Hijacking the Hijackers: Escherichia coli Pathogenicity Islands Redirect Helper Phage Packaging for Their Own Benefit

Abstract

SummaryPhage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

“…For the PICImid (plasmid carrying the PICI packaging system) construction, the cosN and rppA genes (ref. 25 ), which are necessary for the packaging of the island EcCICFT073, were cloned. These were amplified with a primer set of InFpi ara-CosPPi NotI1-f and InFpi araCosPPi NotI1-r.…”

“…The E. coli JP12507 (ref. 25 ), derived by lysogenizing phage Φ80 into E. coli 594 strain, was used for the generation of PICI-based EC-CapsidCas13a(s). To prevent Φ80 from being self-packaged during generation of PICI-based EC-CapsidCas13a, the cosN site necessary for phage DNA packaging was deleted to create JP17091.…”

“…Then, the carrier M13 phage was replaced with the lysogenic phage Φ80 for use with the phage-inducible chromosomal island (PICI) packaging system ( Supplementary Fig. 3) 25,26 , which is more flexible in genome manipulation. Finally, the kanamycin (Km) resistance gene (KanR) was inserted as a selection marker to generate the constructs of PICI-based EC-CapsidCas13a:: KanR_bla IMP-1 and EC-CapsidCas13a::KanR_nontargeting as a nontargeting spacer control (Fig.…”

Molecular Piracy: Redirection of Bacteriophage Capsid Assembly by Mobile Genetic Elements

“…However, the fact that the change is from a large to a small isometric capsid—rather than prolate to isometric—suggests that the size redirection proceeds by yet another still undetermined mechanism. A similar PICI present in E. coli (EcCICFT073) also encodes a capsid protein homolog but appears to be packaged into capsids of normal (large) size, at least when induced by phage lambda [60].…”

Development of CRSIPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

“…S2C). Then, the carrier phage M13 was replaced with the lysogenic phage Φ80 with the use of the PICI (phage-inducible chromosomal island) packaging system (17,18) (Fig. 4A and Fig.…”