Complete Genome Sequence of an Escherichia coli Strain Isolated from Laboratory Mouse Stool for Use as a Chassis for Transgene Delivery to the Murine Microbiome.

Tools to explore functional changes in the microbiome are limited. Here, we report the complete genome sequence of a strain of Escherichia coli that was isolated from murine stool. This sequence will provide essential information to further develop this tool, and similar tools, to explore the complex murine microbiome.

Intestinal transgene delivery with native E. coli chassis allows persistent physiological changes.

Live bacterial therapeutics (LBTs) could reverse diseases by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally raised (CR) hosts have been unsuccessful because engineered microbial organisms (i.e., chassis) have difficulty in colonizing the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts and enables LBT with curative intent.

Diet and feeding pattern modulate diurnal dynamics of the ileal microbiome and transcriptome.

Compositional oscillations of the gut microbiome are essential for normal peripheral circadian rhythms, both of which are disrupted in diet-induced obesity (DIO). Although time-restricted feeding (TRF) maintains circadian synchrony and protects against DIO, its impact on the dynamics of the cecal gut microbiome is modest. Thus, other regions of the gut, particularly the ileum, the nexus for incretin and bile acid signaling, may play an important role in entraining peripheral circadian rhythms. We demonstrate the effect of diet and feeding rhythms on the ileal microbiome composition and transcriptome in mice. The dynamic rhythms of ileal microbiome composition and transcriptome are dampened in DIO. TRF partially restores diurnal rhythms of the ileal microbiome and transcriptome, increases GLP-1 release, and alters the ileal bile acid pool and farnesoid X receptor (FXR) signaling, which could explain how TRF exerts its metabolic benefits. Finally, we provide a web resource for exploration of ileal microbiome and transcriptome circadian data.

Polar Effects of Transposon Insertion into a Minimal Bacterial Genome.

Global transposon mutagenesis is a valuable tool for identifying genes required for cell viability. Here we present a global analysis of the orientation of viable Tn5-Puror (Tn5-puromycin resistance) insertions into the near-minimal bacterial genome of JCVI-syn2.0. Sixteen of the 478 protein-coding genes show a noticeable asymmetry in the orientation of disrupting insertions of Tn5-Puror Ten of these are located in operons, upstream of essential or quasi-essential genes. Inserts transcribed in the same direction as the downstream gene are favored, permitting read-through transcription of the essential or quasi-essential gene. Some of these genes were classified as quasi-essential solely because of polar effects on the expression of downstream genes. Three genes showing asymmetry in Tn5-Puror insertion orientation prefer the orientation that avoids collisions between read-through transcription of Tn5-Puror and transcription of an adjacent gene. One gene (JCVISYN2_0132 [abbreviated here as "_0132"]) shows a strong preference for Tn5-Puror insertions transcribed upstream, away from the downstream nonessential gene _0133. This suggested that expression of _0133 due to read-through from Tn5-Puror is lethal when _0132 function is disrupted by transposon insertion. This led to the identification of genes _0133 and _0132 as a toxin-antitoxin pair. The three remaining genes show read-through transcription of Tn5-Puror directed downstream and away from sizable upstream intergenic regions (199 bp to 363 bp), for unknown reasons. In summary, polar effects of transposon insertion can, in a few cases, affect the classification of genes as essential, quasi-essential, or nonessential and sometimes can give clues to gene function.IMPORTANCE In studies of the minimal genetic requirements for life, we used global transposon mutagenesis to identify genes needed for a minimal bacterial genome. Transposon insertion can disrupt the function of a gene but can also have polar effects on the expression of adjacent genes. In the Tn5-Puror construct used in our studies, read-through transcription from Tn5-Puror can drive expression of downstream genes. This results in a preference for Tn5-Puror insertions transcribed toward a downstream essential or quasi-essential gene within the same operon. Such polar effects can have an impact on the classification of genes as essential, quasi-essential, or nonessential, but this has been observed in only a few cases. Also, polar effects of Tn5-Puror insertion can sometimes give clues to gene function.

Planktonic Marine Archaea.

Archaea are ubiquitous and abundant members of the marine plankton. Once thought of as rare organisms found in exotic extremes of temperature, pressure, or salinity, archaea are now known in nearly every marine environment. Though frequently referred to collectively, the planktonic archaea actually comprise four major phylogenetic groups, each with its own distinct physiology and ecology. Only one group-the marine Thaumarchaeota-has cultivated representatives, making marine archaea an attractive focus point for the latest developments in cultivation-independent molecular methods. Here, we review the ecology, physiology, and biogeochemical impact of the four archaeal groups using recent insights from cultures and large-scale environmental sequencing studies. We highlight key gaps in our knowledge about the ecological roles of marine archaea in carbon flow and food web interactions. We emphasize the incredible uncultivated diversity within each of the four groups, suggesting there is much more to be done.

Taxon-specific aerosolization of bacteria and viruses in an experimental ocean-atmosphere mesocosm.

Ocean-derived, airborne microbes play important roles in Earth's climate system and human health, yet little is known about factors controlling their transfer from the ocean to the atmosphere. Here, we study microbiomes of isolated sea spray aerosol (SSA) collected in a unique ocean-atmosphere facility and demonstrate taxon-specific aerosolization of bacteria and viruses. These trends are conserved within taxonomic orders and classes, and temporal variation in aerosolization is similarly shared by related taxa. We observe enhanced transfer into SSA of Actinobacteria, certain Gammaproteobacteria, and lipid-enveloped viruses; conversely, Flavobacteriia, some Alphaproteobacteria, and Caudovirales are generally under-represented in SSA. Viruses do not transfer to SSA as efficiently as bacteria. The enrichment of mycolic acid-coated Corynebacteriales and lipid-enveloped viruses (inferred from genomic comparisons) suggests that hydrophobic properties increase transport to the sea surface and SSA. Our results identify taxa relevant to atmospheric processes and a framework to further elucidate aerosolization mechanisms influencing microbial and viral transport pathways.

Feathermoss and epiphytic Nostoc cooperate differently: expanding the spectrum of plant-cyanobacteria symbiosis.

Dinitrogen (N2)-fixation by cyanobacteria in symbiosis with feathermosses is the primary pathway of biological nitrogen (N) input into boreal forests. Despite its significance, little is known about the cyanobacterial gene repertoire and regulatory rewiring needed for the establishment and maintenance of the symbiosis. To determine gene acquisitions and regulatory changes allowing cyanobacteria to form and maintain this symbiosis, we compared genomically closely related symbiotic-competent and -incompetent Nostoc strains using a proteogenomics approach and an experimental set up allowing for controlled chemical and physical contact between partners. Thirty-two gene families were found only in the genomes of symbiotic strains, including some never before associated with cyanobacterial symbiosis. We identified conserved orthologs that were differentially expressed in symbiotic strains, including protein families involved in chemotaxis and motility, NO regulation, sulfate/phosphate transport, and glycosyl-modifying and oxidative stress-mediating exoenzymes. The physical moss-cyanobacteria epiphytic symbiosis is distinct from other cyanobacteria-plant symbioses, with Nostoc retaining motility, and lacking modulation of N2-fixation, photosynthesis, GS-GOGAT cycle and heterocyst formation. The results expand our knowledge base of plant-cyanobacterial symbioses, provide a model of information and material exchange in this ecologically significant symbiosis, and suggest new currencies, namely nitric oxide and aliphatic sulfonates, may be involved in establishing and maintaining the cyanobacteria-feathermoss symbiosis.

Web-based bioinformatics workflows for end-to-end RNA-seq data computation and analysis in agricultural animal species.

BACKGROUND: Remarkable advances in Next Generation Sequencing (NGS) technologies, bioinformatics algorithms and computational technologies have significantly accelerated genomic research. However, complicated NGS data analysis still remains as a major bottleneck. RNA-seq, as one of the major area in the NGS field, also confronts great challenges in data analysis. RESULTS: To address the challenges in RNA-seq data analysis, we developed a web portal that offers three integrated workflows that can perform end-to-end compute and analysis, including sequence quality control, read-mapping, transcriptome assembly, reconstruction and quantification, and differential analysis. The first workflow utilizes Tuxedo (Tophat, Cufflink, Cuffmerge and Cuffdiff suite of tools). The second workflow deploys Trinity for de novo assembly and uses RSEM for transcript quantification and EdgeR for differential analysis. The third combines STAR, RSEM, and EdgeR for data analysis. All these workflows support multiple samples and multiple groups of samples and perform differential analysis between groups in a single workflow job submission. The calculated results are available for download and post-analysis. The supported animal species include chicken, cow, duck, goat, pig, horse, rabbit, sheep, turkey, as well as several other model organisms including yeast, C. elegans, Drosophila, and human, with genomic sequences and annotations obtained from ENSEMBL. The RNA-seq portal is freely available from http://weizhongli-lab.org/RNA-seq . CONCLUSIONS: The web portal offers not only bioinformatics software, workflows, computation and reference data, but also an integrated environment for complex RNA-seq data analysis for agricultural animal species. In this project, our aim is not to develop new RNA-seq tools, but to build web workflows for using popular existing RNA-seq methods and make these tools more accessible to the communities.

Design and synthesis of a minimal bacterial genome.

We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling.

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼ 10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.

Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage.

Bacteria in the 16S rRNA clade SAR86 are among the most abundant uncultivated constituents of microbial assemblages in the surface ocean for which little genomic information is currently available. Bioinformatic techniques were used to assemble two nearly complete genomes from marine metagenomes and single-cell sequencing provided two more partial genomes. Recruitment of metagenomic data shows that these SAR86 genomes substantially increase our knowledge of non-photosynthetic bacteria in the surface ocean. Phylogenomic analyses establish SAR86 as a basal and divergent lineage of γ-proteobacteria, and the individual genomes display a temperature-dependent distribution. Modestly sized at 1.25-1.7 Mbp, the SAR86 genomes lack several pathways for amino-acid and vitamin synthesis as well as sulfate reduction, trends commonly observed in other abundant marine microbes. SAR86 appears to be an aerobic chemoheterotroph with the potential for proteorhodopsin-based ATP generation, though the apparent lack of a retinal biosynthesis pathway may require it to scavenge exogenously-derived pigments to utilize proteorhodopsin. The genomes contain an expanded capacity for the degradation of lipids and carbohydrates acquired using a wealth of tonB-dependent outer membrane receptors. Like the abundant planktonic marine bacterial clade SAR11, SAR86 exhibits metabolic streamlining, but also a distinct carbon compound specialization, possibly avoiding competition.