In vitro Modeling of Chicken Cecal Microbiota Ecology and Metabolism Using the PolyFermS Platform

Continuous in vitro fermentation models provide a useful tool for a fast, reproducible, and direct assessment of treatment-related changes in microbiota metabolism and composition independent of the host. In this study, we used the PolyFermS model to mimic the conditions of the chicken cecum and evaluated three nutritive media for in vitro modeling of the chicken cecal microbiota ecology and metabolism. We observed that our model inoculated with immobilized cecal microbiota and fed with a modified Viande Levure medium (mVL-3) reached a high bacterial cell density of up to approximately 10.5 log cells per mL and stable microbiota composition, akin to the host, during 82 days of continuous operation. Relevant bacterial functional groups containing primary fibrolytic (Bacteroides, Bifidobacteriaceae, Ruminococcaceae), glycolytic (Enterococcus), mucolytic (Bacteroides), proteolytic (Bacteroides), and secondary acetate-utilizing butyrate-producing and propionate-producing (Lachnospiraceae) taxa were preserved in vitro. Besides, conserved metabolic and functional Kyoto Encyclopedia of Genes and Genomes pathways were observed between in vitro microbiota and cecal inoculum microbiota as predicted by functional metagenomics analysis. Furthermore, we demonstrated that the continuous inoculation provided by the inoculum reactor generated reproducible metabolic profiles in second-stage reactors comparable to the chicken cecum, allowing for the simultaneous investigation and direct comparison of different treatments with a control. In conclusion, we showed that PolyFermS is a suitable model for mimicking chicken cecal microbiota fermentation allowing ethical and ex vivo screening of environmental factors, such as dietary additives, on chicken cecal fermentation. We report here for the first time a fermentation medium (mVL-3) that closely mimics the substrate conditions in the chicken cecum and supports the growth and metabolic activity of the cecal bacterial akin to the host. Our PolyFermS chicken cecum model is a useful tool to study microbiota functionality and structure ex vivo.

Microbial consortiums of putative degraders of low-density polyethylene-associated compounds in the ocean

Polyethylene (PE) is one of the most abundant plastics in the ocean. The development of a biofilm on PE in the ocean has been reported, yet whether some of the biofilm-forming organisms can biodegrade this plastic in the environment remains unknown. Via metagenomics analysis, we taxonomically and functionally analysed three biofilm communities using low-density polyethylene (LDPE) as their sole carbon source for two years. Several of the taxa that increased in relative abundance over time were closely related to known degraders of alkane and other hydrocarbons. Alkane degradation has been proposed to be involved in PE degradation, and most of the organisms increasing in relative abundance over time harboured genes encoding proteins essential in alkane degradation, such as the genes alkB and CYP153, encoding an alkane monooxygenase and a cytochrome P450 alkane hydroxylase. Weight loss of PE sheets when incubated with these communities and chemical and electron microscopic analyses provided evidence for alteration of the PE surface over time. Taken together, these results provide evidence for the utilization of LDPE-associated compounds by the prokaryotic communities. This study identifies a group of genes potentially involved in the degradation of the LDPE polymeric structure and/or associated plastic additives in the ocean and describes a phylogenetically diverse community of plastic biofilm-dwelling microbes with the potential of utilizing LDPE-associated compounds as carbon and energy source.

Free DNA and Metagenomics Analyses: Evaluation of Free DNA Inactivation Protocols for Shotgun Metagenomics Analysis of Human Biological Matrices

Culture-independent approaches now represent the gold standard for the investigation of both environmental and host-associated complex microbial communities. Nevertheless, despite the great advantages offered by these novel methodologies based on the use of next-generation DNA sequencing approaches, a number of bias sources have been identified. Among the latter, free DNA contained in biological matrices is one of the main sources of inaccuracy in reconstructing the resident microbial population of viable cells. For this reason, the photoreactive DNA-binding dye propidium monoazide (PMAxx™) has been developed by improving standard PMA. This compound binds and inactivates free DNA, thus preventing its amplification and sequencing. While the performances of PMA have been previously investigated, the efficiency with PMAxx™ has been tested mainly for amplicon-based profiling approaches on a limited number of biological matrices. In this study, we validated the performance of PMAxx™ for shotgun metagenomics approaches employing various human-associated matrices. Notably, results revealed that the effectiveness of PMAxx™ in inactivating free DNA of prokaryotes and eukaryotes tends to vary significantly based on the biological matrices analyzed.

A synergistic consortium involved in Rac -dichlorprop degradation as revealed by DNA-stable isotope probing and metagenomics analysis

Rac -dichlorprop, a commonly used phenoxyalkanoic acid herbicide, is frequently detected in environments and poses threats to environmental safety and human health. Microbial consortia are thought to play key roles in Rac -dichlorprop degradation. However, the compositions of the microbial consortia involved in Rac -dichlorprop degradation remain largely unknown. In this study, DNA-stable isotope probing and metagenomics analysis were integrated to reveal the key microbial consortium responsible for Rac -dichlorprop degradation in a Rac -dichlorprop-degrading enrichment. OTU340 ( Sphingobium sp.) and OTU348 ( Sphingopyxis sp.) were significantly enriched in the 13 C- Rac -dichlorprop-labeled heavy DNA fractions. A Rac -dichlorprop degrader, Sphingobium sp. L3, was isolated from the enrichment by traditional enrichment method but with additional supplementation of the antibiotic ciprofloxacin, which was instructed by metagenomics analysis of the associations between Rac -dichlorprop-degraders and antibiotic resistance genes. As revealed by functional profiling of the metagenomes of the heavy DNA, the genes rdpA and sdpA , involved in the initial degradation of the ( R )- and ( S )-enantiomers of dichlorprop respectively, were mostly taxonomically assigned to Sphingobium species, indicating that Sphingopyxis species might harbor novel dichlorprop degrading genes. In addition, taxonomically diverse bacterial genera such as Dyella , Sphingomonas , Pseudomonas , and Achromobacter were presumed to synergistically cooperate with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of Rac -dichlorprop. Importance Understanding of the key microbial consortium involved in the degradation of the phenoxyalkanoic acid herbicide of Rac -dichlorprop is pivotal for design of synergistic consortia used for enhanced bioremediation of herbicide-contaminated sites. However, the composition of microbial consortium and the interactions between community members during the biodegradation of Rac -dichlorprop are unclear. In this study, DNA-SIP and metagenomics analysis were integrated to reveal that the metabolite 2,4-dichlorophenol degraders Dyella , Sphingomonas , Pseudomonas , and Achromobacter synergistically cooperated with the key degraders Sphingobium / Sphingopyxis for enhanced degradation of Rac -dichlorprop. Our study provides new insights into the synergistic degradation of Rac -dichlorprop at the community level and implies the existence of novel degrading genes for Rac -dichlorprop in nature.

DeePhage: distinguishing virulent and temperate phage-derived sequences in metavirome data with a deep learning approach

Background Prokaryotic viruses referred to as phages can be divided into virulent and temperate phages. Distinguishing virulent and temperate phage–derived sequences in metavirome data is important for elucidating their different roles in interactions with bacterial hosts and regulation of microbial communities. However, there is no experimental or computational approach to effectively classify their sequences in culture-independent metavirome. We present a new computational method, DeePhage, which can directly and rapidly judge each read or contig as a virulent or temperate phage–derived fragment. Findings DeePhage uses a “one-hot” encoding form to represent DNA sequences in detail. Sequence signatures are detected via a convolutional neural network to obtain valuable local features. The accuracy of DeePhage on 5-fold cross-validation reaches as high as 89%, nearly 10% and 30% higher than that of 2 similar tools, PhagePred and PHACTS. On real metavirome, DeePhage correctly predicts the highest proportion of contigs when using BLAST as annotation, without apparent preferences. Besides, DeePhage reduces running time vs PhagePred and PHACTS by 245 and 810 times, respectively, under the same computational configuration. By direct detection of the temperate viral fragments from metagenome and metavirome, we furthermore propose a new strategy to explore phage transformations in the microbial community. The ability to detect such transformations provides us a new insight into the potential treatment for human disease. Conclusions DeePhage is a novel tool developed to rapidly and efficiently identify 2 kinds of phage fragments especially for metagenomics analysis. DeePhage is freely available via http://cqb.pku.edu.cn/ZhuLab/DeePhage or https://github.com/shufangwu/DeePhage.

Structural characterization of a Thorarchaeota profilin indicates eukaryotic-like features but with an extended N-terminus

The emergence of the first eukaryotic cell was preceded by evolutionary events which are still highly debatable. Recently, comprehensive metagenomics analysis has uncovered that the Asgard super-phylum is the closest yet known archaea host of eukaryotes. However, it remains to be established if a large number of eukaryotic signature proteins predicated to be encoded by the Asgard super-phylum are functional at least, in the context of a eukaryotic cell. Here, we determined the three-dimensional structure of profilin from Thorarchaeota by nuclear magnetic resonance spectroscopy and show that this profilin has a rigid core with a flexible N-terminus which was previously implicated in polyproline binding. In addition, we also show that thorProfilin co-localizes with eukaryotic actin in cultured HeLa cells. This finding reaffirm the notion that Asgardean encoded proteins possess eukaryotic-like characteristics and strengthen likely existence of a complex cytoskeleton already in a last eukaryotic common ancestor