Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis

ABSTRACTBacterial endophytes that colonizePopulustrees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. Here, we describe a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria fromPopulusroots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phylaArmatimonadetes,Verrucomicrobia, andPlanctomycetes, which were unrepresented in our previous cultivation efforts. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.IMPORTANCEPlant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses of genomes of uncultured bacteria to provide genomics information that will facilitate future cultivation attempts.

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The Genome of the Anaerobic Fungus Orpinomyces sp. Strain C1A Reveals the Unique Evolutionary History of a Remarkable Plant Biomass Degrader

Applied and Environmental Microbiology ◽

10.1128/aem.00821-13 ◽

2013 ◽

Vol 79 (15) ◽

pp. 4620-4634 ◽

Cited By ~ 132

Author(s):

Noha H. Youssef ◽

M. B. Couger ◽

Christopher G. Struchtemeyer ◽

Audra S. Liggenstoffer ◽

Rolf A. Prade ◽

...

Keyword(s):

Single Molecule ◽

Crop Residues ◽

Plant Biomass ◽

Consolidated Bioprocessing ◽

Genomic Analysis ◽

Comparative Genomic ◽

Evolutionary Trajectory ◽

Content Type ◽

Gene Acquisition ◽

Intergenic Regions

ABSTRACTAnaerobic gut fungi represent a distinct early-branching fungal phylum (Neocallimastigomycota) and reside in the rumen, hindgut, and feces of ruminant and nonruminant herbivores. The genome of an anaerobic fungal isolate,Orpinomycessp. strain C1A, was sequenced using a combination of Illumina and PacBio single-molecule real-time (SMRT) technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large noncoding intergenic regions (73.1%), proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in early-branching fungal lineages and/or nonfungal Opisthokonta. These included genes for posttranslational fucosylation, the production of specific intramembrane proteases and extracellular protease inhibitors, the formation of a complete axoneme and intraflagellar trafficking machinery, and a near-complete focal adhesion machinery. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments. This capability, acquired during its separate evolutionary trajectory in the rumen, along with its resilience and invasiveness compared to prokaryotic anaerobes, renders anaerobic fungi promising agents for consolidated bioprocessing schemes in biofuels production.

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Enzyme Activities at Different Stages of Plant Biomass Decomposition in Three Species of Fungus-Growing Termites

Applied and Environmental Microbiology ◽

10.1128/aem.01815-17 ◽

2017 ◽

Vol 84 (5) ◽

Cited By ~ 6

Author(s):

Rafael R. da Costa ◽

Haofu Hu ◽

Bo Pilgaard ◽

Sabine M. E. Vreeburg ◽

Julia Schückel ◽

...

Keyword(s):

Plant Material ◽

Plant Biomass ◽

Fungal Spores ◽

Decomposition Process ◽

Old World ◽

Content Type ◽

Substrate Conversion ◽

Fungus Comb ◽

Dead Plant ◽

Plant Polymer

ABSTRACTFungus-growing termites rely on mutualistic fungi of the genusTermitomycesand gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes producedin situ. Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites.IMPORTANCEFungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished.

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Single-cell genomics-based analysis reveals a vital ecological role of Thiocapsa sp. LSW in the meromictic Lake Shunet, Siberia

Microbial Genomics ◽

10.1099/mgen.0.000712 ◽

2021 ◽

Vol 7 (12) ◽

Author(s):

Yu-Ting Wu ◽

Pei-Wen Chiang ◽

Kshitij Tandon ◽

Denis Yu Rogozin ◽

Andrey G. Degermendzhy ◽

...

Keyword(s):

Single Cell ◽

Anoxygenic Phototrophic Bacteria ◽

Sequencing Analysis ◽

Ecological Role ◽

Meromictic Lakes ◽

Purple Sulfur Bacterium ◽

Single Cell Genomics ◽

Content Type ◽

Link Type

Meromictic lakes usually harbour certain prevailing anoxygenic phototrophic bacteria in their anoxic zone, such as the purple sulfur bacterium (PSB) Thiocapsa sp. LSW (hereafter LSW) in Lake Shunet, Siberia. PSBs have been suggested to play a vital role in carbon, nitrogen and sulfur cycling at the oxic–anoxic interface of stratified lakes; however, the ecological significance of PSBs in the lake remains poorly understood. In this study, we explored the potential ecological role of LSW using a deep-sequencing analysis of single-cell genomics associated with flow cytometry. An approximately 2.7 Mb draft genome was obtained based on the co-assembly of five single-cell genomes. LSW might grow photolithoautotrophically and could play putative roles not only as a carbon fixer and diazotroph, but also as a sulfate reducer/oxidizer in the lake. This study provides insights into the potential ecological role of Thiocapsa sp. in meromictic lakes.

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Unravelling Single Cell Genomics

10.1039/9781849732284 ◽

2010 ◽

Keyword(s):

Single Cell ◽

Single Cell Genomics

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Faculty Opinions recommendation of Genome of the pathogen Porphyromonas gingivalis recovered from a biofilm in a hospital sink using a high-throughput single-cell genomics platform.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718011804.793477013 ◽

2013 ◽

Author(s):

Edward Feil

Keyword(s):

Single Cell ◽

Porphyromonas Gingivalis ◽

High Throughput ◽

Single Cell Genomics

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Faculty Opinions recommendation of Single-cell genomics for dissection of complex malaria infections.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718379792.793518969 ◽

2016 ◽

Author(s):

Frank Seeber

Keyword(s):

Single Cell ◽

Single Cell Genomics

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Faculty Opinions recommendation of Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718878575.793503515 ◽

2017 ◽

Author(s):

Daniel Gray

Keyword(s):

Single Cell ◽

Antigen Expression ◽

Single Cell Genomics ◽

Self Antigen

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Gezielte Zellsortierung in der Einzelzellgenomik

BIOspektrum ◽

10.1007/s12268-021-1569-5 ◽

2021 ◽

Vol 27 (3) ◽

pp. 274-276

Author(s):

Morgan S. Sobol ◽

Anne-Kristin Kaster

Keyword(s):

Dark Matter ◽

Single Cell ◽

Cell Sorting ◽

Genetic Information ◽

Fluorescent Labeling ◽

Single Cell Genomics ◽

Rare Biosphere ◽

Prokaryotic Genomes ◽

Selection Of

AbstractSingle cell genomics (SCG) can provide reliable context for assembled genome fragments on the level of individual prokaryotic genomes and has rapidly emerged as an essential complement to cultivation-based and metagenomics research approaches. Targeted cell sorting approaches, which enable the selection of specific taxa by fluorescent labeling, compatible with subsequent single cell genomics offers an opportunity to access genetic information from rare biosphere members which would have otherwise stayed hidden as microbial dark matter.

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An Evolutionary Link between Natural Transformation and CRISPR Adaptive Immunity

mBio ◽

10.1128/mbio.00309-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 52

Author(s):

Peter Jorth ◽

Marvin Whiteley

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Bacterial Diversity ◽

Natural Transformation ◽

Bacterial Species ◽

Comparative Genomic ◽

Content Type ◽

Immune Systems ◽

Adaptive Immune ◽

Adaptive Immune Systems

ABSTRACTNatural transformation by competent bacteria is a primary means of horizontal gene transfer; however, evidence that competence drives bacterial diversity and evolution has remained elusive. To test this theory, we used a retrospective comparative genomic approach to analyze the evolutionary history ofAggregatibacter actinomycetemcomitans, a bacterial species with both competent and noncompetent sister strains. Through comparative genomic analyses, we reveal that competence is evolutionarily linked to genomic diversity and speciation. Competence loss occurs frequently during evolution and is followed by the loss of clustered regularly interspaced short palindromic repeats (CRISPRs), bacterial adaptive immune systems that protect against parasitic DNA. Relative to noncompetent strains, competent bacteria have larger genomes containing multiple rearrangements. In contrast, noncompetent bacterial genomes are extremely stable but paradoxically susceptible to infective DNA elements, which contribute to noncompetent strain genetic diversity. Moreover, incomplete noncompetent strain CRISPR immune systems are enriched for self-targeting elements, which suggests that the CRISPRs have been co-opted for bacterial gene regulation, similar to eukaryotic microRNAs derived from the antiviral RNA interference pathway.IMPORTANCEThe human microbiome is rich with thousands of diverse bacterial species. One mechanism driving this diversity is horizontal gene transfer by natural transformation, whereby naturally competent bacteria take up environmental DNA and incorporate new genes into their genomes. Competence is theorized to accelerate evolution; however, attempts to test this theory have proved difficult. Through genetic analyses of the human periodontal pathogenAggregatibacter actinomycetemcomitans, we have discovered an evolutionary connection between competence systems promoting gene acquisition and CRISPRs (clustered regularly interspaced short palindromic repeats), adaptive immune systems that protect bacteria against genetic parasites. We show that competentA. actinomycetemcomitansstrains have numerous redundant CRISPR immune systems, while noncompetent bacteria have lost their CRISPR immune systems because of inactivating mutations. Together, the evolutionary data linking the evolution of competence and CRISPRs reveals unique mechanisms promoting genetic heterogeneity and the rise of new bacterial species, providing insight into complex mechanisms underlying bacterial diversity in the human body.

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