Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics

Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment

Energiestoffwechsel von Cytochrom-freien methanogenen Archaea bei Wachstum auf H2 und CO2

Methanogene Archaea sind Organismen, die Methan als Endprodukt ihres anaeroben Energiestoffwechsels bilden. Es gibt zwei stoffwechselphysiologisch unterschiedliche Gruppen: Zum einen die Cytochrom-freien Methanogenen, welche nur H2 und CO2 und/oder Formiat zu Methan umsetzen und zum anderen die Cytochrom-haltigen Methanogenen, die auf Acetat, Methanol und/oder Methylamin spezialisiert sind und von denen nur einige wenige auf H2 und CO2 wachsen können. Da lediglich der Energiestoffwechsel Cytochrom-haltiger Methanogener bei Wachstum auf H2 und CO2 weitgehend verstanden ist, war es das Ziel der vorliegenden Arbeit, den Energiestoffwechsel Cytochrom-freier Methanogener aufzuklären. Dazu wurden zunächst die Wachstumserträge und H2-Schwellenkonzentrationen von zwei Cytochrom-freien Methanogenen bestimmt und mit denen eines Cytochrom-haltigen verglichen. Anschließend wurde die Kopplung des ersten und letzten Schrittes der Methanbildung aus H2 und CO2 auf Enzymebene untersucht

First shotgun metagenomics study of Juan de Fuca deep-sea sediments reveals distinct microbial communities above, within, between, and below sulfate methane transition zones

The marine deep subsurface is home to a vast microbial ecosystem, affecting biogeochemical cycles on a global scale. One of the better-studied deep biospheres is the Juan de Fuca (JdF) Ridge, where hydrothermal fluid introduces oxidants into the sediment from below, resulting in two sulfate methane transition zones (SMTZs). In this study, we present the first shotgun metagenomics study of unamplified DNA from sediment samples from different depths in this stratified environment. Bioinformatic analyses showed a shift from a heterotrophic, Chloroflexota-dominated community above the upper SMTZ to a chemolithoautotrophic Proteobacteria-dominated community below the secondary SMTZ. The reintroduction of sulfate likely enables respiration and boosts active cells that oxidize acetate, iron, and complex carbohydrates to degrade dead biomass in this low-abundance, low-diversity environment. In addition, analyses showed many proteins of unknown function as well as novel metagenome-assembled genomes (MAGs). The study provides new insights into microbial communities in this habitat, enabled by an improved DNA extraction protocol that allows a less biased view of taxonomic composition and metabolic activities, as well as uncovering novel taxa. Our approach presents the first successful attempt at unamplified shotgun sequencing samples from beyond 50 meters below the seafloor and opens new ways for capturing the true diversity and functional potential of deep-sea sediments

How clear is our current view on microbial dark matter? (Re-)assessing public MAG & SAG datasets with MDMcleaner

As of today, the majority of environmental microorganisms remain uncultured and is therefore referred to as ‘microbial dark matter’ (MDM). Hence, genomic insights into these organisms are limited to cultivation-independent approaches such as single-cell- and metagenomics. However, without access to cultured representatives for verifying correct taxon-assignments, MDM genomes may cause potentially misleading conclusions based on misclassified or contaminant contigs, thereby obfuscating our view on the uncultured microbial majority. Moreover, gradual database contaminations by past genome submissions can cause error propagations which affect present as well as future comparative genome analyses. Consequently, strict contamination detection and filtering need to be applied, especially in the case of uncultured MDM genomes. Current genome reporting standards, however, emphasize completeness over purity and the de facto gold standard genome assessment tool, checkM, discriminates against uncultured taxa and fragmented genomes. To tackle these issues, we present a novel contig classification, screening, and filtering workflow and corresponding open-source python implementation called MDMcleaner, which was tested and compared to other tools on mock and real datasets. MDMcleaner revealed substantial contaminations overlooked by current screening approaches and sensitively detects misattributed contigs in both novel genomes and the underlying reference databases, thereby greatly improving our view on ‘microbial dark matter’

Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation

As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as “microbial dark matter.” In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique—out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages—in whole or in part—have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs

Detection of the carbapenemase gene blaVIM-5 in members of the Pseudomonas putida group isolated from polluted Nigerian wetlands

Abstract There are increasing concerns about possible dissemination of clinically relevant antibiotic resistance genes, including genes encoding for carbapenemases in the environment. However, little is known about environmental distribution of antibiotic resistance in Africa. In this study, four polluted urban wetlands in Nigeria were investigated as potential reservoirs of carbapenem-resistant bacteria (CRB). CRB were isolated from the wetlands, characterized by Blue-Carba test, MIC determinations and whole genome sequencing (WGS). Nine of 65 bacterial isolates identified as members of the Pseudomonas putida group (P. plecoglossicida and P. guariconensis, respectively) harboured the metallo-beta-lactamase gene bla VIM-5. WGS revealed the bla VIM-5 in three novel Tn402-like class 1 integron structures containing the cassette arrays aadB|bla VIM-5|bla PSE-1, aadB|bla VIM-5|aadB|bla PSE-1, and bla VIM-5|aadB|tnpA|bla PSE-1|smr2|tnpA, respectively. Strains carrying the aadB|bla VIM-5|bla PSE-1 cassette also carried an identical integron without bla VIM-5. In addition, the strains harboured another Tn402-like class 1 integron carrying bcr2, several multidrug resistance efflux pumps, and at least one of ampC, aph(3”)-lb, aph(6)-ld, tetB, tetC, tetG, floR, and macAB. This is the first report of a carbapenemase gene in bacteria from environmental sources in Nigeria and the first report of bla VIM-5 in environmental bacteria isolates. This result underscores the role of the Nigerian environment as reservoir of bacteria carrying clinically relevant antibiotic resistance genes