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1.
Mol Cell ; 75(5): 1020-1030.e4, 2019 09 05.
Article in English | MEDLINE | ID: mdl-31350119

ABSTRACT

Phage-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.


Subject(s)
Bacteriophages/physiology , DNA, Viral/metabolism , Escherichia coli/virology , Genomic Islands , Virus Assembly/physiology , DNA, Viral/genetics , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
2.
ISME J ; 12(9): 2114-2128, 2018 09.
Article in English | MEDLINE | ID: mdl-29875435

ABSTRACT

Phage-inducible chromosomal islands (PICIs) are a recently discovered family of pathogenicity islands that contribute substantively to horizontal gene transfer, host adaptation and virulence in Gram-positive cocci. Here we report that similar elements also occur widely in Gram-negative bacteria. As with the PICIs from Gram-positive cocci, their uniqueness is defined by a constellation of features: unique and specific attachment sites, exclusive PICI genes, a phage-dependent mechanism of induction, conserved replication origin organization, convergent mechanisms of phage interference, and specific packaging of PICI DNA into phage-like infectious particles, resulting in very high transfer frequencies. We suggest that the PICIs represent two or more distinct lineages, have spread widely throughout the bacterial world, and have diverged much more slowly than their host organisms or their prophage cousins. Overall, these findings represent the discovery of a universal class of mobile genetic elements.


Subject(s)
Bacteriophages/physiology , Genomic Islands , Gram-Negative Bacteria/genetics , Chromosomes, Bacterial , Gene Transfer, Horizontal
3.
ISME J ; 11(4): 1029-1042, 2017 04.
Article in English | MEDLINE | ID: mdl-27959343

ABSTRACT

The SaPIs are a cohesive subfamily of extremely common phage-inducible chromosomal islands (PICIs) that reside quiescently at specific att sites in the staphylococcal chromosome and are induced by helper phages to excise and replicate. They are usually packaged in small capsids composed of phage virion proteins, giving rise to very high transfer frequencies, which they enhance by interfering with helper phage reproduction. As the SaPIs represent a highly successful biological strategy, with many natural Staphylococcus aureus strains containing two or more, we assumed that similar elements would be widespread in the Gram-positive cocci. On the basis of resemblance to the paradigmatic SaPI genome, we have readily identified large cohesive families of similar elements in the lactococci and pneumococci/streptococci plus a few such elements in Enterococcus faecalis. Based on extensive ortholog analyses, we found that the PICI elements in the four different genera all represent distinct but parallel lineages, suggesting that they represent convergent evolution towards a highly successful lifestyle. We have characterized in depth the enterococcal element, EfCIV583, and have shown that it very closely resembles the SaPIs in functionality as well as in genome organization, setting the stage for expansion of the study of elements of this type. In summary, our findings greatly broaden the PICI family to include elements from at least three genera of cocci.


Subject(s)
Bacteriophages/physiology , Gene Expression Regulation, Bacterial/physiology , Genomic Islands , Gram-Positive Cocci/virology , Attachment Sites, Microbiological
4.
Mol Microbiol ; 91(3): 423-37, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24283262

ABSTRACT

Bacteriophages play a major role in spreading mobile genetic elements (MGEs)-encoded genes among bacterial populations. In spite of this, the molecular requirements for building phage transducing particles have not been completely deciphered. Here, we systematically inactivated each ORF from the packaging and lysis modules of the staphylococcal phage ϕ11, used as a model for the Siphoviridae phages infecting Gram-positive bacteria, and determined their functional role in transferring different MGEs including plasmids, staphylococcal pathogenicity islands (SaPIs) and the phage itself. In a previous report, we identified seven of these ORFs as being required for the production of functional phage or SaPI particles. In this report, we have completed the mutational analysis and have identified and characterized 15 additional phage-encoded proteins required for the production of mature phage, SaPI, or transducing particles. Apart from these, we have not yet ascertained any specific function for the six remaining ϕ11 genes, though they are highly conserved among the staphylococcal bacteriophages. To the best of our knowledge, this study represents the first systematic deletion analysis of all the ORFs comprising the morphogenetic and lysis modules of a phage, clearly defining the molecular requirements involved in phage-mediated MGEs transfer.


Subject(s)
Interspersed Repetitive Sequences , Staphylococcus Phages/physiology , Staphylococcus aureus/virology , Transduction, Genetic , Virus Assembly , DNA Mutational Analysis , Gene Deletion , Open Reading Frames , Staphylococcus Phages/genetics
5.
Mol Microbiol ; 85(5): 833-45, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22742067

ABSTRACT

Staphylococcus aureus pathogenicity islands (SaPIs) are a group of related 15-17 kb mobile genetic elements that commonly carry genes for superantigen toxins and other virulence factors. The key feature of their mobility is the induction of SaPI excision and replication by certain phages and their efficient encapsidation into specific small-headed phage-like infectious particles. Previous work demonstrated that chromosomal integration depends on the SaPI-encoded recombinase, Int. However, although involved in the process, Int alone was not sufficient to mediate efficient SaPI excision from chromosomal sites, and we expected that SaPI excision would involve an Xis function, which could be encoded by a helper phage or by the SaPI, itself. Here we report that the latter is the case. In vivo recombination assays with plasmids in Escherichia coli demonstrate that SaPI-coded Xis is absolutely required for recombination between the SaPI att(L) and att(R) sites, and that both sites, as well as their flanking SaPI sequences, are required for SaPI excision. Mutational analysis reveals that Xis is essential for efficient horizontal SaPI transfer to a recipient strain. Finally, we show that the master regulator of the SaPI life cycle, Stl, blocks expression of int and xis by binding to inverted repeats present in the promoter region, thus controlling SaPI excision.


Subject(s)
Genomic Islands/genetics , Staphylococcus aureus/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Blotting, Western , DNA Mutational Analysis , Real-Time Polymerase Chain Reaction , Recombination, Genetic/genetics , Staphylococcus aureus/metabolism
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