A transcriptionally active lipid vesicle encloses the injectedChimalliviridaegenome in early infection

Abstract

Eukaryotic viruses assemble compartments required for genome replication, but no such organelles are known to be essential for prokaryotic viruses. Bacteriophages of the family Chimalliviridae sequester their genomes within a phagegenerated organelle, the phage nucleus, which is enclosed by a lattice of viral protein ChmA. Using the dRfxCas13d-based knockdown system CRISPRi-ART, we show that ChmA is essential for the E. coli phage Goslar life cycle. Without ChmA, infections are arrested at an early stage in which the injected phage genome is enclosed in a membrane-bound vesicle capable of gene expression but not DNA replication. Not only do we demonstrate that the phage nucleus is essential for genome replication, but we also show that the Chimalliviridae early phage infection (EPI) vesicle is a transcriptionally active, phage-generated organelle.

Genome-wide Characterization of Diverse Bacteriophages Enabled by RNA-Binding CRISPRi

“…For instance, alongside this study, CRISPRi-ART has proven critical to discover novel pre-nucleus compartmentalization and RNA export in Chimalliviridae (i.e. nucleus-forming phages) ( 50 , 51 ), whose enclosed genomes are broadly shielded from DNA targeting CRISPRi ( 19 , 20 , 22 , 52 ). Other individual crRNAs targeting essential genes such as those knocking down A2 from phage T5 or 00025 from PTXU04, can be simple experimental approaches to study enigmatic infection states of diverse prokaryotic viruses.…”

Defining the constituents and functions of a lipid-based jumbo phage compartment

“…Overall these results suggest that bacterial membranes are recruited to the injected phage gDNA and protein cargo of ΦKZ-like jumbophages during the early parts of the phage infection cycle. Based on the collective evidence from fluorescence microscopy experiments and previous cryo-ET 6,15 we suggest that the injected phage gDNA and protein cargo associate with lipids derived from the bacterial inner membrane to assemble a small spherical compartment. To be consistent with previous work 15,23 , we refer to this body as the EPI (early phage infection) vesicle.…”

“…Based on the collective evidence from fluorescence microscopy experiments and previous cryo-ET 6,15 we suggest that the injected phage gDNA and protein cargo associate with lipids derived from the bacterial inner membrane to assemble a small spherical compartment. To be consistent with previous work 15,23 , we refer to this body as the EPI (early phage infection) vesicle.…”

“…Following these observations, thin-section transmission electron microscopy studies observed that ΦKZ (infecting P. aeruginosa ) and a related phage SPN3US (infecting S. typhimurium ) generate small round organelles early in the infection 13,14 . More recent studies have observed that cells infected with 201Φ2-1 phage ( P. chlororaphis ) or Goslar phage ( E. coli ) possess “ u nidentified s pherical b odies (USBs)” or “ e arly p hage infection (EPI) vesicles”, which were proposed to contain a lipid bilayer based on cryo-electron tomography (cryo-ET) 6,15 . The function and constituents of this early organelle, whether it derives from the host inner membrane, and whether injected phage DNA or protein are inside remains unclear.…”

An essential and highly selective protein import pathway encoded by nucleus-forming phage

“…Selection against PhiKZ PicA with catalytically-active Cas13a only produced escape mutants with single nucleotide missense mutations (Table 1), suggesting that the gene encoding PicA is essential. To determine how loss of PicA affects E. coli phage Goslar replication, we used the recently developed CRISPRi-ART approach, which utilizes a catalytically-deactivated Ruminococcus flavefaciens Cas13d (dCas13d) (39,40) to selectively knock down Goslar PicA during infection of E. coli. We chose to focus on Goslar since we found that the dCas13d repression system was less effective when used in P. aeruginosa against PhiKZ.…”