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
Infection of humans by enterohemorrhagic
In EHEC strains, Shiga toxins are encoded by genes (called
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 [
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 [
Schematic maps of the
Strain |
Genotype or relevant characteristics | Reference |
---|---|---|
|
|
[ |
|
MG1655 bearing |
[ |
|
MG1655 bearing |
This study, by recombination |
|
MG1655 bearing |
This study, by recombination |
|
MG1655 bearing |
This study, by recombination |
|
MG1655 bearing |
This study, by recombination |
|
MG1655 bearing |
This study, by recombination |
|
MG1655 bearing |
This study, by recombination |
|
MG1655 bearing |
This study, by recombination |
|
MG1655 bearing |
This study, by recombination |
|
MG1655 bearing Φ |
[ |
|
MG1655 bearing Φ |
This study, by recombination |
|
MG1655 bearing Φ |
This study, by recombination |
|
MG1655 bearing Φ |
This study, by recombination |
|
MG1655 bearing Φ |
This study, by recombination |
|
MG1655 bearing Φ |
This study, by recombination |
|
MG1655 bearing Φ |
This study, by recombination |
|
MG1655 bearing Φ |
This study, by recombination |
|
MG1655 but |
[ |
|
MG1655 |
This study, by lysogenization |
|
MG1655 |
This study, by lysogenization |
|
MG1655 |
This study, by lysogenization |
|
MG1655 |
This study, by lysogenization |
|
|
[ |
|
PQ37 bearing |
This study, by lysogenization |
|
PQ37 bearing |
This study, by lysogenization |
|
PQ37 bearing Φ |
This study, by lysogenization |
|
PQ37 bearing Φ |
This study, by lysogenization |
Bacteriophages
The deletion mutants were constructed as described previously [
Primers used for construction of
Primer name | Sequence (5′ |
---|---|
pF_ |
ATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTACAAAGCGAGGAATTAACCCTCACTAAAGGGCG |
|
|
pF_ |
ATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTACAAAGCGAGGAATTAACCCTCACTAAAGGGCG |
|
|
pF_ |
ATATCCGGGTAGGCGCAATCACTTTCGTCTACTCCGTTACAAAGCGAGGAATTAACCCTCACTAAAGGGCG |
|
|
pF_ |
CACAAAGCATCTTCTGTTGAGTTAAGAACGAGTATCGAGATGGCACATAGAATTAACCCTCACTAAAGGGCG |
|
|
pF_ |
AGAGAAACCAGGTATGACAACCACGGAATGCATTTTTCTGGCAGCGGGCTAATTAACCCTCACTAAAGGGCG |
|
|
pF_ |
ACATCATTGATTCAGCATCAGAAATAGAAGAATTACAGCGCAACACAGCAAATTAACCCTCACTAAAGGGCG |
|
|
pF_ |
GAAATTAACTCTCAGGCACTGCGTGAAGCGGCAGAGCAGGCAATGCATGAAATTAACCCTCACTAAAGGGCG |
|
|
pF_ |
ATGAGTATCAATGAGTTAGAGTCTGAGCAAAAAGATTGGGCGTTATCAATAATTAACCCTCACTAAAGGGCG |
|
|
pF_Φ |
TGGTAATGAAGCATCCTCACGATAATATCCGGGTAGGCACGATCACTTTCAATTACCTCACTAAAGGGCG |
|
|
pF_Φ |
TGGTAATGAAGCATCCTCACGATAATATCCGGGTAGGCACGATCACTTTCAATTACCTCACTAAAGGGCG |
|
|
pF_Φ |
TGGTAATGAAGCATCCTCACGATAATATCCGGGTAGGCACGATCACTTTCAATTACCTCACTAAAGGGCG |
|
|
pF_Φ |
TGCACAAAGCATCTCCTGTTGAATTAAGAACGAGTATCGGGATGGCACATAATTAACCCTCACTAAAGGGCG |
|
|
pF_Φ |
TTCTGGCAGCAGGCTTCATATTCTGTGTGCTTATGCTTGCCGACATGGGAAATTAACCCTCACTAAAGGGCG |
|
|
pF_Φ |
TGGCAGACCTCATTGATTCAGCATCAGAAATTGAAGAATTACAGCGCAACAATTAACCCTCACTAAAGGGCGG |
|
|
pF_Φ |
ATCAGGCACTGCGTGAAAAGGCAGAGAAAGCAACTAAAGGAAGCTACATCAATTAACCCTCACTAAAGGGCG |
Lysogenic strains were constructed according to the procedure described previously [
Primers used for PCR.
Primers for bacterial sequences | Primers for phage sequences | ||
---|---|---|---|
Name | Sequence (5′ |
Name | Sequence (5′ |
pF_recA | AGATTTCGACGATACGGCCC | pF_ |
TTTGATTTCAATTTTGTCCCACT |
pR_recA | AACCATCTCTACCGGTTCGC | pR_ |
ACCATGGCATCACAGTATCG |
pF_lexA | ATGGATGGTGACTTGCTGGC | pF_ |
TACCGCTGATTCGTGGAACA |
pR_lexA | TTCGTCATCAATACGTGCGAC | pR_ |
GGGTTCGGGAATGCAGGATA |
pF_ssb | ATCGAAGGTCAGCTGCGTAC | pF_ |
TGCCGTCACTGCATAAACC |
pR_ssb | CGACTTCTGTGGTGTAGCGA | pR_ |
TCTATCGCGACGAAAGTATGC |
pF_recF | CGATACCGGCGCTATACTCC | pF_ |
ATTCTTTGGGACTCCTGGCTG |
pR_recF | TTACGAACAGCTACGCCCG | pR_ |
GTAAATTACGTGACGGATGGAAAC |
pF_rpoD | GAATCTGAAATCGGGCGCAC | pF_ |
CTCGTGATTTCGGTTTGCGA |
pR_rpoD | GTCAACAGTTCAACGGTGCC | pR_ |
AAGCAGCAAATCCCCTGTTG |
pF_rpoH | GCTTTGGTGGTCGCAACTTT | pF_ |
ACCTCAAGCCAGAATGCAGA |
pR_rpoH | TCGCCGTTCACTGGATCAAA | pR_ |
CCAAAGGTGATGCGGAGAGA |
pF_rpoS | TTGCTCTGCGATCTCTTCCG | pF_ |
ATGCGGAAGAGGTAAAGCCC |
pR_rpoS | GAACGTTTACCTGCGAACCG | pR_ |
TGGAATGTGTAAGAGCGGGG |
pF_uvrA | GTCCATATCCGCCACTACCG | pF_ |
TCGCAATGCTTGGAACTGAGA |
pR_uvrA | TTACCCAACGTCTTGCCGAG | pR_ |
CCCTCTTCCACCTGCTGATC |
pF_ftsK | ACAAACCGTTTATCTGCGCG | pF_ |
AATTCTGGCGAATCCTCTGA |
pR_ftsK | ATCTTTACCCAGCACCACGG | pR_ |
GAATTGCATCCGGTTT |
pF_16SrRNA | CCTTACGACCAGGGCTACAC | pF_ |
TTCTGCGGTAAGCACGAAC |
pR_16SrRNA | TTATGAGGTCCGCTTGCTCT | pR_ |
TGCATCAGATAGTTGATAGCCTTT |
|
|
pF_ |
ATCGACCGTTGCAGCAATA |
|
|
pR_ |
GCTCGAACTGACCATAACCAG |
|
|
pF_Φ |
CAGTTGCCGGTATCCCTGT |
|
|
pR_ Φ |
TGAGGCTTTCTTGCTTGTCA |
pF_Φ |
TATCGCGCCGGATGAGTAAG | ||
pR_ Φ |
CGCACAGCTTTGTATAATTTGCG | ||
pF_Φ |
TGCCGTCACTGCATAAACC | ||
pR_ Φ |
TCTATCGCGACGAAAGTATGC | ||
|
|
pF_Φ |
ATTCTTTGGGACTCCTGGCTG |
|
|
pR_ Φ |
GTAAATTACGTGACGGATGGAAAC |
|
|
pF_Φ |
AGGCGTTTCGTGAGTACCTT |
pR_ Φ |
TTACACCGCCCTACTCTAAGC | ||
|
|
pF_Φ |
TGCTGTCTCCTTTCACACGA |
|
|
pR_ Φ |
GCGATGGGTGGCTCAAAATT |
|
|
pF_Φ |
CGAAGGCTTGTGGAGTTAGC |
|
|
pR_ Φ |
GTCTTAGGGAGGAAGCCGTT |
|
|
pF_Φ |
TGATCGCGCAGAAACTGATTTAC |
|
|
pR_ Φ |
GACAGCCAATCATCTTTGCCA |
|
|
pF_Φ |
AAGCGAGTTTGCCACGAT |
|
|
pR_ Φ |
GAACCCGAACTGCTTACCG |
pF_Φ |
GGGAGTGAGGCTTGAGATGG | ||
pR_ Φ |
TACAGAGGTTCTCCCTCCCG | ||
pF_Φ |
GGGTGGATGGTAAGCCTGT | ||
pR_ Φ |
TAACCCGGTCGCATTTTTC |
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
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 [
For the isolation of total RNA, the previously described [
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
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 [
The multiple sequence alignment was performed using the ClustalW algorithm available at the website
Until now, all
Lytic development of bacteriophages
Deletions of individual genes and open reading frames from the
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 ( |
60–90 min | 60 min |
MG1655 ( |
90–120 min | 30–60 min |
MG1655 ( |
60–90 min | 30–60 min |
MG1655 ( |
60–90 min | 30–60 min |
MG1655 ( |
60–90 min | 30–90 min |
MG1655 ( |
60–90 min | 30–60 min |
MG1655 ( |
30–60 min | 0–30 min |
MG1655 ( |
60–90 min | 30–60 min |
MG1655 ( |
60–90 min | 30–60 min |
MG1655 (Φ |
60–90 min | 150–180 min |
MG1655 (Φ |
a | 150–180 min |
MG1655 (Φ |
150–180 min | 150–180 min |
MG1655 (Φ |
120–150 min | 90–120 min |
MG1655 (Φ |
120–150 min | 30–60 min |
MG1655 (Φ |
90–120 min | 30–60 min |
MG1655 (Φ |
90–120 min | 120–150 min |
MG1655 (Φ |
120–150 min | 30–60 min |
We conclude that the genes and open reading frames from the
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 [
Alignment of amino acid sequences of
When testing H2O2- or UV-dependent induction of prophages
Induction of the S.O.S. response in
Since unexpected results were obtained in experiments with hydrogen peroxide-treated
Expression of genes from the S.O.S. regulon in
Indications that overexpression of some genes from the
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 [
Expression of selected bacteriophage genes in
While negative regulation of transcription from cII-dependent promoters by overexpression of the
Experiments analogous to those described in two preceding subsections were performed with lysogenic cells irradiated with UV. Interestingly, in both
Expression of genes from the S.O.S. regulon in
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
Expression of selected bacteriophage genes in
The
The authors declare no conflict of interests.
Katarzyna Licznerska and Aleksandra Dydecka contributed equally to this work.
This work was supported by the National Science Center (Poland) Grant no. 2013/09/B/NZ2/02366 to Alicja Węgrzyn.