Phage-inducible chromosomal islands are ubiquitous within the bacterial universe

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.

Fig. 1

Genome maps for PICIs. Genomes are aligned according to the prophage convention, with the integrase gene (int) at the left end. Genes are colored according to their sequence and function: int is yellow; transcription regulator (stl or rpr (GP), and alpA or merR (GN)) is dark blue; replication genes are purple; encapsidation genes are green, with the terminase small subunit gene (terS) in light green; superantigen and other virulence genes are pink; genes encoding putative phage resistance proteins are black; other accessory genes are red; genes encoding hypothetical proteins are white

Fig. 2

Characterization of GN PICI replication origins. a Comparative map of the replication origins of several E. coli PICIs. The iterons are represented by arrows, and their sequences are shown at left. Note that there are always two sets of iterons flanking an AT rich region, which could be the melting site. b Testing EcCICFT073 pri-rep-ori function. pK03Blue derivative plasmids pri-ori, pri-∆ori or ∆pri-ori were tested for maintenance in DH5α on selective agar at the permissive (30 °C) or restrictive (44 °C) temperature. A One-way ANOVA with Tukey’s multiple comparisons test was performed to compare mean differences within rows. Adjusted p values were as follows: pri-ori 44 °C vs pri-∆ori 44 °C < 0.0001^****, pri-ori 44 °C vs ∆pri-ori 44 °C < 0.0001^****

Fig. 3

Phage induction of EcCICFT073. CFT073 strains were SOS induced with MC (2 μg/ml). Samples were removed at the indicated time points and used to prepare minilysates, which were resolved on an 0.7% agarose gel (upper panel), and Southern blotted (lower panel) with an EcCICFT073 probe. M: Southern blot molecular marker (DNA molecular weight marker VII; Roche)

Fig. 4

Functionality of the EcCICFT073 cos sites. EcCICFT073 cat wt, or its derivatives carrying mutations in the cos1 or/and cos2 sites, were introduced in the lysogenic strains for phages λ or ϕ80. The different strains were MC induced (2 μg/ml) and the transfer of the island quantified. A t-test was performed to compare the wt against the different cos mutants. Adjusted p values were as follows: Phage Lambda EcCICFT073 ∆cos1 = 0.002^**, Phage Lambda EcCICFT073∆cos2 = 0.0266^*, Phage 80 EcCICFT073 ∆cos1 = 0.003^***, Phage ϕ80 EcCICFT073 ∆cos2 = 0.0002^***

Fig. 5

EcCICFT073 interferes with λ reproduction. E. coli strains C600, JP12677 (C600 EcCICFT073 tetA-positive), JP15181 (C600 EcCICFT073 tetA-positive ∆c1499) or JP15182 (C600 EcCICFT073 tetA-positive ∆c1500) were infected with phage λ (∼500 pfu per plate), plated on phage bottom agar, and incubated for 24 h at 37 °C. Plates were stained with 0.1% TTC in LB and photographed

Fig. 6

Characterization of the EcCICFT073 alpA gene. a–c Schematic representation of the different blaZ transcriptional fusions generated. The relevant genes are shown. b–d Miller assay performed using the plasmids represented in (a–c). Results (average ± s.d.) of three independent assays are shown. A 2-way ANOVA with Tukey’s multiple comparisons test was performed to compare mean differences within rows. Adjusted p values were as follows: pJP1290 vs pJP1291 = 0. 0011^**, pJP1288 vs pJP1289 = 0.0001^***, PalpA  < 0.0001^****, Pint  > 0.9999 ^ns. ns, not significant. e EcCICFT073 excision and replication after expression of the cloned alpA gene. A non-lysogenic derivative of strain C600 carrying EcCICFT073 was complemented with plasmid pBAD18 (empty plasmid) or plasmid pJP2037, which carries alpA under the control of the P_BAD promoter. As controls, non-lysogenic derivatives of strain C600 carrying EcCICFT073 ∆int or EcCICFT073 ∆pri were complemented with plasmid pJP2037. One milliliter of each culture (optical density (OD)_600nm = 0.3) was collected 2 h after treatment with 0.02% arabinose and used to prepare standard minilysates, which were resolved on a 0.7% agarose gel, Southern blotted and probed for EcCICFT073 DNA. In these experiments, because no helper phage is present, the excised EcCICFT073 DNA appears as covalently closed circular molecules (CCC). f Helper phages activate alpA transcription. Different lysogenic and non-lysogenic strains, containing plasmid pJP1290, were MC-induced and assayed for β-galactosidase activity. Results (average ± s.d.) of three independent assays are shown. Strains: C600 (non-lysogenic), JP10400 (C600 lysogenic for phage λ), 594 (non-lysogenic), JP12507 (594 lysogenic for phage 80) and JP15151 (594 lysogenic for phage 80 ∆terS). A 2-way ANOVA with Tukey’s multiple comparisons test was performed to compare mean differences within rows. Adjusted p values were as follows; C600 > 0.9999 ^ns, Phage Lambda < 0.0001^****, 594 > 0.9999 ^ns, Phage 80 < 0.0001^****, Phage 80 ∆terS < 0.0001^****. ns, not significant

Fig. 7

Induction of PmCI172 and PmCI86. a DNA extracted from a phage lysates of MC-treated cultures of P. multocida strains 172 or 86. b Southern blot of the DNA shown in panel (a), using a phage- or PmCI-specific probe. c Electron microscopy analysis of the MC-induced P. multocida 172 lysate. Several different fields are shown, containing normal Mu phage particles and PmCI172 particles (arrows), which have smaller heads. Scale bars are 50 nm