Highlights d Screening of human fecal metagenomic samples reveals 249 crAss-like phage genomes d The crAss-like phages were classified into 4 subfamilies composed of 10 candidate genera d A crAss-like phage was propagated in ex vivo human fecal fermentations d Short-tailed phage virions could be visualized by electron microscopy

17CrAssphage is yet to be cultured even though it represents the most abundant virus in 18 the gut microbiota of humans. Recently, sequence based classification was performed on 19 distantly related crAss-like phages from multiple environments, leading to the proposal of a 20 familial level taxonomic group [Yutin N, et al. (2018) Discovery of an expansive 21 bacteriophage family that includes the most abundant viruses from the human gut. Nat 22 Microbiol 3(1):38-46]. Here, we assembled the metagenomic sequencing reads from 702 23 human faecal virome/phageome samples and obtained 98 complete circular crAss-like phage 24 genomes and 145 contigs ≥70kb. In silico comparative genomics and taxonomic analysis was 25 performed, resulting in a classification scheme of crAss-like phages from human faecal 26 microbiomes into 4 candidate subfamilies composed of 10 candidate genera. Moreover, 27 laboratory analysis was performed on faecal samples from an individual harbouring 7 distinct 28 crAss-like phages. We achieved propagation of crAss-like phages in ex vivo human faecal 29 fermentations and visualised Podoviridae virions by electron microscopy. Furthermore, 30 detection of a crAss-like phage capsid protein could be linked to metagenomic sequencing 31 data confirming crAss-like phage structural annotations. 32 33 34 35 36 37 38 39 40 41 3 Significance 42CrAssphage is the most abundant biological entity in the human gut, but it remains 43 uncultured in the laboratory and its host(s) is unknown. CrAssphage was not identified in 44 metagenomic studies for many years as its sequence is so different from anything present in 45 databases. To this day, it can only be detected from sequences assembled from metagenomics 46 or viromic datasets (crAsscross Assembly). In this study, we identified 243 new crAss-like 47 phages from human faecal metagenomic studies. Taxonomic analysis of these crAss-like 48 phages highlighted their extensive diversity within the human microbiome. We also present 49 the first propagation of crAssphage in faecal fermentations and provide the first electron 50 micrographs of this extraordinary bacteriophage. 51 52 53In recent years, increasing numbers of bacteria, archaea, fungi, protists and viruses 54 residing on and within the human body have been associated with various states of human 55 health and disease, including diet, age, weight, inflammatory bowel disease (IBD), diabetes, 56 and cognition (1-7). A relatively small number of eukaryote viruses present in the 57 gastrointestinal tract can target the human host, however, much larger and much more 58 complex populations of viruses that target bacteria (bacteriophages) also reside there. The 59 role of phages in the gut has been a subject of increased interest as initial investigations have 60 revealed substantial differences in bacteriophage populations between healthy and diseased 61 cohorts (7-11). It is likely that phages have an important role in shaping our gut microbiome, 62 but their precise role remains poorly understood. 63In 2014, ...

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Insertion sequences (ISs) are small transposable elements widespread in bacterial genomes, where they play an essential role in chromosome evolution by stimulating recombination and genetic flow. Despite their ubiquity, it is unclear how ISs interact with the host. Here, we report a survey of the orientation patterns of ISs in bacterial chromosomes with the objective of gaining insight into the interplay between ISs and host chromosomal functions. We find that a significant fraction of IS families present a consistent and family-specific orientation bias with respect to chromosomal DNA replication, especially in Firmicutes. Additionally, we find that the transposases of up to nine different IS families with different transposition pathways interact with the β sliding clamp, an essential replication factor, suggesting that this is a widespread mechanism of interaction with the host. Although we find evidence that the interaction with the β sliding clamp is common to all bacterial phyla, it also could explain the observed strong orientation bias found in Firmicutes, because in this group β is asymmetrically distributed during synthesis of the leading or lagging strands. Besides the interaction with the β sliding clamp, other asymmetries also play a role in the biased orientation of some IS families. The utilization of the highly conserved replication sliding clamps suggests a mechanism for host regulation of IS proliferation and also a universal platform for IS dispersal and transmission within bacterial populations and among phylogenetically distant species.

A large part of our current understanding of gene regulation in Gram-positive bacteria is based on Bacillus subtilis , as it is one of the most well studied bacterial model systems. The rapid growth in data concerning its molecular and genomic biology is distributed across multiple annotation resources. Consequently, the interpretation of data from further B. subtilis experiments becomes increasingly challenging in both low- and large-scale analyses. Additionally, B. subtilis annotation of structured RNA and non-coding RNA (ncRNA), as well as the operon structure, is still lagging behind the annotation of the coding sequences. To address these challenges, we created the B. subtilis genome atlas, BSGatlas, which integrates and unifies multiple existing annotation resources. Compared to any of the individual resources, the BSGatlas contains twice as many ncRNAs, while improving the positional annotation for 70 % of the ncRNAs. Furthermore, we combined known transcription start and termination sites with lists of known co-transcribed gene sets to create a comprehensive transcript map. The combination with transcription start/termination site annotations resulted in 717 new sets of co-transcribed genes and 5335 untranslated regions (UTRs). In comparison to existing resources, the number of 5′ and 3′ UTRs increased nearly fivefold, and the number of internal UTRs doubled. The transcript map is organized in 2266 operons, which provides transcriptional annotation for 92 % of all genes in the genome compared to the at most 82 % by previous resources. We predicted an off-target-aware genome-wide library of CRISPR–Cas9 guide RNAs, which we also linked to polycistronic operons. We provide the BSGatlas in multiple forms: as a website (https://rth.dk/resources/bsgatlas/), an annotation hub for display in the UCSC genome browser, supplementary tables and standardized GFF3 format, which can be used in large scale -omics studies. By complementing existing resources, the BSGatlas supports analyses of the B. subtilis genome and its molecular biology with respect to not only non-coding genes but also genome-wide transcriptional relationships of all genes.

23 Viral (meta)genomics is a rapidly growing field of study that is hampered by an inability to annotate 24 the majority of viral sequences; therefore, the development of new bioinformatic approaches is very 25 important. Here, we present a new automatic de novo genome annotation pipeline, called VIGA, to 26 annotate prokaryotic and eukaryotic viral sequences from (meta)genomic studies. VIGA was 27 benchmarked on a database of known viral genomes and a viral metagenomics case study. VIGA 28 generated the most accurate outputs according to the number of coding sequences and their 29 coordinates, outputs also had a lower number of non-informative annotations compared to other 30 programs. 31

Enhancing yield during bacterial enzyme production could have positive economic and environmental impacts. For cell factories, such improvements in yields could potentially be obtained by fine-tuning the metabolic processes and their regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Complex operons that are regulated by a wide variety of transcription factors, non-coding and structured RNAs add to the challenge of finding yield-affecting candidates. Surprisingly, we found that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation (75% of novel RNA predictions had absolute logFC > 2). Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expressed annotations, including both novel candidate and prior annotated ncRNAs, we selected 53 non-coding candidates. The targeting with CRISPRi knockdowns transcription in a genomic region on both the sense and the antisense strand. Thus, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.

Background In nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such environments. Pseudomonas aeruginosa , an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood. Results In this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K + was elicited by high concentration of Na + in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions. Conclusions The individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa , a relevant environmental and pathogenic bacterium, survives to long-term salt stress. Electronic supplementary material The online version of this article (10.1186/s12866-019-1499-2) contains supplementary material, which is available to authorized users.