Oxidative Medicine and Cellular Longevity
Volume 2016 (2016), Article ID 8453135, 14 pages
http://dx.doi.org/10.1155/2016/8453135

Research Article

The Role of the Exo-Xis Region in Oxidative Stress-Mediated Induction of Shiga Toxin-Converting Prophages

Katarzyna Licznerska,¹ Aleksandra Dydecka,¹ Sylwia Bloch,¹ Gracja Topka,¹ Bożena Nejman-Faleńczyk,¹ Alicja Węgrzyn,² and Grzegorz Węgrzyn¹

¹Department of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland
²Laboratory of Molecular Biology (Affiliated with the University of Gdańsk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Wita Stwosza 59, 80-308 Gdańsk, Poland

Received 11 August 2015; Revised 22 September 2015; Accepted 28 September 2015

Academic Editor: Luciano Saso

Copyright © 2016 Katarzyna Licznerska et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Previous studies indicated that these genetic elements could be involved in the regulation of lysogenization and prophage induction processes. The effects were dramatic in Shiga toxin-converting phage after treatment with oxidative stress-inducing agent, hydrogen peroxide, while they were less pronounced in bacteriophage λ and in both phages irradiated with UV. The hydrogen peroxide-caused prophage induction was found to be RecA-dependent. Importantly, in hydrogen peroxide-treated E. coli cells lysogenic for either λ or , deletion of the exo-xis region resulted in a significant decrease in the levels of expression of the S.O.S. regulon genes. Moreover, under these conditions, a dramatic decrease in the levels of expression of phage genes crucial for lytic development (particularly xis, exo, N, cro, O, Q, and R) could be observed in -, but not in λ-bearing cells. We conclude that genes located in the exo-xis region are necessary for efficient expression of both host S.O.S regulon in lysogenic bacteria and regulatory genes of Shiga toxin-converting bacteriophage .

1. Introduction

Infection of humans by enterohemorrhagic Escherichia coli (EHEC) strains causes hemorrhagic colitis, and in some patients it may result in various complications, including, the most severe of them, the hemolytic-uremic syndrome and neurological dysfunctions [1–3]. The main causes of EHEC-mediated complications are Shiga toxins, produced by the infecting bacteria [4]. The severity of EHEC infections and significance of the medical problem related to them are exemplified by local outbreaks, occurring in various geographical regions around the world. One of the most famous of them took place in 2011 in Germany, where over 4,000 patients developed severe symptoms, and 54 died [5–10].

In EHEC strains, Shiga toxins are encoded by genes (called stx genes) located in genomes of prophages [11, 12]. The phages bearing stx genes are referred to as Shiga toxin-converting bacteriophages, and all of them belong to the family of lambdoid phages (with phage λ serving as a paradigm) [12]. stx genes are present between antiterminator gene and the genes coding for proteins causing cell lysis; thus, in the lysogenic state, these genes are not transcribed [13–15] and Shiga toxins are not produced. Their expression is possible only after prophage induction [11, 12] which usually requires activation of the bacterial S.O.S. response, mediated by RecA protein, though RecA-independent induction of Shiga toxin-converting prophages by EDTA has also been reported [16].

During infection of human intestine by EHEC, the oxidative stress appears to be the most likely condition causing the bacterial S.O.S. response and subsequent induction of Shiga toxin-converting prophages [17]. In fact, it was demonstrated that hydrogen peroxide (which is produced by neutrophils as a response to infection) enhanced production of Shiga toxins by EHEC [18] due to oxidative stress-mediated induction of Shiga toxin-converting prophages [19, 20].

Since many antibiotics not only kill bacteria and inhibit their growth but also induce prophage lytic development, their use is not recommended when EHEC infection is confirmed or even suspected (reviewed in [12]). Therefore, there are serious problems with treatment of patients, indicating that searching for new targets of potential therapies against Shiga toxin-producing bacteria is important. One might consider that such therapies should be focused on inhibition of Shiga toxin-converting prophage induction which would impair production of the EHEC virulence factor. All lambdoid phages, including Shiga toxin-converting bacteriophages, contain the region in their genomes which is dispensable for the development under standard laboratory conditions [14, 15]. Inside this part of the phage genome, there is an evolutionarily conserved fragment, located between exo and xis genes and transcribed from promoter, called the exo-xis region (Figure 1). This region encompasses several genes and open reading frames whose functions in phage development are largely unknown, and only a few articles are available in the literature that focused on them. Nevertheless, some interesting observations have been reported. Namely, induction of expression of genes from the exo-xis region resulted in synchronization of the host cell cycle [21] and inhibition of host DNA replication [22]. Moreover, overexpression of these genes impaired lysogenization of E. coli by bacteriophage [23] and enhanced induction of prophages and (one of Shiga toxin-converting phages) [24]. Ea8.5 protein, encoded by a gene located in the exo-xis region, contains a fused homeodomain/zinc finger fold [25] which suggests a regulatory role for this protein. Interestingly, prophage induction with mitomycin C or hydrogen peroxide caused different expression patterns of genes from the exo-xis region; such differences were observed in both phages, and [26]. In this work, we used the deletion mutants to investigate the role of the exo-xis region in induction of and prophages under oxidative stress conditions.

Figure 1: Schematic maps of the exo-xis regions of bacteriophages and . Genes from the exo-xis region are marked as thick grey arrows, and other genes are shown as thick open arrows. Directionality of transcription from promoter is indicated as thin dashed arrow. terminator is marked as black oval.

2. Materials and Methods

2.1. Bacteria and Bacteriophages

E. coli MG1655 strain [27] and its derivatives, used in this work, are listed in Table 1. Bacteria were routinely cultured in the Luria-Bertani (LB) medium at 30°C (most experiments) or 37°C (lysogenization and recombination procedures during construction of strains and SOS ChromoTest, according to the instructions of kits’ manufacturers), under aerobic conditions. Where appropriate, the following antibiotics were added: chloramphenicol up to 20 μg/mL, kanamycin up to 50 μg/mL, and/or tetracycline up to 12.5 μg/mL.

Table 1: Escherichia coli strains.

Strain

Genotype or relevant characteristics
Reference

E. coli MG1655
   ilvG rfb-50 rph-1
[27]

E. coli MG1655 (λ)
MG1655 bearing λ prophage
[24]

E. coli MG1655 (λΔexo-xis)
MG1655 bearing λ prophage with deletion of all orfs and genes localized between genes exo and xis
This study, by recombination

E. coli MG1655 (λΔorfs)
MG1655 bearing λ prophage with deletion of orf60a, orf63, orf61, and orf73
This study, by recombination

E. coli MG1655 (λΔorf60a)
MG1655 bearing λ prophage with deletion of orf60a
This study, by recombination

E. coli MG1655 (λΔorf63)
MG1655 bearing λ prophage with deletion of orf63
This study, by recombination

E. coli MG1655 (λΔorf61)
MG1655 bearing λ prophage with deletion of orf61
This study, by recombination

E. coli MG1655 (λΔorf73)
MG1655 bearing λ prophage with deletion of orf73
This study, by recombination

E. coli MG1655 (λΔea22)
MG1655 bearing λ prophage with deletion of ea22
This study, by recombination

E. coli MG1655 (λΔea8.5)
MG1655 bearing λ prophage with deletion of ea8.5
This study, by recombination

E. coli MG1655 (Φ)
MG1655 bearing Φ prophage
[24]

E. coli MG1655 (ΦΔexo-xis)
MG1655 bearing Φ prophage with deletion of all orfs and genes localized between genes exo and xis
This study, by recombination

E. coli MG1655 (ΦΔorfs)
MG1655 bearing Φ prophage with deletion of 4 orfs being homologues of λorf60a, λorf63, λorf61, and λorf73
This study, by recombination

E. coli MG1655 (ΦΔorf60a)
MG1655 bearing Φ prophage with deletion of vb__9c, the homologue of λorf60a
This study, by recombination

E. coli MG1655 (ΦΔorf63)
MG1655 bearing Φ prophage with deletion of vb__8c, the homologue of λorf63
This study, by recombination

E. coli MG1655 (ΦΔorf61)
MG1655 bearing Φ prophage with deletion of vb__7c, the homologue of λorf61
This study, by recombination

E. coli MG1655 (ΦΔorf73)
MG1655 bearing Φ prophage with deletion of the sequence of putative C4 zinc finger protein, the homologue of λorf73
This study, by recombination

E. coli MG1655 (ΦΔea22)
MG1655 bearing Φ prophage with deletion of vb__6c, the analogue of λea22
This study, by recombination

E. coli MG1655recA13
MG1655 but recA13
[33]

E. coli MG1655recA13 (λ)
MG1655recA13 bearing λ prophage
This study, by lysogenization

E. coli MG1655recA13 (λΔexo-xis)
MG1655recA13 bearing λ prophage with deletion of all orfs and genes localized between genes exo and xis
This study, by lysogenization

E. coli MG1655recA13 (Φ)
MG1655recA13 bearing Φ prophage
This study, by lysogenization

E. coli MG1655recA13 (ΦΔexo-xis)
MG1655recA13 bearing Φ prophage with deletion of all orfs and genes localized between genes exo and xis
This study, by lysogenization

E. coli PQ37
sfiA::Mud(Ap lac) cts, lacΔU169, mal ⁺, galE, galY, PhoC, rfa, F⁻, thr, leu, his, pyrD, thi, trp::MUC ⁺, srl300::Tn10, rpoB, uvrA ⁺
[31]

E. coli PQ37 (λ)
PQ37 bearing λ prophage
This study, by lysogenization

E. coli PQ37 (λΔexo-xis)
PQ37 bearing λ prophage with deletion of all orfs and genes localized between genes exo and xis
This study, by lysogenization

E. coli PQ37 (Φ)
PQ37 bearing Φ prophage
This study, by lysogenization

E. coli PQ37 (ΦΔexo-xis)
PQ37 bearing Φ prophage with deletion of all orfs and genes localized between genes exo and xis
This study, by lysogenization

Bacteriophages papa (from our collection) [26] and (Δstx2::cat) [28] were employed in this study. Phage suspensions were stored in the TM buffer (10 mM Tris-HCl, 10 mM MgSO₄, pH 7.2) at 4°C.

The deletion mutants were constructed as described previously [29], by using the Quick and Easy E. coli Gene Deletion Kit (from Gene Bridges). The deletion of the indicated region was performed according to the manufacturer’s protocol using primers listed in Table 2. In the first step, the targeted sequence has been replaced with the FRT-flanked kanamycin resistance cassette, and the selection marker was subsequently removed in the FLP-recombinase step, leaving only 87 nucleotides of the cassette in the place of the original sequence. Each deletion was confirmed by DNA sequencing.

Table 2: Primers used for construction of E. coli strains.

Primer name
Sequence (5′3′)

pF_λ_exo-xis = pF_λ_orf60a
pR_λ_exo-xis
ATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTACAAAGCGAGGAATTAACCCTCACTAAAGGGCG
AGGCGGCTTGGTGTTCTTTCAGTTCTTCAATTCGAATATTGGTTACGTCTTAATACGACTCACTATAGGGCTC

pF_λ_orfs = pF_λ_orf60a
pR_λ_orfs = pR_λ_orf73
ATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTACAAAGCGAGGAATTAACCCTCACTAAAGGGCG
ACCTCTCTGTTTACTGATAAGTTCCAGATCCTCCTGGCAACTTGCACAAGTAATACGACTCACTATAGGGCTC

pF_λ_orf60a
pR_λ_orf60a
ATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTACAAAGCGAGGAATTAACCCTCACTAAAGGGCG
AGATGCTTTGTGCATACAGCCCCTCGTTTATTATTTATCTCCTCAGCCAGTAATACGACTCACTATAGGGCTC

pF_λ_orf63
pR_λ_orf63
CACAAAGCATCTTCTGTTGAGTTAAGAACGAGTATCGAGATGGCACATAGAATTAACCCTCACTAAAGGGCG
TCATACCTGGTTTCTCTCATCTGCTTCTGCTTTCGCCACCATCATTTCCATAATACGACTCACTATAGGGCTC

pF_λ_orf61
pR_λ_orf61
AGAGAAACCAGGTATGACAACCACGGAATGCATTTTTCTGGCAGCGGGCTAATTAACCCTCACTAAAGGGCG
TTATCCGGAAACTGCTGTCTGGCTTTTTTTGATTTCAGAATTAGCCTGACTAATACGACTCACTATAGGGCTC

pF_λ_orf73
pR_λ_orf73
ACATCATTGATTCAGCATCAGAAATAGAAGAATTACAGCGCAACACAGCAAATTAACCCTCACTAAAGGGCG
ACCTCTCTGTTTACTGATAAGTTCCAGATCCTCCTGGCAACTTGCACAAGTAATACGACTCACTATAGGGCTC

pF_λ_ea22
pR_λ_ea22
GAAATTAACTCTCAGGCACTGCGTGAAGCGGCAGAGCAGGCAATGCATGAAATTAACCCTCACTAAAGGGCG
GTCAGACATCATATGCAGATACTCACCTGCATCCTGAACCCATTGACCTCCTAATACGACTCACTATAGGGCTC

pF_λ_ea8.5
pR_λ_ea8.5
ATGAGTATCAATGAGTTAGAGTCTGAGCAAAAAGATTGGGCGTTATCAATAATTAACCCTCACTAAAGGGCG
TAATCATCTATATGTTTTGTACAGAGAGGGCAAGTATCGTTTCCACCGTATAATACGACTCACTATAGGGCTC

pF_Φ_exo-xis = pF__orf60a
pR_Φ_exo-xis
TGGTAATGAAGCATCCTCACGATAATATCCGGGTAGGCACGATCACTTTCAATTACCTCACTAAAGGGCG
TGGCAATATGCTTTTCCTTCTCAATTTCCGCTTTAATCATATGCAGTTCGTAATACGACTCACTATAGGGCTC

pF_Φ_orfs = pF_Φ_orf60a
pR_Φ_orfs = pR_Φ_orf73
TGGTAATGAAGCATCCTCACGATAATATCCGGGTAGGCACGATCACTTTCAATTACCTCACTAAAGGGCG
CTTCGAACCTCTCTGTTTACTGATAAGCTCCAGATCCTCCTGGCAACTTGTAATACGACTCACTATAGGGCTC

pF_Φ_orf60a
pR_Φ_orf60a
TGGTAATGAAGCATCCTCACGATAATATCCGGGTAGGCACGATCACTTTCAATTACCTCACTAAAGGGCG
GGAGATGCTTTGTGCATACAGCCCCTCGTTATTATTTATCTCTTCAGCCTAATACGACTCACTATAGGGCTC

pF_Φ_orf63
pR_Φ_orf63
TGCACAAAGCATCTCCTGTTGAATTAAGAACGAGTATCGGGATGGCACATAATTAACCCTCACTAAAGGGCG
CATCATTTCCAGCTTTTGTGAAAGGGATGTGGCTAACGTATGAAATTCTTTAATACGACTCACTATAGGGCTC

pF_Φ_orf61
pR_Φ_orf61
TTCTGGCAGCAGGCTTCATATTCTGTGTGCTTATGCTTGCCGACATGGGAAATTAACCCTCACTAAAGGGCG
CACCGTTCCTTAAAGACGCCGTTTAACATGCCGATCGCCAGACTTAAATGTACGACTCACTATAGGGCTC

pF_Φ_orf73
pR_Φ_orf73
TGGCAGACCTCATTGATTCAGCATCAGAAATTGAAGAATTACAGCGCAACAATTAACCCTCACTAAAGGGCGG
CTTCGAACCTCTCTGTTTACTGATAAGCTCCAGATCCTCCTGGCAACTTGTAATACGACTCACTATAGGGCTC

pF_Φ_ea22  
pR_Φ_ea22
ATCAGGCACTGCGTGAAAAGGCAGAGAAAGCAACTAAAGGAAGCTACATCAATTAACCCTCACTAAAGGGCG
ATCCAGTGTGACGGTTTCCACGACGCACCAGGAATTATCCACCCATCATTATACGACTCACTATAGGGCTC

Lysogenic strains were constructed according to the procedure described previously [24], with slight modifications. Briefly, host bacteria were cultured to A₆₀₀ of 0.5 in LB medium supplemented with MgSO₄ and CaCl₂ (to final concentrations of 10 mM each) at 37°C with shaking. At this point, one milliliter of the culture was withdrawn and centrifuged (10 min, 2000 ×g). Pellet was washed twice with TCM buffer (10 mM Tris-HCl, pH 7.2, 10 mM MgSO₄, 10 mM CaCl₂) and then suspended in 1 mL of the same buffer. Next, bacteria were incubated for 30 min at 30°C and mixed with phage suspensions at multiplicity of infection (m.o.i.) = 5. Mixtures of bacteria and phages were incubated in TMC buffer for 30 min at 30°C; then serial dilutions were prepared in TM buffer (10 mM Tris-HCl, 10 mM MgSO₄; pH 7.2), and the mixture was plated onto LB agar. Plates were incubated at 37°C overnight. Lysogens were verified by sensitivity to UV irradiation and confirmed by PCR with primers designed to amplify phage sequence (Table 3).

Table 3: Primers used for PCR.

Primers for bacterial sequences
Primers for phage sequences

Name
Sequence (5′3′)
Name
Sequence (5′3′)

pF_recA
AGATTTCGACGATACGGCCC
pF_λ_int
TTTGATTTCAATTTTGTCCCACT

pR_recA
AACCATCTCTACCGGTTCGC
pR_ λ_int
ACCATGGCATCACAGTATCG

pF_lexA
ATGGATGGTGACTTGCTGGC
pF_λ_xis
TACCGCTGATTCGTGGAACA

pR_lexA
TTCGTCATCAATACGTGCGAC
pR_ λ_xis
GGGTTCGGGAATGCAGGATA

pF_ssb
ATCGAAGGTCAGCTGCGTAC
pF_λ_exo
TGCCGTCACTGCATAAACC

pR_ssb
CGACTTCTGTGGTGTAGCGA
pR_ λ_exo
TCTATCGCGACGAAAGTATGC

pF_recF
CGATACCGGCGCTATACTCC
pF_λ_cIII
ATTCTTTGGGACTCCTGGCTG

pR_recF
TTACGAACAGCTACGCCCG
pR_ λ_cIII
GTAAATTACGTGACGGATGGAAAC

pF_rpoD
GAATCTGAAATCGGGCGCAC
pF_λ_N
CTCGTGATTTCGGTTTGCGA

pR_rpoD
GTCAACAGTTCAACGGTGCC
pR_ λ_N
AAGCAGCAAATCCCCTGTTG

pF_rpoH
GCTTTGGTGGTCGCAACTTT
pF_λ_cI
ACCTCAAGCCAGAATGCAGA

pR_rpoH
TCGCCGTTCACTGGATCAAA
pR_ λ_cI
CCAAAGGTGATGCGGAGAGA

pF_rpoS
TTGCTCTGCGATCTCTTCCG
pF_λ_cro
ATGCGGAAGAGGTAAAGCCC

pR_rpoS
GAACGTTTACCTGCGAACCG
pR_ λ_cro
TGGAATGTGTAAGAGCGGGG

pF_uvrA
GTCCATATCCGCCACTACCG
pF_λ_cII
TCGCAATGCTTGGAACTGAGA

pR_uvrA
TTACCCAACGTCTTGCCGAG
pR_ λ_cII
CCCTCTTCCACCTGCTGATC

pF_ftsK
ACAAACCGTTTATCTGCGCG
pF_λ_O
AATTCTGGCGAATCCTCTGA

pR_ftsK
ATCTTTACCCAGCACCACGG
pR_ λ_O
GAATTGCATCCGGTTT

pF_16SrRNA
CCTTACGACCAGGGCTACAC
pF_λ_Q
TTCTGCGGTAAGCACGAAC

pR_16SrRNA
TTATGAGGTCCGCTTGCTCT
pR_ λ_Q
TGCATCAGATAGTTGATAGCCTTT

pF_λ_R
ATCGACCGTTGCAGCAATA

pR_ λ_R
GCTCGAACTGACCATAACCAG

pF_Φ_int
CAGTTGCCGGTATCCCTGT

pR_ Φ_int
TGAGGCTTTCTTGCTTGTCA

pF_Φ_xis
TATCGCGCCGGATGAGTAAG

pR_ Φ_xis
CGCACAGCTTTGTATAATTTGCG

pF_Φ_exo
TGCCGTCACTGCATAAACC

pR_ Φ_exo
TCTATCGCGACGAAAGTATGC

pF_Φ_cIII
ATTCTTTGGGACTCCTGGCTG

pR_ Φ_cIII
GTAAATTACGTGACGGATGGAAAC

pF_Φ_N
AGGCGTTTCGTGAGTACCTT

pR_ Φ_N
TTACACCGCCCTACTCTAAGC

pF_Φ_cI
TGCTGTCTCCTTTCACACGA

pR_ Φ_cI
GCGATGGGTGGCTCAAAATT

pF_Φ_cro
CGAAGGCTTGTGGAGTTAGC

pR_ Φ_cro
GTCTTAGGGAGGAAGCCGTT

pF_Φ_cII
TGATCGCGCAGAAACTGATTTAC

pR_ Φ_cII
GACAGCCAATCATCTTTGCCA

pF_Φ_O
AAGCGAGTTTGCCACGAT

pR_ Φ_O
GAACCCGAACTGCTTACCG

pF_Φ_Q
GGGAGTGAGGCTTGAGATGG

pR_ Φ_Q
TACAGAGGTTCTCCCTCCCG

pF_Φ_R
GGGTGGATGGTAAGCCTGT

pR_ Φ_R
TAACCCGGTCGCATTTTTC

2.2. Phage Lytic Development after Prophage Induction

Bacteria lysogenic with tested phages were cultured in LB medium at 30°C to A₆₀₀ of 0.1. Two induction agents were tested: H₂O₂ (1 mM) and UV irradiation (50 J/m²; this dose was achieved by 20 sec incubation of the bacterial suspensions in Petri dishes under UV lamp hanged 17 cm above the laboratory table). At indicated times after induction, samples of bacterial cultures were harvested, and 30 μL of chloroform was added to 0.5 mL of each sample. The mixture was vortexed and centrifuged for 5 min in a microcentrifuge. Then, serial dilutions were prepared in TM buffer, and phage titer (number of phages per mL) was determined by spotting 2.5 μL of each dilution of the phage lysate on a freshly prepared LB agar (1.5%) or LB agar with 2.5 μg/mL chloramphenicol (according to a procedure described previously [30]), with a poured mixture of 1 mL of the indicator E. coli MG1655 strain culture and 2 mL of 0.7% nutrient agar (prewarmed to 45°C), supplemented with MgSO₄ and CaCl₂ (to a final concentration of 10 mM each). When full-plate titration was used, 0.1 mL of phage lysate dilutions was plated onto LB agar. Plates were incubated at 37°C overnight. Analogous experiment but without induction agents (control experiments), which allows estimation of effects of spontaneous prophage induction, was performed with each lysogenic strain. The relative phage titer, expressed as plaque forming units (pfu)/mL, was calculated by subtracting the values obtained in the control experiment from the values determined in the main experiment, and as a consequence it represents the ratio of phage titers in induced and noninduced cultures. Each experiment was repeated three times.

2.3. The S.O.S. Assay

The S.O.S. assay was performed using the SOS-ChromoTest Kit (Environmental Bio-Detection Products Inc.), following the manufacturer’s protocol and using provided 4-nitro-quinoline oxide (4-NQO) as a positive reference standard, and 1 mM H₂O₂ and UV irradiation (50 J/m²) as tested inducers of the S.O.S. response [31, 32]. In the case of UV light irradiation, the production of -galactosidase was evaluated immediately after the exposure, without 2 h incubation at 37°C (recommended by the manufacturer), as, without this modification, the visual detection of the blue color was not possible due to rapid S.O.S. response after UV irradiation. Before use, the SOS-ChromoTest bacterial strain (E. coli PQ37, provided with the kit) was lysogenized by following phages: λ, λΔexo-xis, , or Δexo-xis, according to procedure described above.

2.4. Preparation of RNA and cDNA from Bacteria

For the isolation of total RNA, the previously described [26] procedure was employed. Briefly, the prophage induction was performed with 1 mM H₂O₂ or UV irradiation (at the dose of 50 J/m²). Following induction, the samples were withdrawn at indicated times and the growth of bacteria was inhibited by the addition of NaN₃ (Sigma-Aldrich) to a final concentration of 10 mM. Total RNA was isolated from 10⁹ bacterial cells by using the High Pure RNA Isolation Kit (Roche Applied Science). Bacterial genomic DNA carryover was removed by incubation with TURBO DNase from TURBO DNA-free Kit (Life Technologies) for 60 min at 37°C, according to the manufacturer’s guidelines. To evaluate the quality and quantity of the isolated RNA, a NanoDrop spectrophotometer was employed, considering the absorbance ratio (which should be 1.8 ≤ A260/A280 ≤ 2.0). Moreover, band patterns of total RNA were visualized by electrophoresis. The absence of DNA from RNA samples was controlled by PCR amplification and by real-time PCR amplification (all analyzed genes were tested). RNA preparations were stored at −80°C. cDNA was obtained with Transcriptor Reverse Transcriptase and random hexamer primers (Roche Applied Science), using total RNA samples (1.25 μg) as templates. cDNA reaction mixtures were diluted 10-fold for use in real-time PCR.

2.5. Real-Time PCR Assay and Data Analysis

The patterns of genes’ expression were determined by quantitative real-time reverse transcription-PCR (qRT-PCR), using the LightCycler 480 Real-Time PCR System (Roche Applied Science) and cDNA samples from lysogenic bacteria. Transcripts of tested phage and bacterial genes were compared in parallel to 16S rRNA housekeeping gene (according to a procedure described previously [34]), whose expression was found to be constant. Primers were developed by Primer3web version 4.0.0 and produced by Sigma-Aldrich or GENOMED. The transcriptional analysis of phage and bacterial genes from lysogenic strains was performed with primers presented in Table 3. Real-time PCR amplifications were carried out for 55 cycles in 20 μL reaction volume, using LightCycler 480 SYBR Green I Master (Roche Applied Science) as a fluorescent detection dye. Reactions were performed in Roche 96-well plates containing 10 μL 2x SYBR Green I Master Mix, 6.25 ng/μL cDNA, and 200 nM of each gene-specific primer (Table 3). Relative quantification assays were performed with cDNA of 16S rRNA and phage/bacterial genes multiplex assay. All templates were amplified using the following program: incubation at 95°C for 5 min, followed by 55 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s. No template control was included with each run. The specificity of amplified products was examined by melting curve analysis immediately after the final PCR cycle and confirmed by gel electrophoresis. Each experiment was conducted in triplicate.

The relative changes in gene expression revealed by quantitative real-time PCR experiments were analyzed using the calibrator, normalizing relative quantification method with efficiency correction (called the E-Method). This method has been used and described in detail previously [26, 29, 35]. The values obtained at time 0 (representing the conditions of spontaneous prophage induction) were used as calibrators. Thus, the following equation was employed to calculate the final results:

where is target and is reference.

2.6. In Silico Analyses

The multiple sequence alignment was performed using the ClustalW algorithm available at the website http://www.genome.jp/tools/clustalw/. The Pfam protein families database [36], available at the website http://pfam.xfam.org/, was used to identify protein domains.

3. Results and Discussion

3.1. Deletion of the Exo-Xis Region Impairs but Not Prophage Induction after Treatment with Hydrogen Peroxide

Until now, all in vivo studies on effects of the exo-xis region on host or phage development were performed with the use of strains overexpressing genes from this region [21–24, 26]. In this work, we have constructed a series of bacteriophage and mutants with deletions of either the whole exo-xis region or individual genes or open reading frames (Table 1). When wild-type and prophages were induced by UV irradiation (employed in this work as positive control conditions causing effective prophage induction) or hydrogen peroxide treatment of the lysogenic cells, efficiencies of induction and further phage lytic development were comparable in both phages, though some differences were observed in the duration of the lag phase of the phage development (Figure 2). Induction of Δexo-xis mutant with UV irradiation was similar to that observed for the wild-type λ, and treatment with hydrogen peroxide caused only a slight delay in the mutant phage development. The decrease in the phage titer at later times of the experiments is characteristic for λ and most probably arises from adsorption of the progeny virions on fragments of disrupted cell envelopes [15, 24]. However, induction of Δexo-xis prophage by UV irradiation was less efficient than that of the wild-type , and induction of the mutant by hydrogen peroxide was severely impaired (Figure 2). More detailed analyses, based on the full-plate phage titration method, allowing detection of 10 pfu/mL (see Section 2 for details), indicated that the number of pfu per mL of Δexo-xis phage after induction with hydrogen peroxide was at the same range (10³/mL) as that measured without specific induction (i.e., representing efficiency of spontaneous prophage induction). Nevertheless, the titer of Δexo-xis measured at 240 and 360 min after induction was and , respectively, that is, still 3-4 times higher than that without induction, which was and , respectively (note that the titer of the wild-type after prophage induction was several orders of magnitude higher than that without induction, Figure 2).

(a)

(b)

Figure 2: Lytic development of bacteriophages (a) and (b), either wild-type (open symbols) or Δexo-xis (closed symbols), after induction of lysogenic E. coli MG1655 with UV irradiation (50 J/m², circles) or hydrogen peroxide (1 mM, triangles). The presented results are mean values from 3 independent experiments (biological samples), with error bars indicating SD. Statistically significant differences ( in t-test) between wild-type and Δexo-xis phages were found at times 270, 300, and 360 min of experiments with hydrogen peroxide and at 270, 300, 330, and 360 min of experiments with UV for and at times 120, 240, 270, 300, 330, and 360 min of experiments with hydrogen peroxide and at 270, 300, 330, and 360 min of experiments with UV for .

Deletions of individual genes and open reading frames from the exo-xis region in did not affect significantly the phage titer. However, such deletions resulted in delays in prophage induction by hydrogen peroxide (Table 4). Interestingly, when prophage induction was stimulated by UV irradiation, such effect was not observed, and in some cases even more rapid induction of the mutant prophages occurred. In bacteriophage λ, only slight effects of deletions of individual genes and open reading frames were detected (Table 4).

Table 4: Duration of the lag phase of the phage lytic development after prophage induction with either hydrogen peroxide (1 mM) or UV irradiation (50 J/m²).

Strain
The time range of the switch from lag to log phase

H₂O₂ (1 mM)
UV (50 J/m²)

MG1655 (λ)
60–90 min
60 min

MG1655 (λΔexo-xis)
90–120 min
30–60 min

MG1655 (λΔorfs)
60–90 min
30–60 min

MG1655 (λΔorf60a)
60–90 min
30–60 min

MG1655 (λΔorf63)
60–90 min
30–90 min

MG1655 (λΔorf61)
60–90 min
30–60 min

MG1655 (λΔorf73)
30–60 min
0–30 min

MG1655 (λΔea22)
60–90 min
30–60 min

MG1655 (λΔea8.5)
60–90 min
30–60 min

MG1655 (Φ)
60–90 min
150–180 min

MG1655 (ΦΔexo-xis)
a
150–180 min

MG1655 (ΦΔorfs)
150–180 min
150–180 min

MG1655 (ΦΔorf60a)
120–150 min
90–120 min

MG1655 (ΦΔorf63)
120–150 min
30–60 min

MG1655 (ΦΔorf61)
90–120 min
30–60 min

MG1655 (ΦΔorf73)
90–120 min
120–150 min

MG1655 (ΦΔea22)
120–150 min
30–60 min

The value was not determined due to a very low efficiency of prophage induction under these conditions (as shown in Figure 2).

We conclude that the genes and open reading frames from the exo-xis region play important roles in the regulation of lambdoid prophage induction, as deletions of the whole region or single loci caused significant changes in efficiency and timing of this process. The effects of mutations are more pronounced in Shiga toxin-converting phage than in λ and in lysogenic E. coli cells treated with hydrogen peroxide than in UV-irradiated ones. Thus, the exo-xis region seems to be particularly important for phage under conditions of the oxidative stress, the most likely conditions causing Shiga toxin-converting prophage induction during infection with EHEC.

3.2. Hydrogen Peroxide-Mediated Prophage Induction Is a RecA-Dependent Process

Efficient induction of lambdoid prophages is a RecA-dependent process due to a molecular mimicry between the phage cI repressor and the host-encoded LexA repressor which is self-cleaved after stimulation by the activated form of RecA protein under the S.O.S. response conditions [12–15]. Such a mimicry is well known for bacteriophage λ cI protein and LexA [12, 13], and we found that both domain structure and amino acid residues crucial for the self-cleavage are also conserved in cI repressor of phage (Figure 3) (note that cI sequence of is identical to that of another Shiga toxin-converting bacteriophage, 933 W [37]). Nevertheless, since RecA-independent induction of Shiga toxin-converting prophages has also been reported [16], we asked whether hydrogen peroxide-caused prophage induction depends on the activation of the S.O.S. response.

Figure 3: Alignment of amino acid sequences of E. coli LexA protein and cI repressors of bacteriophages and . Specific protein domains are indicated by grey background. Self-cleavage sites are underlined (two amino acid residues between which the cleavage occurs). The active sites of the peptidase domains are framed. Symbols under the sequence alignment indicate conserved sequence (), conservative mutations (:), semiconservative mutations (.), and nonconservative mutations ().

When testing H₂O₂- or UV-dependent induction of prophages λ, λΔexo-xis, , and Δexo-xis in recA13 mutant host, in the assays analogous to those presented in Figure 2, pfu/mL values were at the levels of those estimated for the uninduced controls (, , , and  pfu/mL for λ, λΔexo-xis, , and Δexo-xis, resp.). Therefore, we conclude that induction of the investigated prophages under conditions of the oxidative stress (treatment with hydrogen peroxide) strongly depends on RecA function. Indeed, in cells lysogenic for λ or and treated with UV light or hydrogen peroxide, efficient induction of the S.O.S. response was evident, as estimated with the SOS ChromoTest (Figure 4). Intriguingly, while induction of the S.O.S. response by hydrogen peroxide in λΔexo-xis lysogen was comparable to that in λ lysogen, the signal in the SOS ChromoTest in Δexo-xis lysogen was considerably weaker than in the analogous experiment with lysogen (Figure 4). No such difference could be observed in UV-irradiated bacteria (Figure 4).

Figure 4: Induction of the S.O.S. response in E. coli PQ37 lysogenic with , Δexo-xis, , or Δexo-xis, treated with 4-NQO (4-nitro-quinoline oxide, positive control), H₂O₂ (1 mM), or UV (50 J/m²), using the SOS ChromoTest. β-Galactosidase activity (identified by the blue spots) represents induction of the S.O.S. regulon. Alkaline phosphatase activity (identified by yellow spots) evaluates viability of tested bacteria. Quantification of β-galactosidase activity was performed by densitometry, using the ImageJ software (available at http://imagej.nih.gov/ij/index.html). The results (in arbitrary units reflecting value = 1 ascribed to samples with no inductor), presented as numbers inside the corresponding spots, are mean values from three measurements (with SD < 10% in each case). All these values were significantly ( in t-test) higher than that in the control experiments with no inductor. When Δexo-xis mutants were compared to wild-type phages, the only significant difference ( in t-test) occurred between and Δexo-xis lysogens induced with hydrogen peroxide.

3.3. Deletion of the Exo-Xis Region Negatively Influences Expression of Genes from the S.O.S. Regulon in Hydrogen Peroxide-Treated Lysogenic Bacteria

Since unexpected results were obtained in experiments with hydrogen peroxide-treated Δexo-xis lysogenic cells (Figure 4), we aimed to investigate the phenomenon of a less efficient induction of the S.O.S. response in more detail. Thus, expression of genes from the S.O.S. regulon was tested by reverse transcription quantitative real-time PCR in E. coli cells lysogenic for λ, λΔexo-xis, , and Δexo-xis and treated with hydrogen peroxide. In both λ and , deletion of the exo-xis region caused a significant reduction in the mRNA levels of most of the S.O.S. regulon genes relative to wild-type prophages, with exceptions of rpoS gene in both phages and ssb, uvrA, and ftsK genes in , especially at later times after the treatment (Figure 5). Interestingly, in the case of wild-type lysogenic cells, the enhanced expression of particular genes from the S.O.S. regulon persisted longer, in most cases until 16 min after induction, whereas in the deletion mutant it decreases after 4 min (Figure 5). The impairment in expression of genes from the S.O.S. regulon (in particular recA and lexA genes, encoding the main regulators of the S.O.S. response) in the absence of the exo-xis region was more pronounced in than in λ. Moreover, induction of the S.O.S. regulon occurred significantly earlier in and Δexo-xis lysogens than in cells bearing λ and λΔexo-xis prophages (Figure 5). These results might explain, at least partially, effects of deletions of exo-xis genes on prophage induction, demonstrated in Figure 2 and Table 4, particularly delayed induction of prophage devoid of certain genes and open reading frames, and less pronounced effects of their lack in λ than in .

Figure 5: Expression of genes from the S.O.S. regulon in E. coli MG1655 lysogenic with , Δexo-xis, , or Δexo-xis, at indicated times after treatment with 1 mM H₂O₂, as estimated by reverse transcription quantitative real-time PCR. The values obtained with untreated cells were used as calibrators and were subtracted from the values determined at particular time points; thus, the presented values indicate the induction of expression of tested genes. The presented results are mean values from 3 independent experiments (biological samples), with error bars indicating SD. The additional panel for E. coli () represents the results with different scale.

Indications that overexpression of some genes from the exo-xis region of λ can influence host cell cycle and DNA replication have been reported previously [21, 22]. Suggestions that some genes of prophage may affect host growth were also published [38]. However, the results described in this subsection demonstrate for the first time that the exo-xis region can significantly modulate one of global cellular responses, the S.O.S. response, after treatment with hydrogen peroxide.

3.4. Expression of Crucial Phage Genes Is Dramatically Decreased after Treatment of Lysogenic Cells with Hydrogen Peroxide in the Absence of the Exo-Xis Region in Prophage

Expression of phage genes, crucial for the regulatory processes and lytic development, has been tested under the same conditions as described in the preceding subsection. The specific conditions and time after addition of hydrogen peroxide into the cell culture at which samples were withdrawn were chosen on the basis of similar experiments reported previously [26]. Interestingly, different effects of the deletion of the exo-xis region were observed for phages λ and . In λ, deletion of genes and open reading frames located between exo and xis genes did not cause considerable effects on mRNA levels for xis, exo, and Q, whereas expression of int, cIII, N, cI, cro, cII, O, and R was enhanced upon treatment with hydrogen peroxide (Figure 6). Completely different results were obtained when and Δexo-xis lysogens were studied; namely, expression of all tested genes was drastically impaired in hydrogen peroxide-treated bacteria in the absence of the exo-xis region on the prophage (Figure 6).

Figure 6: Expression of selected bacteriophage genes in E. coli MG1655 lysogenic with or , either wild-type (blue columns) or Δexo-xis (yellow columns) at 160 min after treatment with 1 mM H₂O₂, as estimated by reverse transcription quantitative real-time PCR. The values obtained with untreated cells were used as calibrators and were subtracted from the values determined at particular time points; thus, the presented values indicate the induction of expression of tested genes. The presented results are mean values from 3 independent experiments (biological samples), with error bars indicating SD. The additional panel for E. coli () represents the results of Δexo-xis variant with different scale, due to very small values measured. Statistically significant differences (in t-test) are marked as follows: , , and .

While negative regulation of transcription from cII-dependent promoters by overexpression of the exo-xis region has been reported previously in phage λ [23], this study demonstrated for the first time significant effects of this region on expression of a battery of phage genes under conditions of the oxidative stress. The results presented in Figure 6 for phage λ are compatible with those published previously (though obtained with different methods) [23], as overexpression of the exo-xis region had opposite effects to those observed in its absence. On the other hand, severely impaired expression of all tested phage genes in Δexo-xis was unexpected. However, these results (Figure 6) can explain a strong defect in the induction of Δexo-xis prophage (and perhaps further lytic development) by hydrogen peroxide (Figure 2). Similarly, drastic differences between effects of Δexo-xis mutations on hydrogen peroxide-mediated prophage induction between λ and (Figure 2) can be ascribed to opposite regulation of expression of phage genes in the absence of the exo-xis region.

3.5. Effects of the Exo-Xis Region on Expression of Host and Phage Gene in UV-Irradiated Lysogenic Cells

Experiments analogous to those described in two preceding subsections were performed with lysogenic cells irradiated with UV. Interestingly, in both λ and phages, deletion of the exo-xis region caused only moderate effects on expression of most genes from the S.O.S. regulon (Figure 7), contrary to hydrogen peroxide-treated bacteria where the differences were significantly higher (compare Figures 5 and 7). The exceptions in UV-irradiated cells were rpoD, rpoH, and rpoS genes in λ and rpoH and rpoS in , whose expressions were at considerably lower level in the absence of the exo-xis region (Figure 7). One should also note that the induction of the S.O.S. regulon with UV irradiation was quicker than that with hydrogen peroxide. These results indicate that the influence of the exo-xis region on the S.O.S. response is particularly well pronounced under conditions of the oxidative stress.

Figure 7: Expression of genes from the S.O.S. regulon in E. coli MG1655 lysogenic with , Δexo-xis, , or Δexo-xis, at indicated times after UV irradiation (50 J/m²), as estimated by reverse transcription quantitative real-time PCR. The values obtained with untreated cells were used as calibrators and were subtracted from the values determined at particular time points; thus, the presented values indicate the induction of expression of tested genes. The presented results are mean values from 3 independent experiments (biological samples), with error bars indicating SD.

Unlike the S.O.S. regulon expression, levels of mRNAs of bacteriophage genes in UV-irradiated cells were affected similarly to those in hydrogen peroxide-treated lysogenic bacteria by the absence of the exo-xis region (Figure 8). Again, although some differences were observed between λ and λΔexo-xis, the differences between and Δexo-xis were dramatic. This indicates that the influence of the exo-xis region on expression of phage genes after prophage induction does not depend on the induction agent.

Figure 8: Expression of selected bacteriophage genes in E. coli MG1655 lysogenic with or , either wild-type (blue columns) or Δexo-xis (yellow columns) at 160 min after UV irradiation (50 J/m²), as estimated by reverse transcription quantitative real-time PCR. The values obtained with untreated cells were used as calibrators and were subtracted from the values determined at particular time points; thus, the presented values indicate the induction of expression of tested genes. The presented results are mean values from 3 independent experiments (biological samples), with error bars indicating SD. The additional panel for E. coli () represents the results of Δexo-xis variant with different scale, due to very small values measured. Statistically significant differences (in t-test) are marked as follows: , , and .

4. Conclusions

The exo-xis region is necessary for effective, RecA-dependent induction of Shiga toxin-converting bacteriophage under conditions of the oxidative stress. In hydrogen peroxide-treated E. coli, this region positively influences expression of the S.O.S. regulon in both and lysogens and expression of phage genes crucial for lytic development (particularly xis, exo, N, cro, O, Q, and R) in , but not in . Since the oxidative stress appears to be the major cause of induction of Shiga toxin-converting prophages during infections of human intestine by enterohemorrhagic E. coli (EHEC), the exo-xis region and/or products of its expression might be considered as potential targets for anti-EHEC drugs.

Conflict of Interests

The authors declare no conflict of interests.

Authors’ Contribution

Katarzyna Licznerska and Aleksandra Dydecka contributed equally to this work.

Acknowledgment

This work was supported by the National Science Center (Poland) Grant no. 2013/09/B/NZ2/02366 to Alicja Węgrzyn.

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