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September 22, 2019  |  

Genomic insights into the acid adaptation of novel methanotrophs enriched from acidic forest soils.

Soil acidification is accelerated by anthropogenic and agricultural activities, which could significantly affect global methane cycles. However, detailed knowledge of the genomic properties of methanotrophs adapted to acidic soils remains scarce. Using metagenomic approaches, we analyzed methane-utilizing communities enriched from acidic forest soils with pH 3 and 4, and recovered near-complete genomes of proteobacterial methanotrophs. Novel methanotroph genomes designated KS32 and KS41, belonging to two representative clades of methanotrophs (Methylocystis of Alphaproteobacteria and Methylobacter of Gammaproteobacteria), were dominant. Comparative genomic analysis revealed diverse systems of membrane transporters for ensuring pH homeostasis and defense against toxic chemicals. Various potassium transporter systems, sodium/proton antiporters, and two copies of proton-translocating F1F0-type ATP synthase genes were identified, which might participate in the key pH homeostasis mechanisms in KS32. In addition, the V-type ATP synthase and urea assimilation genes might be used for pH homeostasis in KS41. Genes involved in the modification of membranes by incorporation of cyclopropane fatty acids and hopanoid lipids might be used for reducing proton influx into cells. The two methanotroph genomes possess genes for elaborate heavy metal efflux pumping systems, possibly owing to increased heavy metal toxicity in acidic conditions. Phylogenies of key genes involved in acid adaptation, methane oxidation, and antiviral defense in KS41 were incongruent with that of 16S rRNA. Thus, the detailed analysis of the genome sequences provides new insights into the ecology of methanotrophs responding to soil acidification.

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September 22, 2019  |  

Accurate determination of bacterial abundances in human metagenomes using full-length 16S sequencing reads

DNA sequencing of PCR-amplified marker genes, especially but not limited to the 16S rRNA gene, is perhaps the most common approach for profiling microbial communities. Due to technological constraints of commonly available DNA sequencing, these approaches usually take the form of short reads sequenced from a narrow, targeted variable region, with a corresponding loss of taxonomic resolution relative to the full length marker gene. We use Pacific Biosciences single-molecule, real-time circular consensus sequencing to sequence amplicons spanning the entire length of the 16S rRNA gene. However, this sequencing technology suffers from high sequencing error rate that needs to be addressed in order to take full advantage of the longer sequence. Here, we present a method to model the sequencing error process using a generalized pair hidden Markov chain model and estimate bacterial abundances in microbial samples. We demonstrate, with simulated and real data, that our model and its associated estimation procedure are able to give accurate estimates at the species (or subspecies) level, and is more flexible than existing methods like SImple Non-Bayesian TAXonomy (SINTAX).

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September 22, 2019  |  

High-resolution characterization of the human microbiome.

The human microbiome plays an important and increasingly recognized role in human health. Studies of the microbiome typically use targeted sequencing of the 16S rRNA gene, whole metagenome shotgun sequencing, or other meta-omic technologies to characterize the microbiome’s composition, activity, and dynamics. Processing, analyzing, and interpreting these data involve numerous computational tools that aim to filter, cluster, annotate, and quantify the obtained data and ultimately provide an accurate and interpretable profile of the microbiome’s taxonomy, functional capacity, and behavior. These tools, however, are often limited in resolution and accuracy and may fail to capture many biologically and clinically relevant microbiome features, such as strain-level variation or nuanced functional response to perturbation. Over the past few years, extensive efforts have been invested toward addressing these challenges and developing novel computational methods for accurate and high-resolution characterization of microbiome data. These methods aim to quantify strain-level composition and variation, detect and characterize rare microbiome species, link specific genes to individual taxa, and more accurately characterize the functional capacity and dynamics of the microbiome. These methods and the ability to produce detailed and precise microbiome information are clearly essential for informing microbiome-based personalized therapies. In this review, we survey these methods, highlighting the challenges each method sets out to address and briefly describing methodological approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

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September 22, 2019  |  

A single-cell genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 µm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.

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September 22, 2019  |  

Genomics and host specialization of honey bee and bumble bee gut symbionts.

Gilliamella apicola and Snodgrassella alvi are dominant members of the honey bee (Apis spp.) and bumble bee (Bombus spp.) gut microbiota. We generated complete genomes of the type strains G. apicola wkB1(T) and S. alvi wkB2(T) (isolated from Apis), as well as draft genomes for four other strains from Bombus. G. apicola and S. alvi were found to occupy very different metabolic niches: The former is a saccharolytic fermenter, whereas the latter is an oxidizer of carboxylic acids. Together, they may form a syntrophic network for partitioning of metabolic resources. Both species possessed numerous genes [type 6 secretion systems, repeats in toxin (RTX) toxins, RHS proteins, adhesins, and type IV pili] that likely mediate cell-cell interactions and gut colonization. Variation in these genes could account for the host fidelity of strains observed in previous phylogenetic studies. Here, we also show the first experimental evidence, to our knowledge, for this specificity in vivo: Strains of S. alvi were able to colonize their native bee host but not bees of another genus. Consistent with specific, long-term host association, comparative genomic analysis revealed a deep divergence and little or no gene flow between Apis and Bombus gut symbionts. However, within a host type (Apis or Bombus), we detected signs of horizontal gene transfer between G. apicola and S. alvi, demonstrating the importance of the broader gut community in shaping the evolution of any one member. Our results show that host specificity is likely driven by multiple factors, including direct host-microbe interactions, microbe-microbe interactions, and social transmission.

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September 22, 2019  |  

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

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September 22, 2019  |  

Crosstalk between gut microbiota and Sirtuin-3 in colonic inflammation and tumorigenesis.

Colorectal cancer (CRC) is a disease involving a variety of genetic and environmental factors. Sirtuin-3 (Sirt3) is expressed at a low level in cancer tissues of CRC, but it is unclear how Sirt3 modulates colonic tumorigenesis. In this study, we found that gut microbiota play a central role in the resistance to CRC tumor formation in wild-type (WT) mice through APC (Adenomatous Polyposis Coli)-mutant mouse microbiota transfer via Wnt signaling. We also found that Sirt3-deficient mice were hypersusceptible to colonic inflammation and tumor development through altered intestinal integrity and p38 signaling, respectively. Furthermore, susceptibility to colorectal tumorigenesis was aggravated by initial commensal microbiota deletion via Wnt signaling. Mice with Sirt3-deficient microbiota transfer followed by chemically induced colon tumorigenesis had low Sirt3 expression compared to WT control microbiome transfer, mainly due to a decrease in Escherichia/Shigella, as well as an increase in Lactobacillus reuteri and Lactobacillus taiwanensis. Collectively, our data revealed that Sirt3 is an anti-inflammatory and tumor-suppressing gene that interacts with the gut microbiota during colon tumorigenesis.

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September 22, 2019  |  

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality. Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hyphomonadaceae strain UKL13-1 (3,501,508 bp and 56.12% GC), incertae sedis Betaproteobacterium strain UKL13-2 (3,387,087 bp and 54.98% GC), and incertae sedis Bacteroidetes strain UKL13-3 (3,236,529 bp and 37.33% GC). Each genome consists of a single circular chromosome with no identified plasmids. When compared with binned Illumina assemblies of the same three genomes, there was ~7% discrepancy in total genome length. Gaps where Illumina assemblies broke were often due to repetitive elements. Within these missing sequences were essential genes and genes associated with a variety of functional categories. Annotated gene content reveals that both Proteobacteria are aerobic anoxygenic phototrophs, with Betaproteobacterium UKL13-2 potentially capable of phototrophic oxidation of sulfur compounds. Both proteobacterial genomes contain transporters suggesting they are scavenging fixed nitrogen from A. flos-aquae in the form of ammonium. Bacteroidetes UKL13-3 has few completely annotated biosynthetic pathways, and has a comparatively higher proportion of unannotated genes. The genomes were detected in only a few other freshwater metagenomes, suggesting that these bacteria are not ubiquitous in freshwater systems. Our results indicate that long-read sequencing is a viable method for sequencing dominant members from low-diversity microbial communities, and should be considered for environmental metagenomics when conditions meet these requirements.

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September 22, 2019  |  

Diversified microbiota of meconium is affected by maternal diabetes status.

This study was aimed to assess the diversity of the meconium microbiome and determine if the bacterial community is affected by maternal diabetes status.The first intestinal discharge (meconium) was collected from 23 newborns stratified by maternal diabetes status: 4 mothers had pre-gestational type 2 diabetes mellitus (DM) including one mother with dizygotic twins, 5 developed gestational diabetes mellitus (GDM) and 13 had no diabetes. The meconium microbiome was profiled using multi-barcode 16S rRNA sequencing followed by taxonomic assignment and diversity analysis.All meconium samples were not sterile and contained diversified microbiota. Compared with adult feces, the meconium showed a lower species diversity, higher sample-to-sample variation, and enrichment of Proteobacteria and reduction of Bacteroidetes. Among the meconium samples, the taxonomy analyses suggested that the overall bacterial content significantly differed by maternal diabetes status, with the microbiome of the DM group showing higher alpha-diversity than that of no-diabetes or GDM groups. No global difference was found between babies delivered vaginally versus via Cesarean-section. Regression analysis showed that the most robust predictor for the meconium microbiota composition was the maternal diabetes status that preceded pregnancy. Specifically, Bacteroidetes (phyla) and Parabacteriodes (genus) were enriched in the meconium in the DM group compared to the no-diabetes group.Our study provides evidence that meconium contains diversified microbiota and is not affected by the mode of delivery. It also suggests that the meconium microbiome of infants born to mothers with DM is enriched for the same bacterial taxa as those reported in the fecal microbiome of adult DM patients.

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September 22, 2019  |  

Metagenomic binning of a marine sponge microbiome reveals unity in defense but metabolic specialization.

Marine sponges are ancient metazoans that are populated by distinct and highly diverse microbial communities. In order to obtain deeper insights into the functional gene repertoire of the Mediterranean sponge Aplysina aerophoba, we combined Illumina short-read and PacBio long-read sequencing followed by un-targeted metagenomic binning. We identified a total of 37 high-quality bins representing 11 bacterial phyla and two candidate phyla. Statistical comparison of symbiont genomes with selected reference genomes revealed a significant enrichment of genes related to bacterial defense (restriction-modification systems, toxin-antitoxin systems) as well as genes involved in host colonization and extracellular matrix utilization in sponge symbionts. A within-symbionts genome comparison revealed a nutritional specialization of at least two symbiont guilds, where one appears to metabolize carnitine and the other sulfated polysaccharides, both of which are abundant molecules in the sponge extracellular matrix. A third guild of symbionts may be viewed as nutritional generalists that perform largely the same metabolic pathways but lack such extraordinary numbers of the relevant genes. This study characterizes the genomic repertoire of sponge symbionts at an unprecedented resolution and it provides greater insights into the molecular mechanisms underlying microbial-sponge symbiosis.

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September 22, 2019  |  

Genomic microdiversity of Bifidobacterium pseudocatenulatum underlying differential strain-level responses to dietary carbohydrate intervention.

The genomic basis of the response to dietary intervention of human gut beneficial bacteria remains elusive, which hinders precise manipulation of the microbiota for human health. After receiving a dietary intervention enriched with nondigestible carbohydrates for 105 days, a genetically obese child with Prader-Willi syndrome lost 18.4% of his body weight and showed significant improvement in his bioclinical parameters. We obtained five isolates (C1, C15, C55, C62, and C95) of one of the most abundantly promoted beneficial species, Bifidobacterium pseudocatenulatum, from a postintervention fecal sample. Intriguingly, these five B. pseudocatenulatum strains showed differential responses during the dietary intervention. Two strains were largely unaffected, while the other three were promoted to different extents by the changes in dietary carbohydrate resources. The differential responses of these strains were consistent with their functional clustering based on the COGs (Clusters of Orthologous Groups), including those involved with the ABC-type sugar transport systems, suggesting that the strain-specific genomic variations may have contributed to the niche adaption. Particularly, B. pseudocatenulatum C15, which had the most diverse types and highest gene copy numbers of carbohydrate-active enzymes targeting plant polysaccharides, had the highest abundance after the dietary intervention. These studies show the importance of understanding genomic diversity of specific members of the gut microbiota if precise nutrition approaches are to be realized.IMPORTANCE The manipulation of the gut microbiota via dietary approaches is a promising option for improving human health. Our findings showed differential responses of multiple B. pseudocatenulatum strains isolated from the same habitat to the dietary intervention, as well as strain-specific correlations with bioclinical parameters of the host. The comparative genomics revealed a genome-level microdiversity of related functional genes, which may have contributed to these differences. These results highlight the necessity of understanding strain-level differences if precise manipulation of gut microbiota through dietary approaches is to be realized. Copyright © 2017 Wu et al.

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September 22, 2019  |  

The non-specific adenine DNA methyltransferase M.EcoGII.

We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo – using a high copy pRRS plasmid vector and a methylation-deficient E. coli host-extensive in vivo adenine methylation activity is revealed. M.EcoGII methylates adenine residues in any DNA sequence context and this activity extends to dA and rA bases in either strand of a DNA:RNA-hybrid oligonucleotide duplex and to rA bases in RNAs prepared by in vitro transcription. Using oligonucleotide and bacteriophage M13mp18 virion DNA substrates, we find that M.EcoGII also methylates single-stranded DNA in vitro and that this activity is only slightly less robust than that observed using equivalent double-stranded DNAs. In vitro assays, using purified recombinant M.EcoGII enzyme, demonstrate that up to 99% of dA bases in duplex DNA substrates can be methylated thereby rendering them insensitive to cleavage by multiple restriction endonucleases. These properties suggest that the enzyme could also be used for high resolution mapping of protein binding sites in DNA and RNA substrates.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019  |  

Characterization of the SN35N strain-specific exopolysaccharide encoded in the whole circular genome of a plant-derived Lactobacillus plantarum.

Lactobacillus plantarum SN35N, which has been previously isolated from pear, secretes exopolysaccharide (EPS). The aim of the present study is to characterize the EPS chemically and to find the EPS-biosynthesizing gene cluster. The present study demonstrates that the strain produces an acidic EPS carrying phosphate residue, which is composed of glucose, galactose, and mannose at a molecular ratio of 15.0?:?5.7?:?1.0. We also show that acidic EPS strongly inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35), promoting an inflammatory reaction. In the present study, we also determined the complete genome sequence of the SN35N strain, demonstrating that the genome is a circular DNA with 3267626?bp, and the number of predicted coding genes is 3146, with a GC content of 44.51%. In addition, the strain harbors four plasmids, designated pSN35N-1, -2, -3, and -4. Although four EPS-biosynthesizing genes, designated lpe1, lpe2, lpe3, and lpe4, are present in the SN35N chromosomal DNA, another EPS gene cluster, lpe5, is located in the pSN35N-3 plasmid, composed of 35425?bp. EPS low-producing mutants, which were obtained by treating SN35N cells with novobiocin, lost the lpe5 gene cluster in the plasmid-curing experiment, suggesting that the gene cluster for the biosynthesis of acidic EPS is present in the plasmid. The present study shows the chemical characterization of the acidic EPS and its inhibitory effect to the hyaluronidase.

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September 22, 2019  |  

In situ analyses directly in diarrheal stool reveal large variations in bacterial load and active toxin expression of enterotoxigenic Escherichia coli and Vibrio cholerae.

The bacterial pathogens enterotoxigenicEscherichia coli(ETEC) andVibrio choleraeare major causes of diarrhea. ETEC causes diarrhea by production of the heat-labile toxin (LT) and heat-stable toxins (STh and STp), whileV. choleraeproduces cholera toxin (CT). In this study, we determined the occurrence and bacterial doses of the two pathogens and their respective toxin expression levels directly in liquid diarrheal stools of patients in Dhaka, Bangladesh. By quantitative culture and real-time quantitative PCR (qPCR) detection of the toxin genes, the two pathogens were found to coexist in several of the patients, at concentrations between 102and 108bacterial gene copies per ml. Even in culture-negative samples, gene copy numbers of 102to 104of either ETEC orV. choleraetoxin genes were detected by qPCR. RNA was extracted directly from stool, and gene expression levels, quantified by reverse transcriptase qPCR (RT-qPCR), of the genes encoding CT, LT, STh, and STp showed expression of toxin genes. Toxin enzyme-linked immunosorbent assay (ELISA) confirmed active toxin secretion directly in the liquid diarrhea. Analysis of ETEC isolates by multiplex PCR, dot blot analysis, and genome sequencing suggested that there are genetic ETEC profiles that are more commonly found as dominating single pathogens and others that are coinfectants with lower bacterial loads. The ETEC genomes, including assembled genomes of dominating ETEC isolates expressing LT/STh/CS5/CS6 and LT/CS7, are provided. In addition, this study highlights an emerging important ETEC strain expressing LT/STp and the novel colonization factor CS27b. These findings have implications for investigations of pathogenesis as well as for vaccine development. IMPORTANCEThe cause of diarrheal disease is usually determined by screening for several microorganisms by various methods, and sole detection is used to assign the agent as the cause of disease. However, it has become increasingly clear that many infections are caused by coinfections with several pathogens and that the dose of the infecting pathogen is important. We quantified the absolute numbers of enterotoxigenicE. coli(ETEC) andVibrio choleraedirectly in diarrheal fluid. We noted several events where both pathogens were found but also a large dose dependency. In three samples, we found ETEC as the only pathogen sought for. These isolates belonged to globally distributed ETEC clones and were the dominating species in stool with active toxin expression. This suggests that certain superior virulent ETEC lineages are able to outcompete the gut microbiota and be the sole cause of disease and hence need to be specifically monitored.

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September 22, 2019  |  

Candidatus Nitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermophilic archaeon with a highly mobile genome.

Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first enrichment of a thermophilic representative with a sequenced genome, which facilitates the search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota.CandidatusNitrosocaldus cavascurensis has been enriched from a hot spring in Ischia, Italy. It grows optimally at 68°C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 23 h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome ofCa.N. cavascurensis harbors anamoAXCB gene cluster encoding ammonia monooxygenase and genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although abona fidegene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the potential importance of this compound for AOA metabolism.Ca.N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.

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