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September 22, 2019  |  

Comparative genome and transcriptome analysis reveals distinctive surface characteristics and unique physiological potentials of Pseudomonas aeruginosa ATCC 27853.

Pseudomonas aeruginosa ATCC 27853 was isolated from a hospital blood specimen in 1971 and has been widely used as a model strain to survey antibiotics susceptibilities, biofilm development, and metabolic activities of Pseudomonas spp.. Although four draft genomes of P. aeruginosa ATCC 27853 have been sequenced, the complete genome of this strain is still lacking, hindering a comprehensive understanding of its physiology and functional genome.Here we sequenced and assembled the complete genome of P. aeruginosa ATCC 27853 using the Pacific Biosciences SMRT (PacBio) technology and Illumina sequencing platform. We found that accessory genes of ATCC 27853 including prophages and genomic islands (GIs) mainly contribute to the difference between P. aeruginosa ATCC 27853 and other P. aeruginosa strains. Seven prophages were identified within the genome of P. aeruginosa ATCC 27853. Of the predicted 25 GIs, three contain genes that encode monoxoygenases, dioxygenases and hydrolases that could be involved in the metabolism of aromatic compounds. Surveying virulence-related genes revealed that a series of genes that encode the B-band O-antigen of LPS are lacking in ATCC 27853. Distinctive SNPs in genes of cellular adhesion proteins such as type IV pili and flagella biosynthesis were also observed in this strain. Colony morphology analysis confirmed an enhanced biofilm formation capability of ATCC 27853 on solid agar surface compared to Pseudomonas aeruginosa PAO1. We then performed transcriptome analysis of ATCC 27853 and PAO1 using RNA-seq and compared the expression of orthologous genes to understand the functional genome and the genomic details underlying the distinctive colony morphogenesis. These analyses revealed an increased expression of genes involved in cellular adhesion and biofilm maturation such as type IV pili, exopolysaccharide and electron transport chain components in ATCC 27853 compared with PAO1. In addition, distinctive expression profiles of the virulence genes lecA, lasB, quorum sensing regulators LasI/R, and the type I, III and VI secretion systems were observed in the two strains.The complete genome sequence of P. aeruginosa ATCC 27853 reveals the comprehensive genetic background of the strain, and provides genetic basis for several interesting findings about the functions of surface associated proteins, prophages, and genomic islands. Comparative transcriptome analysis of P. aeruginosa ATCC 27853 and PAO1 revealed several classes of differentially expressed genes in the two strains, underlying the genetic and molecular details of several known and yet to be explored morphological and physiological potentials of P. aeruginosa ATCC 27853.


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.


September 22, 2019  |  

Transcriptional fates of human-specific segmental duplications in brain.

Despite the importance of duplicate genes for evolutionary adaptation, accurate gene annotation is often incomplete, incorrect, or lacking in regions of segmental duplication. We developed an approach combining long-read sequencing and hybridization capture to yield full-length transcript information and confidently distinguish between nearly identical genes/paralogs. We used biotinylated probes to enrich for full-length cDNA from duplicated regions, which were then amplified, size-fractionated, and sequenced using single-molecule, long-read sequencing technology, permitting us to distinguish between highly identical genes by virtue of multiple paralogous sequence variants. We examined 19 gene families as expressed in developing and adult human brain, selected for their high sequence identity (average >99%) and overlap with human-specific segmental duplications (SDs). We characterized the transcriptional differences between related paralogs to better understand the birth-death process of duplicate genes and particularly how the process leads to gene innovation. In 48% of the cases, we find that the expressed duplicates have changed substantially from their ancestral models due to novel sites of transcription initiation, splicing, and polyadenylation, as well as fusion transcripts that connect duplication-derived exons with neighboring genes. We detect unannotated open reading frames in genes currently annotated as pseudogenes, while relegating other duplicates to nonfunctional status. Our method significantly improves gene annotation, specifically defining full-length transcripts, isoforms, and open reading frames for new genes in highly identical SDs. The approach will be more broadly applicable to genes in structurally complex regions of other genomes where the duplication process creates novel genes important for adaptive traits.© 2018 Dougherty et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.


September 22, 2019  |  

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested.Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes.MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.


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.


September 22, 2019  |  

Metagenomic approaches to assess bacteriophages in various environmental niches.

Bacteriophages are ubiquitous and numerous parasites of bacteria and play a critical evolutionary role in virtually every ecosystem, yet our understanding of the extent of the diversity and role of phages remains inadequate for many ecological niches, particularly in cases in which the host is unculturable. During the past 15 years, the emergence of the field of viral metagenomics has drastically enhanced our ability to analyse the so-called viral ‘dark matter’ of the biosphere. Here, we review the evolution of viral metagenomic methodologies, as well as providing an overview of some of the most significant applications and findings in this field of research.


September 22, 2019  |  

Comparative genomic analysis of Sulfurospirillum cavolei MES reconstructed from the metagenome of an electrosynthetic microbiome.

Sulfurospirillum spp. play an important role in sulfur and nitrogen cycling, and contain metabolic versatility that enables reduction of a wide range of electron acceptors, including thiosulfate, tetrathionate, polysulfide, nitrate, and nitrite. Here we describe the assembly of a Sulfurospirillum genome obtained from the metagenome of an electrosynthetic microbiome. The ubiquity and persistence of this organism in microbial electrosynthesis systems suggest it plays an important role in reactor stability and performance. Understanding why this organism is present and elucidating its genetic repertoire provide a genomic and ecological foundation for future studies where Sulfurospirillum are found, especially in electrode-associated communities. Metabolic comparisons and in-depth analysis of unique genes revealed potential ecological niche-specific capabilities within the Sulfurospirillum genus. The functional similarities common to all genomes, i.e., core genome, and unique gene clusters found only in a single genome were identified. Based upon 16S rRNA gene phylogenetic analysis and average nucleotide identity, the Sulfurospirillum draft genome was found to be most closely related to Sulfurospirillum cavolei. Characterization of the draft genome described herein provides pathway-specific details of the metabolic significance of the newly described Sulfurospirillum cavolei MES and, importantly, yields insight to the ecology of the genus as a whole. Comparison of eleven sequenced Sulfurospirillum genomes revealed a total of 6246 gene clusters in the pan-genome. Of the total gene clusters, 18.5% were shared among all eleven genomes and 50% were unique to a single genome. While most Sulfurospirillum spp. reduce nitrate to ammonium, five of the eleven Sulfurospirillum strains encode for a nitrous oxide reductase (nos) cluster with an atypical nitrous-oxide reductase, suggesting a utility for this genus in reduction of the nitrous oxide, and as a potential sink for this potent greenhouse gas.


September 22, 2019  |  

Characteristics of ARG-carrying plasmidome in the cultivable microbial community from wastewater treatment system under high oxytetracycline concentration.

Studies on antibiotic production wastewater have shown that even a single antibiotic can select for multidrug resistant bacteria in aquatic environments. It is speculated that plasmids are an important mechanism of multidrug resistance (MDR) under high concentrations of antibiotics. Herein, two metagenomic libraries were constructed with plasmid DNA extracted from cultivable microbial communities in a biological wastewater treatment reactor supplemented with 0 (CONTROL) or 25 mg/L of oxytetracycline (OTC-25). The OTC-25 plasmidome reads were assigned to 72 antibiotic resistance genes (ARGs) conferring resistance to 13 types of antibiotics. Dominant ARGs, encoding resistance to tetracycline, aminoglycoside, sulfonamide, and multidrug resistance genes, were enriched in the plasmidome under 25 mg/L of oxytetracycline. Furthermore, 17 contiguous multiple-ARG carrying contigs (carrying =?2 ARGs) were discovered in the OTC-25 plasmidome, whereas only nine were found in the CONTROL. Mapping of the OTC-25 plasmidome reads to completely sequenced plasmids revealed that the conjugative IncU resistance plasmid pFBAOT6 of Aeromonas caviae, carrying multidrug resistance transporter (pecM), tetracycline resistance genes (tetA, tetR), and transposase genes, might be a potential prevalent resistant plasmid in the OTC-25 plasmidome. Additionally, two novel resistant plasmids (containing contig C301682 carrying multidrug resistant operon mexCD-oprJ and contig C301632 carrying the tet36 and transposases genes) might also be potential prevalent resistant plasmids in the OTC-25 plasmidome. This study will be helpful to better understand the role of plasmids in the development of MDR in water environments under high antibiotic concentrations.


September 22, 2019  |  

Single-cell RNAseq for the study of isoforms-how is that possible?

Single-cell RNAseq and alternative splicing studies have recently become two of the most prominent applications of RNAseq. However, the combination of both is still challenging, and few research efforts have been dedicated to the intersection between them. Cell-level insight on isoform expression is required to fully understand the biology of alternative splicing, but it is still an open question to what extent isoform expression analysis at the single-cell level is actually feasible. Here, we establish a set of four conditions that are required for a successful single-cell-level isoform study and evaluate how these conditions are met by these technologies in published research.


September 22, 2019  |  

Diverse antibiotic resistance genes in dairy cow manure.

Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected to intensive antibiotic use, such as pigs and chickens. Cow manure has received less attention, although it is commonly used in crop production. Here, we report the discovery of novel and diverse antibiotic resistance genes in the cow microbiome, demonstrating that it is a significant reservoir of antibiotic resistance genes. The genomic resource presented here lays the groundwork for understanding the dispersal of antibiotic resistance from the agroecosystem to other settings.


September 22, 2019  |  

Multiplex amplicon sequencing for microbe identification in community-based culture collections.

Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.


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.


September 22, 2019  |  

Resolving the complexity of human skin metagenomes using single-molecule sequencing.

Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation.The species comprising a microbial community are often difficult to deconvolute due to technical limitations inherent to most short-read sequencing technologies. Here, we leverage new advances in sequencing technology, single-molecule sequencing, to significantly improve reconstruction of a complex human skin microbial community. With this long-read technology, we were able to reconstruct and annotate a closed, high-quality genome of a previously uncharacterized skin species. We demonstrate that hybrid approaches with short-read technology are sufficiently powerful to reconstruct even single-nucleotide polymorphism level variation of species in this a community. Copyright © 2016 Tsai et al.


September 22, 2019  |  

Prey range and genome evolution of Halobacteriovorax marinus predatory bacteria from an estuary

Halobacteriovorax strains are saltwater-adapted predatory bacteria that attack Gram-negative bacteria and may play an important role in shaping microbial communities. To understand how Halobacteriovorax strains impact ecosystems and develop them as biocontrol agents, it is important to characterize variation in predation phenotypes and investigate Halobacteriovorax genome evolution. We isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island using Vibrio from the same site as prey. Small, fast-moving, attack-phase BE01 cells attach to and invade prey cells, consistent with the intraperiplasmic predation strategy of the H. marinus type strain, SJ. BE01 is a prey generalist, forming plaques on Vibrio strains from the estuary, Pseudomonas from soil, and Escherichia coli. Genome analysis revealed extremely high conservation of gene order and amino acid sequences between BE01 and SJ, suggesting strong selective pressure to maintain the genome in this H. marinus lineage. Despite this, we identified two regions of gene content difference that likely resulted from horizontal gene transfer. Analysis of modal codon usage frequencies supports the hypothesis that these regions were acquired from bacteria with different codon usage biases than H. marinus. In one of these regions, BE01 and SJ carry different genes associated with mobile genetic elements. Acquired functions in BE01 include the dnd operon, which encodes a pathway for DNA modification, and a suite of genes involved in membrane synthesis and regulation of gene expression that was likely acquired from another Halobacteriovorax lineage. This analysis provides further evidence that horizontal gene transfer plays an important role in genome evolution in predatory bacteria. IMPORTANCE Predatory bacteria attack and digest other bacteria and therefore may play a role in shaping microbial communities. To investigate phenotypic and genotypic variation in saltwater-adapted predatory bacteria, we isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island, assayed whether it could attack different prey bacteria, and sequenced and analyzed its genome. We found that BE01 is a prey generalist, attacking bacteria from different phylogenetic groups and environments. Gene order and amino acid sequences are highly conserved between BE01 and the H. marinus type strain, SJ. By comparative genomics, we detected two regions of gene content difference that likely occurred via horizontal gene transfer events. Acquired genes encode functions such as modification of DNA, membrane synthesis and regulation of gene expression. Understanding genome evolution and variation in predation phenotypes among predatory bacteria will inform their development as biocontrol agents and clarify how they impact microbial communities.


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