Primers used for site-directed mutagenesis.

<p>The asterisk (*) indicate position of deleted nucleotide in forward primer. Analogous deletion was introduced in the reverse primer but is not shown for clarity of presentation.</p><p>Primers used for site-directed mutagenesis.</p

Expression patterns of genes from the <i>exo-xis</i> region, as well as <i>int, N, cro, Q</i> and <i>R</i> genes of bacteriophage λ (panel A) and Ф24_B (panel B) infecting <i>E. coli</i> MG1655 host at 30°C.

<p>Levels of transcripts corresponding to particular genes or <i>ORF</i>s were determined at following times after infection: 2.5 (violet), 5 (green), 7.5 (red), 10 (white), 20 (black) and 30 (orange) minutes in case of phage λ and 10 (white), 20 (black), 30 (orange), 40 (gray), 50 (yellow), and 60 (blue) minutes in case of phage 24_B. The presented results are mean values from 3 experiments with error bars indicating SD.</p

Predicted promoters for the <i>orf73</i> coding region of bacteriophages λ and Ф24_B.

<p>Predicted promoters for the <i>orf73</i> coding region of bacteriophages λ and Ф24_B.</p

Survival of <i>E. coli</i> MG1655 cells lysogenic for λ (panels A and B) or Ф24_B (panels C and D) bacteriophages after prophage induction with 0.2 µg/ml mitomycin C (panels A and C) or 1 mM H₂O₂ (panels B and D) at 30°C.

<p>Host cells contained either the pJW0tet vector (open squares) or a plasmid containing the <i>exo-xis</i> region from with λ (pGAW3773tet; panels A and B) or Ф24_B (pSBe.x.r; panels C and D) (closed squares). The presented results are mean values from 3 experiments with error bars indicating SD.</p

Relative level of DNA of λ (panels A and B) or Ф24_B (panels C and D) bacteriophages after prophage induction with 0.2 µg/ml mitomycin C (panels A and C) or 1 mM H₂O₂ (panels B and D) in <i>E. coli</i> MG1655 host at 30°C.

<p>Host cells contained either the pJW0tet vector (open squares) or a plasmid containing the <i>exo-xis</i> region from with λ (pGAW3773tet; panels A and B) or Ф24_B (pSBe.x.r; panels C and D) (closed squares). The presented results are mean values from 3 experiments with error bars indicating SD.</p

Expression patterns of genes from the <i>exo-xis</i> region, as well as <i>int, N, cro, Q</i> and <i>R</i> genes of bacteriophage λ (panels A and B) and Ф24_B (panels C and D) after prophage induction with 0.2 µg/ml mitomycin C (panels A and C) or 1 mM H₂O₂ (panels B and D) in <i>E. coli</i> MG1655 host at 30°C.

<p>Levels of transcripts corresponding to particular genes or <i>ORF</i>s were determined at following times after induction: 64 (red), 128 (gray), 136 (orange), 144 (dark blue), 152 (yellow), 160 (green), 176 (maroon) and 192 (light blue) minutes. The presented results are mean values from 3 experiments with error bars indicating SD.</p

Primers used in the real-time PCR assay.

<p>Primers used in the real-time PCR assay.</p

Predicted terminators in the <i>exo-xis</i> regions of bacteriophages λ and Ф24_B.

<p>Secondary structures are indicated, where loops are in <i>italic</i> font and stems in <b><u>bold underlined</u></b> font. The sequences of predicted terminators t₁ and t₂ are exactly the same in the case of both phages λ and Ф24_B.</p><p>Predicted terminators in the <i>exo-xis</i> regions of bacteriophages λ and Ф24_B.</p

Maps of <i>exo-xis</i> regions and other genes of bacteriophages λ and Ф24_B analyzed in this work (accession numbers: GI:9626243 and GI:307604077 respectively).

<p>Dark arrows with continuous outside lines represent highly conserved genes and open reading frames (over 70% nucleotide as well as amino acid sequence identity). Dark arrows with punctuated outside lines represent highly conserved (above 70% sequence identity) open reading frames present in genomes of λ and 933W phages, available in the NCBI database but uncharacterized in annotations. The presence of <i>orf73</i> in the λ <i>exo-xis</i> region was indicated by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108233#pone.0108233-Sergueev1" target="_blank">[48]</a>. Light arrows represent genes and open reading frames with low level of sequence identity (<38%). Note the high homology between λ and Ф24_B<i>exo-orf73</i> regions and low level of identity of other analyzed genes. Arrows indicate positions of promoters predicted with BPROM program. The localizations and −10 and −35 sequences of predicted promoters <i>p</i>_{1_λ} and <i>p</i>_{1_Φ24B} are exactly the same (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108233#pone-0108233-t003" target="_blank">Table 3</a>). Schematic steam-loop structures [•] indicate localizations of predicted transcription terminators, found on the basis of nucleotide seuquence analysis with ARNold software. The localizations and sequences of predicted terminators t₁ and t₂ are exactly the same in case of both phages λ and Ф24_B (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108233#pone-0108233-t004" target="_blank">Table 4</a>). Note that in the case of Ф24_B phage, some ORFs from the <i>exo-xis</i> region: <i>vb_24B_9c, vb_24B_8c, vb_24B_7c</i>, putative C4 zinc finger protein and <i>vb_24B_6c</i> are homologues of λ <i>orf60a, orf63, orf61, orf73</i> and gene <i>ea22</i> respectively. For clarity of this work, only the names of λ ORFs were used.</p

Inhibition of Shiga toxin-converting bacteriophage development by novel antioxidant compounds

<p>Oxidative stress may be the major cause of induction of Shiga toxin-converting (Stx) prophages from chromosomes of Shiga toxin-producing <i>Escherichia coli</i> (STEC) in human intestine. Thus, we aimed to test a series of novel antioxidant compounds for their activities against prophage induction, thus, preventing pathogenicity of STEC. Forty-six compounds (derivatives of carbazole, indazole, triazole, quinolone, ninhydrine, and indenoindole) were tested. Fifteen of them gave promising results and were further characterized. Eleven compounds had acceptable profiles in cytotoxicity tests with human HEK-293 and HDFa cell lines. Three of them (selected for molecular studies) prevent the prophage induction at the level of expression of specific phage genes. In bacterial cells treated with hydrogen peroxide, expression of genes involved in the oxidative stress response was significantly less efficient in the presence of the tested compounds. Therefore, they apparently reduce the oxidative stress, which prevents induction of Stx prophage in <i>E. coli.</i></p