During the past decade, environmental research has demonstrated that archaea are abundant and widespread in nature and play important ecological roles at a global scale. Currently, however, the majority of archaeal lineages cannot be cultivated under laboratory conditions and are known exclusively or nearly exclusively through metagenomics. A similar trend extends to the archaeal virosphere, where isolated representatives are available for a handful of model archaeal virus-host systems. Viral metagenomics provides an alternative way to circumvent the limitations of culture-based virus discovery and offers insight into the diversity, distribution, and environmental impact of uncultured archaeal viruses. Presently, metagenomics approaches have been successfully applied to explore the viromes associated with various lineages of extremophilic and mesophilic archaea, including Asgard archaea (Asgardarchaeota), ANME-1 archaea (Methanophagales), thaumarchaea (Nitrososphaeria), altiarchaea (Altiarchaeota), and marine group II archaea (Poseidoniales). Here, we provide an overview of methods widely used in archaeal virus metagenomics, covering metavirome preparation, genome annotation, phylogenetic and phylogenomic analyses, and archaeal host assignment. We hope that this summary will contribute to further exploration and characterization of the enigmatic archaeal virome lurking in diverse environments.

BACKGROUND: Clade 5 Clostridioides difficile diverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11 C. difficile isolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates.
RESULTS: Isolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes.
CONCLUSIONS: We concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.

CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide-a consensus PAM-within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations.IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a \"directionality\" in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.

Due to recent advances in next-generation sequencing over the past decade, our understanding of the human microbiome and its relationship to health and disease has increased dramatically. Yet, our insights into the human virome, and its interplay with important microbes that impact human health, is relatively limited. Prokaryotic and eukaryotic viruses are present throughout the human body, comprising a large and diverse population which influences several niches and impacts our health at various body sites. The presence of prokaryotic viruses like phages, has been documented at many different body sites, with the human gut being the richest ecological niche. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins constitute the adaptive immune system of bacteria, which prevents attack by invasive nucleic acid. CRISPR-Cas systems function by uptake and integration of foreign genetic element sequences into the CRISPR array, which constitutes a genomic archive of iterative vaccination events. Consequently, CRISPR spacers can be investigated to reconstruct interplay between viruses and bacteria, and metagenomic sequencing data can be exploited to provide insights into host-phage interactions within a niche. Here, we show how the CRISPR spacer content of commensal and pathogenic bacteria can be used to determine the evidence of their phage exposure. This framework opens new opportunities for investigating host-virus dynamics in metagenomic data, and highlights the need to dedicate more efforts for virome sampling and sequencing.

CRISPR-Cas are nucleic acid-based prokaryotic immune systems. CRISPR arrays accumulate spacers from foreign DNA and provide resistance to mobile genetic elements containing identical or similar sequences. Thus, the set of spacers present in a given bacterium can be regarded as a record of encounters of its ancestors with genetic invaders. Such records should be specific for different lineages and change with time, as earlier acquired spacers get obsolete and are lost. Here, we studied type I-E CRISPR spacers of Escherichia coli from extinct pachyderm. We find that many spacers recovered from intestines of a 42 000-year-old mammoth match spacers of present-day E. coli. Present-day CRISPR arrays can be reconstructed from palaeo sequences, indicating that the order of spacers has also been preserved. The results suggest that E. coli CRISPR arrays were not subject to intensive change through adaptive acquisition during this time.