‐effective option for any laboratory interested in performing high‐throughput 16S rRNA gene sequencing.
Innovation in next‐generation DNA sequencing technology continues to contribute to the democratization of 16S rRNA gene sequencing, which is now often carried out on the Illumina MiSeq and HiSeq platforms. Here, we describe a 16S rRNA gene sequencing protocol for the latest benchtop high‐throughput sequencer, the Illumina MiniSeq, which provides comparable quantity and quality of data, but at significantly reduced cost compared to larger and more expensive sequencers. This opens the opportunity for smaller labs to perform their own 16S sequencing independently.
The deep subseafloor sedimentary biosphere is one of the largest ecosystems on Earth, where microbes subsist under energy-limited conditions over long timescales. It remains poorly understood how ...mechanisms of microbial metabolism promote increased fitness in these settings. We discovered that the candidate bacterial phylum “
Candidatus
Atribacteria” dominated a deep-sea subseafloor ecosystem, where it exhibited increased transcription of genes associated with acetogenic fermentation and reproduction in million-year-old sediment. We attribute its improved fitness after burial in the seabed to its capabilities to derive energy from increasingly oxidized metabolites via a bacterial microcompartment and utilize a potentially reversible Wood-Ljungdahl pathway to help meet anabolic and catabolic requirements for growth. Our findings show that “
Ca
. Atribacteria” can perform all the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in anoxic sediments that are millions of years old.
ABSTRACT
How microbial metabolism is translated into cellular reproduction under energy-limited settings below the seafloor over long timescales is poorly understood. Here, we show that microbial abundance increases an order of magnitude over a 5 million-year-long sequence in anoxic subseafloor clay of the abyssal North Atlantic Ocean. This increase in biomass correlated with an increased number of transcribed protein-encoding genes that included those involved in cytokinesis, demonstrating that active microbial reproduction outpaces cell death in these ancient sediments. Metagenomes, metatranscriptomes, and 16S rRNA gene sequencing all show that the actively reproducing community was dominated by the candidate phylum “
Candidatus
Atribacteria,” which exhibited patterns of gene expression consistent with fermentative, and potentially acetogenic, metabolism. “
Ca.
Atribacteria” dominated throughout the 8 million-year-old cored sequence, despite the detection limit for gene expression being reached in 5 million-year-old sediments. The subseafloor reproducing “
Ca.
Atribacteria” also expressed genes encoding a bacterial microcompartment that has potential to assist in secondary fermentation by recycling aldehydes and, thereby, harness additional power to reduce ferredoxin and NAD
+
. Expression of genes encoding the Rnf complex for generation of chemiosmotic ATP synthesis were also detected from the subseafloor “
Ca
. Atribacteria,” as well as the Wood-Ljungdahl pathway that could potentially have an anabolic or catabolic function. The correlation of this metabolism with cytokinesis gene expression and a net increase in biomass over the million-year-old sampled interval indicates that the “
Ca
. Atribacteria” can perform the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in millions-of-years-old anoxic sediments.
IMPORTANCE
The deep subseafloor sedimentary biosphere is one of the largest ecosystems on Earth, where microbes subsist under energy-limited conditions over long timescales. It remains poorly understood how mechanisms of microbial metabolism promote increased fitness in these settings. We discovered that the candidate bacterial phylum “
Candidatus
Atribacteria” dominated a deep-sea subseafloor ecosystem, where it exhibited increased transcription of genes associated with acetogenic fermentation and reproduction in million-year-old sediment. We attribute its improved fitness after burial in the seabed to its capabilities to derive energy from increasingly oxidized metabolites via a bacterial microcompartment and utilize a potentially reversible Wood-Ljungdahl pathway to help meet anabolic and catabolic requirements for growth. Our findings show that “
Ca
. Atribacteria” can perform all the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in anoxic sediments that are millions of years old.
Benthic environments harbor highly diverse and complex microbial communities that control carbon fluxes, but the role of specific uncultivated microbial groups in organic matter turnover is poorly ...understood. In this study, quantitative DNA stable isotope probing (DNA-qSIP) was used for the first time to link uncultivated populations of bacteria and archaea to carbon turnover in lacustrine surface sediments. After 1-week incubations in the dark with
Cbicarbonate, DNA-qSIP showed that ammonia-oxidizing archaea (AOA) were the dominant active chemolithoautotrophs involved in the production of new organic matter. Natural
C-labeled organic matter was then obtained by incubating sediments in the dark for 2.5 months with
Cbicarbonate, followed by extraction and concentration of high-molecular-weight (HMW) (>50-kDa) organic matter. qSIP showed that the labeled organic matter was turned over within 1 week by 823 microbial populations (operational taxonomic units OTUs) affiliated primarily with heterotrophic
,
,
, and
However, several OTUs affiliated with the candidate microbial taxa
,
,
,
,
,
, and
, groups known only from genomic signatures, also contributed to biomass turnover. Of these 823 labeled OTUs, 52% (primarily affiliated with
) also became labeled in 1-week incubations with
Cbicarbonate, indicating that they turned over carbon faster than OTUs that were labeled only in incubations with
C-labeled HMW organic matter. These taxa consisted primarily of uncultivated populations within the
,
,
, and
, highlighting their ecological importance. Our study helps define the role of several poorly understood, uncultivated microbial groups in the turnover of benthic carbon derived from "dark" primary production.
Little is known about the ecological role of uncultivated microbial populations in carbon turnover in benthic environments. To better understand this, we used quantitative stable isotope probing (qSIP) to quantify the abundance of diverse, specific groups of uncultivated bacteria and archaea involved in autotrophy and heterotrophy in a benthic lacustrine habitat. Our results provide quantitative evidence for active heterotrophic and autotrophic metabolism of several poorly understood microbial groups, thus demonstrating their relevance for carbon turnover in benthic settings. Archaeal ammonia oxidizers were significant drivers of
"dark" primary production supporting the growth of heterotrophic bacteria. These findings expand our understanding of the microbial populations within benthic food webs and the role of uncultivated microbes in benthic carbon turnover.
Ammonia-oxidizing archaea (AOA) dominate microbial communities throughout oxic subseafloor sediment deposited over millions of years in the North Atlantic Ocean. Rates of nitrification correlated ...with the abundance of these dominant AOA populations, whose metabolism is characterized by ammonia oxidation, mixotrophic utilization of organic nitrogen, deamination, and the energetically efficient chemolithoautotrophic hydroxypropionate/hydroxybutyrate carbon fixation cycle. These AOA thus have the potential to couple mixotrophic and chemolithoautotrophic metabolism via mixotrophic deamination of organic nitrogen, followed by oxidation of the regenerated ammonia for additional energy to fuel carbon fixation. This metabolic feature likely reduces energy loss and improves AOA fitness under energy-starved, oxic conditions, thereby allowing them to outcompete other taxa for millions of years.
Mounting evidence suggests that animals and their associated bacteria interact via intricate molecular mechanisms, and it is hypothesized that disturbances to the microbiome influence animal ...development. Here, we show that the loss of a key photosymbiont (i.e., bleaching) upon shading correlates with a stark body-plan reorganization in the common aquarium cyanosponge Lendenfeldia chondrodes. The morphological changes observed in shaded sponges include the development of a thread-like morphology that contrasts with the flattened, foliose morphology of control specimens. The microanatomy of shaded sponges markedly differed from that of control sponges, with shaded specimens lacking a well-developed cortex and choanosome. Also, the palisade of polyvacuolar gland-like cells typical in control specimens was absent in shaded sponges. The morphological changes observed in shaded specimens are coupled with broad transcriptomic changes and include the modulation of signaling pathways involved in animal morphogenesis and immune response, such as the Wnt, transforming growth factor β (TGF-β), and TLR-ILR pathways. This study provides a genetic, physiological, and morphological assessment of the effect of microbiome changes on sponge postembryonic development and homeostasis. The correlated response of the sponge host to the collapse of the population of symbiotic cyanobacteria provides evidence for a coupling between the sponge transcriptomic state and the state of its microbiome. This coupling suggests that the ability of animals to interact with their microbiomes and respond to microbiome perturbations has deep evolutionary origins in this group.
Beneath the seafloor, microbial life subsists in isolation from the surface world under persistent energy limitation. The nature and extent of genomic evolution in subseafloor microbes have been ...unknown. Here, we show that the genomes of
bacterial populations cultured from million-year-old subseafloor sediments evolve in clonal populations by point mutation, with a relatively low rate of homologous recombination and elevated numbers of pseudogenes. Ratios of nonsynonymous to synonymous substitutions correlate with the accumulation of pseudogenes, consistent with a role for genetic drift in the subseafloor strains but not in type strains of
isolated from the surface world. Consistent with this, pangenome analysis reveals that the subseafloor bacterial genomes have a significantly lower number of singleton genes than the type strains, indicating a reduction in recent gene acquisitions. Numerous insertion-deletion events and pseudogenes were present in a flagellar operon of the subseafloor bacteria, indicating that motility is nonessential in these million-year-old subseafloor sediments. This genomic evolution in subseafloor clonal populations coincided with a phenotypic difference: all subseafloor isolates have a lower rate of growth under laboratory conditions than the Thalassospira xiamenensis type strain. Our findings demonstrate that the long-term physical isolation of
, in the absence of recombination, has resulted in clonal populations whereby reduced access to novel genetic material from neighbors has resulted in the fixation of new mutations that accumulate in genomes over millions of years.
The nature and extent of genomic evolution in subseafloor microbial populations subsisting for millions of years below the seafloor are unknown. Subseafloor populations have ultralow metabolic rates that are hypothesized to restrict reproduction and, consequently, the spread of new traits. Our findings demonstrate that genomes of cultivated bacterial strains from the genus
isolated from million-year-old abyssal sediment exhibit greatly reduced levels of homologous recombination, elevated numbers of pseudogenes, and genome-wide evidence of relaxed purifying selection. These substitutions and pseudogenes are fixed into the population, suggesting that the genome evolution of these bacteria has been dominated by genetic drift. Thus, reduced recombination, stemming from long-term physical isolation, resulted in small clonal populations of
that have accumulated mutations in their genomes over millions of years.
Biological invasion is one of the main threats to native biodiversity. For a species to become invasive, it must be voluntarily or involuntarily introduced by humans into a nonnative habitat. Mammals ...were among first taxa to be introduced worldwide for game, meat, and labor, yet the number of species introduced in the Neotropics remains unknown. In this data set, we make available occurrence and abundance data on mammal species that (1) transposed a geographical barrier and (2) were voluntarily or involuntarily introduced by humans into the Neotropics. Our data set is composed of 73,738 historical and current georeferenced records on alien mammal species of which around 96% correspond to occurrence data on 77 species belonging to eight orders and 26 families. Data cover 26 continental countries in the Neotropics, ranging from Mexico and its frontier regions (southern Florida and coastal-central Florida in the southeast United States) to Argentina, Paraguay, Chile, and Uruguay, and the 13 countries of Caribbean islands. Our data set also includes neotropical species (e.g., Callithrix sp., Myocastor coypus, Nasua nasua) considered alien in particular areas of Neotropics. The most numerous species in terms of records are from Bos sp. (n = 37,782), Sus scrofa (n = 6,730), and Canis familiaris (n = 10,084); 17 species were represented by only one record (e.g., Syncerus caffer, Cervus timorensis, Cervus unicolor, Canis latrans). Primates have the highest number of species in the data set (n = 20 species), partly because of uncertainties regarding taxonomic identification of the genera Callithrix, which includes the species Callithrix aurita, Callithrix flaviceps, Callithrix geoffroyi, Callithrix jacchus, Callithrix kuhlii, Callithrix penicillata, and their hybrids. This unique data set will be a valuable source of information on invasion risk assessments, biodiversity redistribution and conservation-related research. There are no copyright restrictions. Please cite this data paper when using the data in publications. We also request that researchers and teachers inform us on how they are using the data.
Registry-based studies have identified risk factors for chronic kidney disease (CKD) and for progression to end-stage renal disease. However, usually, these studies do not incorporate sequential ...measurements of kidney function and provide little information on the prognosis of individual patients. The aim of this study is to identify which combinations of demographic and clinical characteristics are useful to discriminate patients with a differential annual decline in glomerular filtration rate (GFR).
This observational retrospective study includes patients enlisted in the registry of the Prevention of Progressive Renal Insufficiency Project of Emilia-Romagna region (Italy) from July 2004 to June 2010, with at least four serum creatinine measurements. Classification tree analysis (CTA) was used to identify subgroups of patients with a different annual GFR decline using demographic and laboratory data collected at study entry.
The CTA procedure generated seven mutually exclusive groups. Among patients with proteinuria, those with a baseline estimated GFR (eGFR) of >33 mL/min/1.73 m(2) exhibited the fastest illness progression in the study population (-3.655 mL/min/1.73 m(2)), followed by patients with a baseline eGFR of <33 mL/min/1.73 m(2) and a baseline serum phosphorus of >4.3 mg/dL (-2.833 mL/min/1.73 m(2)). Among patients without proteinuria, those aged <67 years exhibited a significantly faster progression, which was even faster for the subgroup with diabetes. Among patients aged >67 years, females had on average a stable eGFR over time, with a large variability.
It is possible to rely on a few variables typically accessible in routine clinical practice to stratify patients with a different CKD progression rate. Stratification can be used to guide decisions about the follow-up schedule, treatments to slow progression of kidney disease, prevent its complications and to begin planning for dialysis and transplantation.
The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently ...phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD50 of 2 × 102 CFU, whereas the LD50 for the unencapsulated FP23 was greater than 107 CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.
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