Novel canine high-quality metagenome-assembled genomes, prophages, and host-associated plasmids by long-read metagenomics together with Hi-C proximity ligation

Long-read metagenomics facilitates the assembly of high-quality metagenome-assembled genomes (HQ MAGs) out of complex microbiomes. It provides highly contiguous assemblies by spanning repetitive regions, complete ribosomal genes, and mobile genetic elements. Hi-C proximity ligation data bins the long contigs and their associated extra-chromosomal elements to their bacterial host. Here, we characterized a canine fecal sample combining a long-read metagenomics assembly with Hi-C data, and further correcting frameshift errors. We retrieved 27 HQ MAGs and seven medium-quality (MQ) MAGs considering MIMAG criteria. All the long-read canine MAGs improved previous short-read MAGs from public datasets regarding contiguity of the assembly, presence, and completeness of the ribosomal operons, and presence of canonical tRNAs. This trend was also observed when comparing to representative genomes from a pure culture (short-read assemblies). Moreover, Hi-C data linked six potential plasmids to their bacterial hosts. Finally, we identified 51 bacteriophages integrated into their bacterial host, providing novel host information for eight viral clusters that included Gut Phage Database viral genomes. Even though three viral clusters were species-specific, most of them presented a broader host range. In conclusion, long-read metagenomics retrieved long contigs harboring complete assembled ribosomal operons, prophages, and other mobile genetic elements. Hi-C binned together the long contigs into HQ and MQ MAGs, some of them representing closely related species. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAGs discovery and assignment of extra-chromosomal elements to their bacterial host.

Three Ribosomal Operons of Escherichia coli Contain Genes Encoding Small RNAs That Interact With Hfq and CsrA in vitro

Three out of the seven ribosomal RNA operons in Escherichia coli end in dual terminator structures. Between the two terminators of each operon is a short sequence that we report here to be an sRNA gene, transcribed as part of the ribosomal RNA primary transcript by read-through of the first terminator. The sRNA genes (rrA, rrB and rrF) from the three operons (rrnA, rrnB and rrnD) are more than 98% identical, and pull-down experiments show that their transcripts interact with Hfq and CsrA. Deletion of rrA, B, F, as well as overexpression of rrB, only modestly affect known CsrA-regulated phenotypes like biofilm formation, pgaA translation and glgC translation, and the role of the sRNAs in vivo may not yet be fully understood. Since RrA, B, F are short-lived and transcribed along with the ribosomal RNA components, their concentration reflect growth-rate regulation at the ribosomal RNA promoters and they could function to fine-tune other growth-phase-dependent processes in the cell. The primary and secondary structure of these small RNAs are conserved among species belonging to different genera of Enterobacteriales.

Effect of antibiotics on the cellulolytic and nitrification activity of gray forest soil

Aim. To investigate the effect of certain antibiotics - tylosin, oxytetracycline and benzylpenicillin - on the potential nitrifying and cellulolytic activity of gray forest soil using laboratory model research methods.Material and Methods. The object of the research was agricultural gray forest medium loamy soil. The study was carried out by conducting laboratory model experiments. The analysed samples were incubated at 27°C and in the absence of illumination for 30 days and then subsequently analysed for cellulolytic activity (by the application method) and nitrification activity (by the potentiometric method). The taxonomic composition of the bacterial community of the studied soil was established based on analysis of amplicon libraries of fragments of ribosomal operons of 16S rRNA genes by the NGS method.Results. The largest number of nitrification organisms in the soil studied were archaea of the family Nitrososphaeraceae which are autotrophic ammonium oxidants. Most resistant to the effects of the antibiotics used was cellulolytic activity which was suppressed only by the addition of tylosin and its admixture with oxytetracycline. The nitrification activity of the soil varied depending on the concentration and preparations applied, the greatest inhibitory effect being exerted by tylosin. Antibiotic mixtures slightly enhanced the nitrification process at 50-100 mg/kg and were suppressed in the range of 150-700 mg/kg. Conclusion. Once in the soil, the antibiotics studied are capable of both stimulating and inhibiting enzymatic processes. Mixtures of antibiotics rather than their individual applications produce the greatest impact. In medium loamy gray forest soil the presence of antibiotics is more dangerous to nitrification activity.

Rates of gene conversions between Escherichia coli ribosomal operons

Due to their universal presence and high sequence conservation, ribosomal RNA (rRNA) sequences are used widely in phylogenetics for inferring evolutionary relationships between microbes and in metagenomics for analyzing the composition of microbial communities. Most microbial genomes encode multiple copies of rRNA genes to supply cells with sufficient capacity for protein synthesis. These copies typically undergo concerted evolution that keeps their sequences identical, or nearly so, due to gene conversion, a type of intragenomic recombination that changes one copy of a homologous sequence to exactly match another. Widely varying rates of rRNA gene conversion have previously been estimated by comparative genomics methods and using genetic reporter assays. To more directly measure rates of rRNA intragenomic recombination, we sequenced the seven Escherichia coli rRNA operons in 15 lineages that were evolved for ∼13,750 generations with frequent single-cell bottlenecks that reduce the effects of selection. We identified 38 gene conversion events and estimated an overall rate of intragenomic recombination within the 16S and 23S genes between rRNA copies of 3.6 × 10−4 per genome per generation or 8.6 × 10−6 per rRNA operon per homologous donor operon per generation. This rate varied only slightly from random expectations at different sites within the rRNA genes and between rRNA operons located at different positions in the genome. Our accurate estimate of the rate of rRNA gene conversions fills a gap in our quantitative understanding of how ribosomal sequences and other multicopy elements diversify and homogenize during microbial genome evolution.

Rates of gene conversions between Escherichia coli ribosomal operons

ABSTRACTDue to their universal presence and high sequence conservation, rRNA sequences are used widely in phylogenetics for inferring evolutionary relationships between microbes and in metagenomics for analyzing the composition of microbial communities. Most microbial genomes encode multiple copies of ribosomal RNA (rRNA) genes to supply cells with sufficient capacity for protein synthesis. These copies typically undergo concerted evolution that keeps their sequences identical, or nearly so, due to gene conversion, a type of intragenomic recombination that changes one copy of a homologous sequence to exactly match another. Widely varying rates of rRNA gene conversion have previously been estimated by comparative genomics methods and using genetic reporter assays. To more directly measure rates of rRNA intragenomic recombination, we sequenced the seven Escherichia coli rRNA operons in 15 lineages of cells that were evolved for ~13,750 generations with frequent single-cell bottlenecks that reduce the effects of selection. We identified 34 gene conversion events and estimate an overall rate of intragenomic recombination events between rRNA copies of 3.2 × 10−4 per generation or 5.3 × 10−5 per potential donor sequence. This rate varied only slightly from random expectations between different portions of the rRNA genes and between rRNA operons located at different locations in the genome. This accurate estimate of the rate of rRNA gene conversions fills a gap in our quantitative understanding of how ribosomal sequences and other multicopy elements diversify and homogenize during microbial genome evolution.

socru: typing of genome-level order and orientation around ribosomal operons in bacteria

Rearrangements of large genome fragments occur in bacteria between repeat sequences and can impact on growth and gene expression. Homologous recombination resulting in inversion between indirect repeats and excision/translocation between direct repeats enables these structural changes. One form of rearrangement occurs around ribosomal operons, found in multiple copies across many bacteria, but identification of these rearrangements by sequencing requires reads of several thousand bases to span the ribosomal operons. With long-read sequencing aiding the routine generation of complete bacterial assemblies, we have developed socru, a typing method for the order and orientation of genome fragments between ribosomal operons. It allows for a single identifier to convey the order and orientation of genome-level structure and we have successfully applied this typing to 433 of the most common bacterial species. In a focused analysis, we observed the presence of multiple structural genotypes in nine bacterial pathogens, underscoring the importance of routinely assessing this form of variation alongside traditional single-nucleotide polymorphism (SNP) typing.

Species-level evaluation of the human respiratory microbiome

Background Changes to human respiratory tract microbiome may contribute significantly to the progression of respiratory diseases. However, there are few studies examining the relative abundance of microbial communities at the species level along the human respiratory tract. Findings Bronchoalveolar lavage, throat swab, mouth rinse, and nasal swab samples were collected from 5 participants. Bacterial ribosomal operons were sequenced using the Oxford Nanopore MinION to determine the relative abundance of bacterial species in 4 compartments along the respiratory tract. More than 1.8 million raw operon reads were obtained from the participants with ∼600,000 rRNA reads passing quality assurance/quality control (70–95% identify; >1,200 bp alignment) by Discontiguous MegaBLAST against the EZ BioCloud 16S rRNA gene database. Nearly 3,600 bacterial species were detected overall (>750 bacterial species within the 5 dominant phyla: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteria. The relative abundance of bacterial species along the respiratory tract indicated that most microbes (95%) were being passively transported from outside into the lung. However, a small percentage (<5%) of bacterial species were at higher abundance within the lavage samples. The most abundant lung-enriched bacterial species were Veillonella dispar and Veillonella atypica while the most abundant mouth-associated bacterial species were Streptococcus infantis and Streptococcus mitis. Conclusions Most bacteria detected in lower respiratory samples do not seem to colonize the lung. However, >100 bacterial species were found to be enriched in bronchoalveolar lavage samples (compared to mouth/nose) and may play a substantial role in lung health.

Taxonomic resolution of the ribosomal RNA operon in bacteria: implications for its use with long-read sequencing

DNA barcoding through the use of amplified regions of the ribosomal operon, such as the 16S gene, is a routine method to gain an overview of the microbial taxonomic diversity within a sample without the need to isolate and culture the microbes present. However, bacterial cells usually have multiple copies of this ribosomal operon, and choosing the ‘wrong’ copy could provide a misleading species classification. While this presents less of a problem for well-characterized organisms with large sequence databases to interrogate, it is a significant challenge for lesser known organisms with unknown copy number and diversity. Using the entire length of the ribosomal operon, which encompasses the 16S, 23S, 5S and internal transcribed spacer regions, should provide greater taxonomic resolution but has not been well explored. Here, we use publicly available reference genomes and explore the theoretical boundaries when using concatenated genes and the full-length ribosomal operons, which has been made possible by the development and uptake of long-read sequencing technologies. We quantify the issues of both copy choice and operon length in a phylogenetic context to demonstrate that longer regions improve the phylogenetic signal while maintaining taxonomic accuracy.

Socru: Typing of genome level order and orientation in bacteria

SummaryGenome rearrangements occur in bacteria between repeat sequences and impact growth and gene expression. Homologous recombination can occur between ribosomal operons, which are found in multiple copies in many bacteria. Inversion between indirect repeats and excision/translocation between direct repeats enable structural genome rearrangement. To identify what these rearrangements are by sequencing, reads of several thousand bases are required to span the ribosomal operons. With long read sequencing aiding the routine generation of complete bacterial assemblies, we have developed socru, a typing method for the order and orientation of genome fragments between ribosomal operons, defined against species-specific baselines. It allows for a single identifier to convey the order and orientation of genome level structure and 434 of the most common bacterial species are supported. Additionally, socru can be used to identify large scale misassemblies.Availability and implementationSocru is written in Python 3, runs on Linux and OSX systems and is available under the open source license GNU GPL 3 from https://github.com/quadram-institute-bioscience/[email protected]