Fig 1.
Whole genome comparisons of the four integrated prophages.
Each LF82 phage (in bold) is compared with tBLASTx to its closest relative in databases (up), as well as to an ICTV classified prototype (down), and displayed using the R Genoplot package. Gene color code: green, capsid; light blue, connector; dark blue, tail; red, integration and excision; orange, DNA metabolism; yellow, transcriptional regulators; pink, lysis; grey, hypothetical. Morons identified by our transcriptomic analysis are shown in black (predicted function) or in arrows with thick black border (unknown or putative function).
Table 1.
Prophage genes expressed at least 5-fold above local background in strain LF82 grown in LB medium (overnight culture).
Fig 2.
Shotgun sequencing of the viral particles generated by the five LF82 prophages in an in vitro culture in rich medium.
Nucleotide coverage as a function of LF82 chromosome or Cyrano phage-plasmid coordinates obtained after the mapping of the sequencing reads from the virome of an overnight LF82 culture. The coverages associated with the prophage regions and the bacterial chromosome are shown in dark and light grey respectively. The average coverage linked to bacterial DNA contamination of the virome sequencing is represented in light blue (7.6 reads/bp) for the integrated prophages.
Fig 3.
PCR quantification of viral particles generated by the five LF82 prophages, in rich medium with or without antibiotic, from in vitro LF82 culture.
Phages produced from LF82 cells cultured in the presence or absence of an antibiotic, as indicated, were quantified. Each dot corresponds to one biological replicate. Black or white dots correspond to replicates that are respectively more or less abundant than their associated LF82 genomic DNA contamination. Only values corresponding to black dots are used to calculate the mean (vertical line). The statistical difference (t-test) between antibiotic treated and untreated cultures is indicated by one (p-value < 0.05), two (p-value < 0.01) or three (p-value < 0.005) asterisks.
Fig 4.
Transmission electron microscopy photographs of the virions produced by LF82.
Gally and Cyrano were imaged directly from LF82 culture supernatants. Perceval and Tritos phages were visualized after their purification and propagation on MAC1403 as an indicative strain. Asterisks indicate vesicles. Scale bars are 50 μm long.
Table 2.
Prophage boundaries on E. coli LF82 chromosome.
Fig 5.
Gally performs lateral transduction of over 200 kb of adjacent chromosomal DNA, including Perceval.
A. Zoom of mapped reads within the chromosomal region to the right of Gally prophage containing Perceval. The abrupt increase in reads inside Gally corresponds to the pac region, from which the packaging is initiated. Red bars indicate 40.1 kb steps of decreasing coverage from the pac site of Gally prophage, which could correspond to DNA packaged by a headful mechanism. B. PCR quantification of Perceval virions produced from an in vitro culture in rich medium of a wild-type or a Gally-deleted strain of E. coli LF82. Each black dot corresponds to one biological replicate and black horizontal lines indicate the mean values for each condition. The statistical difference (t-test) between the two strains is indicated by three asterisks (p-value = 0.0006).
Fig 6.
The survival of LF82 treated in vitro with ciprofloxacin is affected in the presence of the Gally prophage, contrary to what is observed after THP-1 macrophages infection.
A. Optical density of in vitro cultures of wild-type (grey lines) or ΔGally (black lines) LF82 bacteria was monitored after addition (dashed lines) or not (continuous lines) of ciprofloxacin at the MIC. Experiments were performed three times and quantifications shown are from one representative experiment. B. and C. Survival (CFU/mL) of the wild-type or ΔGally strains, after 6 (B) or 24 (C) hours in THP-1 macrophage was compared to the initial amount of endocytosed LF82 bacteria (CFU/mL at 1-hour P.I. as reference). Black dots represent values from three biological replicates obtained after independent macrophage infections. Horizontal black lines represent mean values. NS: not significant (t-test, p-values > 0.6).
Fig 7.
Transcription of the Gally prophage in macrophages.
Comparison of coverage by RNA-Seq fragments along Gally genome between THP1 macrophages infected by LF82 at 6h P.I. (MB6, dark red) and LF82 bacteria grown in LB to stationary phase (BLB, orange). Data from two biological replicates are shown for each condition along the region corresponding to the prophage (from 998,954 to 1,037,635 bp) in E. coli LF82 genome. From top to bottom: transcription profiles on + and − strands, expressed in log2(fpkm+5); vertical arrows indicating genes detected as differentially expressed (q-value ≤ 0.01 and |log2FC| ≥ 1), pointing upward for up-regulated in macrophages, downward for down-regulated; genome annotation (names for selected genes). The vertical distance between horizontal dotted lines in the transcription profile panels correspond to a log2FC of 1.
Fig 8.
Gally and Cyrano are not induced in LF82 upon macrophage infection.
A. Comparison of phages/bacteria ratios obtained for Gally and Cyrano, after cultures in vitro in Lennox and Lennox + ciprofloxacin (MIC), and at 6 hours P.I. in macrophages. Black or white dots correspond to replicates that are respectively more or less abundant in phages than their associated LF82 genomic DNA contamination. Only values corresponding to black dots are used to calculate the means (vertical lines). Statistical differences (Mann-Whitney test) between ciprofloxacin-treated (Lennox+cip) or macrophage infection (Macrophages 6H P.I.), and untreated cultures (Lennox) is indicated by two (p-value < 0.01) or three (p-value < 0.005) asterisks. B. Gally phage induction was followed by the MCP-GFP fusion production in in vitro ciprofloxacin-treated LF82 cultures. Snapshots of LF82 bacteria (strain MAC2606) 60 minutes after ciprofloxacin treatment (+cip) or of untreated cells (-cip) are shown. Scale bars correspond to 5 μm. C. Confocal imaging of THP-1 macrophages at 6 hours P.I. with the LF82-pPrpsm-mCherry (OEC2425) (left panel) or LF82-pPrpsm-mCherry Gally mcp-GFP (OEC2481) (right panel). White framed areas correspond to zooms of the white dashed framed parts. Scale bars indicate 5 or 20 μm, as indicated.
Table 3.
Strains used in this study.
Table 4.
Oligonucleotides used in this study.