Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense.

Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.

The Human Gut Virome Is Highly Diverse, Stable, and Individual Specific.

The human gut contains a vast array of viruses, mostly bacteriophages. The majority remain uncharacterized, and their roles in shaping the gut microbiome and in impacting on human health remain poorly understood. We performed longitudinal metagenomic analysis of fecal viruses in healthy adults that reveal high temporal stability, individual specificity, and correlation with the bacterial microbiome. Using a database-independent approach that uses most of the sequencing data, we uncovered the existence of a stable, numerically predominant individual-specific persistent personal virome. Clustering of viral genomes and de novo taxonomic annotation identified several groups of crAss-like and Microviridae bacteriophages as the most stable colonizers of the human gut. CRISPR-based host prediction highlighted connections between these stable viral communities and highly predominant gut bacterial taxa such as Bacteroides, Prevotella, and Faecalibacterium. This study provides insights into the structure of the human gut virome and serves as an important baseline for hypothesis-driven research.

Megaphages infect Prevotella and variants are widespread in gut microbiomes.

Bacteriophages (phages) dramatically shape microbial community composition, redistribute nutrients via host lysis and drive evolution through horizontal gene transfer. Despite their importance, much remains to be learned about phages in the human microbiome. We investigated the gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups and Danish pigs; many of these microbiomes contain phages belonging to a clade with genomes >540 kilobases in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phages. We refer to these as Lak phages. CRISPR spacer targeting indicates that Lak phages infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed and up to 35 putative transfer RNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in the relative abundance of phage subpopulations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phages are widespread in gut communities that contain the Prevotella species, and conclude that megaphages, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.

The bacteriophages of ruminal prevotellas.

Rumen bacteriophage-lyzed bacterial strains of the genus Prevotella were isolated and preliminarily characterized. The strain TCl-1 the species P. bryantii was the only prevotella strain successfully infected with filter sterilized rumen fluid from a black-and-white Holstein cow. Two types of plaques were observed, both rather small and turbid. Preliminary electron microscopy observation showed that several morphologically different bacteriophages were present in these plaques. The plaque eluates were further used for the infection of other prevotella strains. The plaques produced by the bacteriophages were observed with two strains, i.e. P. bryantii B(1)4 and P. brevis GA33. The bacteriophages from both strains were examined by transmission electron microscopy and several morphologically different bacteriophages were observed, among others also a large virion with an icosahedral head with the diameter of approximately 120 nm. The bacteriophage was identified in plaques of bacterial cells of the strain GA33 and has an approximately 800 nm long helical tail, which places it among the largest ruminal bacteriophages described to date. Other bacteriophages from the same indicator strain as well as from P. bryantii B(1)4 strain were smaller and tail structures were not observed in all of them.

Cloning and DNA sequence analysis of the region containing attP of the temperate phage phi AR29 of Prevotella ruminicola AR29.

Phage phi AR29 was shown to exist as a prophage integrated into the chromosome of Prevotella ruminicola AR29. By DNA hybridization studies, the point of integrative recombination on the phage genome (attP) was located on a 4.5 kb EcoRV fragment. After preliminary mapping with restriction endonucleases, a 2.8 kb EcoRV/HindIII fragment was isolated, cloned in Escherichia coli and sequenced. DNA hybridization localized the attP site to the vicinity of an internal DraI site. Sequence analysis showed the presence of several direct and inverted repeats around the attP site, with consensus core sequences similar to the integrase binding sites of phage lambda. Two open reading frames are present adjacent to attP (ORF1 and ORF2). The predicted polypeptide product of ORF1 has a region of structural similarity to known integrases. Although the predicted product of ORF2 shows at best weak homology with known excisionases, no other ORFs occur in the sequence upstream from ORF1, leaving ORF2 as the most likely candidate for this role. However, if ORF2 does represent an xis gene, then this putative integration module would possess a notable difference from that of other temperate phages in the inversion of the positions of int and xis relative to attP. The proposed phi AR29 integration module is being used to develop phage-based integrative vector systems for the genetic manipulation of rumen bacteria.