Influence of the Escherichia coli oxyR gene function on lambda prophage maintenance

Abstract

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of lambda prophage. Here, we demonstrate that H2O2-mediated lambda prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a lambda lysogen in the presence of H2O2. On the other hand, stimulation of the p(M) promoter by cI857 overproduced from a multicopy plasmid was decreased in the DeltaoxyR mutant in the presence of H2O2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the p(M) promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the p(M) promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced lambda prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating lambda prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.

Fig. 1

Schematic map of the p _M-p _R promoter region of bacteriophage λ genome. The OR3 sequence is underlined and the putative OxyR binding site is framed

Fig. 2

λpapa prophage induction in MG1655 wild-type lysogenic strain (open symbols) and its ΔoxyR::kan derivative (closed symbols) after treatment with 1 mM (final concentration) hydrogen peroxide at time 0 (circles) or without such a treatment (squares). Cultures were grown with agitation of flask cultures (at 200 rpm) before addition of H₂O₂ and without agitation after the induction. Number of plaque forming units was estimated in each sample, and the results are expressed as number of phages per cell

Fig. 3

Binding of OxyR to the p _M promoter. a Comparison of the efficiency of OxyR binding to p _M and oxyR promoter regions. Gel mobility shift assays were performed as described in “Materials and methods” using the 258-bp PCR fragment encompassing pM and the 297-bp fragment of oxyR promoter region. Biding reactions were run on a 5% polyacrylamide gel. b EMSA analysis of OxyR binding to p_M promoter region. Gel mobility shift assays were performed as described in “Materials and methods” using 79-bp double-stranded oligos, containing wild-type (lanes 6–10) or mutated sequence (lanes 1–5) of a putative oxyR box. Reactions were run on a 6% polyacrylamide gel

Fig. 4

DNase I footprinting of OxyR binding to the p _M promoter region. OxyR protein, at indicated concentrations, was bound to a labeled DNA fragment, and DNase I digestion was performed as described in “Materials and methods”. Putative OxyR binding site was depicted. A site of enhanced DNA cleavage by DNase I, observed in the presence of OxyR, was marked by an arrow

Fig. 5

DNase I footprinting analysis of the cI repressor interaction with operator sites of p _M-p _R in the presence of the OxyR protein. OxyR, at indicated concentrations, was bound to a labeled DNA fragment. After 20 min incubation at 37°C, the cI repressor was added (as indicated), and incubation was continued for 10 min. DNase I footprinting was performed as described in “Materials and methods”. Operator sites for cI as well as OxyR binding site are marked. a and b represent different variants of the same experiment

Fig. 6

Primer extension analysis of the abundance of cI mRNA in oxyR ⁺ (open symbols) or ΔoxyR::kan (closed symbols) bacteria lysogenic for λ, either untreated (squares) or treated with 1 mM H₂O₂ at time 0 (circles). The analysis was performed as described in “Materials and methods”. Results shown are mean values ± SD

Fig. 7

Stimulation of the p _M promoter activity by the cI protein in the oxyR ⁺ host (open columns) and its ΔoxyR::kan derivative (closed columns) bearing the p _M-lacZ fusion and pGW857 plasmid. Bacteria were grown at 43°C and shifted to 30°C at time 0. At this time, hydrogen peroxide (HP) was added to half of each culture to final concentration of 1 mM. β-galactosidase activity was measured (in Miller units, MU) 30 min after the temperature shift. From obtained values, corresponding activities of β-galactosidase measured at time 0 (about 200–300 Miller units in all cases) were subtracted. Results presented are mean values from three experiments with SD indicated

Fig. 8

Neighbor-joining tree of repressor proteins of eight lambdoid coliphages: λ (GenBank protein identification number: AAA96581, H19B (AAD04644), 933 W (AAD25430), ϕ80 (CAA31471), HK022 (CAA34222), Nil2 (CAC95084), HK97 (NP_037735) and N15 (NP_046933), and five related bacteriophages infecting enterobacteria: L (a Salmonella typhimurium phage; CAA63999), ST104 (a Salmonella typhimurium phage; YP_006379), Sf6 (a Shigella flexneri phage; AAQ12228), PY54 (a Yersinia enterocolitica phage; CAD91799) and APSE-2 (a Candidatus Hamiltonella defense phage; ABA29387). The phages in which a potential OxyR binding site was identified were underlined, twice if the results of both methods coincided, once if a site was identified using only MatInspector, but not Target Explorer. A letter W marks the interior branches judged significant by weighted least squares likelihood ratio test (Sanjuan andWróbel 2005); the bootstrap values were also marked over the corresponding branches