SetR_ICE391, a negative transcriptional regulator of the integrating conjugative element 391 mutagenic response

Abstract

The integrating conjugative element ICE391 (formerly known as IncJ R391) harbors an error-prone DNA polymerase V ortholog, polV_ICE391, encoded by the ICE391 rumAB operon. polV and its orthologs have previously been shown to be major contributors to spontaneous and DNA damage-induced mutagenesis in vivo. As a result, multiple levels of regulation are imposed on the polymerases so as to avoid aberrant mutagenesis. We report here, that the mutagenesis-promoting activity of polV_ICE391 is additionally regulated by a transcriptional repressor encoded by SetR_ICE391, since Escherichia coli expressing SetR_ICE391 demonstrated reduced levels of polV_ICE391–mediated spontaneous mutagenesis relative to cells lacking SetR_ICE391. SetR_ICE391 regulation was shown to be specific for the rumAB operon and in vitro studies with highly purified SetR_ICE391 revealed that under alkaline conditions, as well as in the presence of activated RecA, SetR_ICE391 undergoes a self-mediated cleavage reaction that inactivates repressor functions. Conversely, a non-cleavable SetR_ICE391 mutant capable of maintaining repressor activity, even in the presence of activated RecA, exhibited low levels of polV_ICE391-dependent mutagenesis. Electrophoretic mobility shift assays revealed that SetR_ICE391 acts as a transcriptional repressor by binding to a site overlapping the −35 region of the rumAB operon promoter. Our study therefore provides evidence indicating that SetR_ICE391 acts as a transcriptional repressor of the ICE391-encoded mutagenic response.

1. Introduction

DNA damaging agents cause DNA replication blocking lesions that induce the expression of multiple genes whose gene products act to minimize the effects of the damage through DNA repair and, following extensive damage, DNA damage tolerance mechanisms. In Escherichia coli these mechanisms form the SOS response and their regulation is dependent on the interplay of the LexA and RecA proteins. LexA is the negative transcriptional regulator of more than 40 SOS response genes that function in DNA repair, mutagenesis, and the regulation of cell division [1–3]. As the representative member of the clan SF serine peptidases, LexA possesses a catalytic serine-lysine dyad capable of executing self-cleavage at the Ala84-Gly85 bond within LexA [4,5]. The process of LexA self-cleavage relies on an interaction with RecA. After DNA damage, DNA polymerase III encounters a DNA lesion that it is unable to traverse and dissociates from the damaged strand and reinitiates further downstream. The resulting single-stranded DNA gap is bound by RecA creating a nucleoprotein filament that is often referred to as RecA*. LexA binds avidly to RecA* [6] and in doing so, undergoes a self-cleavage reaction that inactivates LexA’s ability to serve as a transcriptional repressor and leads to the induction of the SOS response [7,8].

The SOS response is structured so as to react to a range of DNA damage by first initiating the expression of genes capable of processing DNA damage in an error-free manner, such as the genes responsible for nucleotide excision repair. Following extensive DNA damage, the error-free response is overwhelmed dictating expression of genes that make-up the mutagenic aspect of the SOS response. It is the affinity of LexA for its binding site, or LexA box, upstream of each SOS-regulated gene which allows for the temporal control of the SOS response; the higher affinity of LexA for the LexA box, the greater damage necessary for expression of the associated gene(s) [1,2]. Of the 40 or so LexA-regulated genes, three encode the catalytic subunits of DNA polymerases, polII (polB); polIV (dinB); and polV (umuC). All three polymerase have been shown to facilitate translesion DNA synthesis (TLS) [9–13]. polII and polIV generally do so in an error-free manner [11,14–17]. However, polV-dependent TLS is generally error-prone and is the major contributor to induced mutagenesis after DNA damage [18,19].

polV is a multi-subunit DNA polymerase. It is composed of two subunits of UmuD’, the RecA*-stimulated autodigestion product of UmuD [20–22], and one subunit of UmuC (UmuD’₂C) [23,24]. However, polV appears to have limited catalytic activity, at least in vitro [25]. The catalytic activity of polV is, however, dramatically stimulated in the presence of RecA* and ATP [25,26]. It is believed that a RecA molecule and ATP are transferred from RecA* to polV to form a higher order structure termed “polV Mut” (consisting of UmuD’₂C-RecA and ATP) that is the functionally and mutagenically active form of the enzyme that facilitates error-prone TLS [26–28].

Orthologs of umuDC, such as mucAB and rumAB, are also negatively regulated by LexA, require RecA*-stimulated self-cleavage of the UmuD-like protein and entail an interaction of the UmuD’- and UmuC-like proteins in a stoichiometry resembling UmuD’₂C (and probably RecA and ATP), in order to perform TLS [29–31]. Unlike the chromosomally encoded umuDC operon, mucAB and rumAB are readily transferred between species as components of mobile genetic elements. rumAB movement between species is mediated via the Integrative Conjugative Element (ICE), ICE391 (formerly known as IncJ R391), and much like the conjugative plasmid, R46, which harbors mucAB [32,33], it also includes the transfer of drug resistance genes. The rumAB operon is of unique interest because of the weak rumAB-mediated mutator activity promoted by the native ICE391 [34,35] when compared to the robust spontaneous and induced mutation frequency of the sub-cloned rumAB [29,31,36].

ICE391 is a member of the SXT/R391 family of ICEs [37]. All ICEs exist integrated into the host chromosome and encode all the genes necessary for conjugation and integration/excision, as there is no circular replicative form like that seen with conjugative plasmids. SXT and ICE391 are each composed of approximately 90 kb of DNA and are closely related elements demonstrating 95% nucleotide identity over 65 kb of DNA [38]. Although a greater knowledge of SXT biology exists, the degree of identity between these ICEs, for the most part, allows inference that ICE391 likely acts in a related manner. For example, regulation of the genes involved in conjugation, integration and excision are highly conserved between SXT and ICE391. The amino acid sequence of the SXT regulators SetC, SetD and SetR and the surrounding regulatory DNA sequences are 95–100% identical to those of ICE391. SetR_SXT (this designation will be used to differentiate SetR_SXT from the identical SetR_ICE391) is a homolog of the λcI-like repressor proteins and retains the amino acids needed for self-cleavage in the presence of RecA*. It is therefore not surprising that induction of the SOS response inactivates SetR_SXT repressor function, allowing for the expression of SetC and SetD, which are activators of conjugation, excision and integration [39,40]. SetR_SXT also appears to auto-regulate its own expression in a manner similar to the λcI repressor [41]. This exit strategy, during times of stress, is reminiscent of prophage excision from the host chromosome after DNA damage [42].

ICE391 harbors the LexA-regulated rumAB operon, which is responsible for the production of the DNA polV ortholog, RumA’₂B (polV_ICE391) [31]; however, the limited mutability of the native ICE391 compared to the sub-cloned rumAB operon [31,34,35], implicates other trans-acting regulatory factors, possibly encoded on ICE391, beyond the host LexA protein.

Our research has identified SetR_ICE391 as a strong candidate for the transcriptional repression of polV_ICE391 expression. Based on the role of SetR_SXT as a transcriptional repressor of setCD, and its homology to the cI-like repressors, including LexA, and the conserved nature of the operators bound by LexA and those bound by SetR_SXT, we investigated whether SetR_ICE391 might be involved in the negative regulation of the ICE391 encoded rumAB operon.

2. Materials and methods

2.1. Bacterial strains and plasmids

Bacterial strains and plasmids used in this study are listed in Table 1. The SetR_ICE391 expressing plasmid, pAMG1, was constructed by amplifying setR via PCR from ICE391 with the primers SetRNdeI5′ (5′-ATC CAC ATA TGA AAA CTT TAT CCG AAC G-3′, the setR start codon is in bold and the NdeI site is underlined) and SetRBamHI3′ (5′-AAA GGA TCC CTC TCA GCG TGC GCC AAT GC-3′, BamHI site underlined) (Sigma). The resulting ~730bp DNA fragment was digested with NdeI and BamHI and the setR_ICE391 fragment was cloned into the similarly digested vector pMal-p2X (New England Biolabs) to form the IPTG-inducible SetR_ICE391 producing plasmid pAMG1. The SetR_ICE391 over-producing plasmid, pDH9, was generated by synthesizing an E. coli codon optimized setR_ICE391 gene (Genscript) and sub-cloning the synthesized gene into NdeI and XhoI restriction enzyme sites of pET22b+ (EMD Millipore). Plasmids pRW154 (umuDC), pRW144 (mucAB) and pRW290 (rumAB) have been described elsewhere [36,43]. pRLH421 is a low-copy number plasmid harboring an ~ 21.5 kb partial EcoRI digest of episomal ICE391 (Genbank U13633). A 493 bp PmeI to Bsu36I setR_ICE391 gene fragment (Genscript) encoding the SetR_ICE391(G94R) non-cleavable variant was sub-cloned into pRHL421 to generate pJM1300. pJM1317 and pJM1318 were constructed by cloning either the wild-type setR_ICE391 gene, or the setR_ICE391(G94R) gene (Genscript) into pRW290 from PstI to BamHI.

Table 1.

E.coli strains and plasmids used in this study.

Strain	Relevant Genotype	Source or Reference
RW120a	ΔumuDC596::cat	[36]
RW518a	ΔumuDC596::cat lexA3(Ind−)	LGI stocks
RW546a	ΔumuDC596::cat lexA51 (Def)	[31]
RW570a	ΔumuDC596::ermGT lexA51 (Def)	LGI stocks
MVG114a	ΔumuDC596::ermGT lexA51 (Def) recA718	LGI stocks
RW1290	λ(DE3) ΔumuDC596::ermGT ΔdinB61::ble ΔpolB::Spec ΔrecA306 srlD300::Tn10	LGI stocks
Plasmid	Relevant Characteristics	Source or Reference
pRW154	Low-copy-number, SpcR, encoding umuDC	[36]
pRW144	Low-copy-number, SpcR, encoding mucAB	[36]
pRW290	Low-copy-number, SpcR, encoding rumAB	[43]
pRLH421	Low-copy-number, SpcR, ~21.5 kb fragment of ICE391	Genbank: U13633
pJM1300	Low-copy-number, SpcR, ~21.5 kb fragment of ICE391 with setRICE391(G94R)	This study
pJM1317	Low-copy-number, SpcR, encoding rumAB and wild-type setRICE391	This study
pJM1318	Low-copy-number, SpcR, encoding rumAB and setRICE391(G94R)	This study
pDH9	Medium-copy-number, AmpR, expressing SetRICE391 from an IPTG-inducible T7 promoter	This study
pAMG1	Medium-copy-number, AmpR, expressing SetRICE391 from an IPTG-inducible Ptac promoter	This study

^aFull genotype: thr-1 araD139 Δ(gpt-proA)62 lacY1 tsx-33 glnV44 galK2 hisG4 rpsL31 xyl-5 mtl-1 argE3 thi-1 sulA211.

Where noted, the following antibiotics were used for selection; Zeocin (25 μg ml⁻¹), Tetracycline (15 μg ml⁻¹), Spectinomycin (50 μg ml⁻¹) and Ampicillin (100 μg ml⁻¹).

2.2. Qualitative analysis of spontaneous reversion of the hisG4(oc)

The E. coli strains were transformed with plasmids expressing SetRICE391 (pRLH421, pJM1317, pAMG1), the non-cleavable SetRICE391(G94R) (pJM1300, pJM1318), RumAB (pRW290) or the cloning vector (pMal-p2X). The data reported in Fig. 3 also included using compatible low copy, spectinomycin resistant plasmids expressing UmuDC (pRW154) or MucAB (pRW144) to assess the specificity of SetR_ICE391 activity. Three antibiotic resistant isolates from each transformation were grown in LB medium containing the proper antibiotics overnight at 37 °C. Five hundred microliters of each overnight culture were centrifuged and the pellet suspended in an equal volume of SM buffer. Next, spontaneous mutagenesis was determined by plating 100μl in triplicate on Davis and Mingioli minimal agar plates [44] plus glucose (0.4% w/v); agar (1.0% w/v); proline, threonine, valine, leucine, and isoleucine (all at 100 μg ml⁻¹); thiamine (0.25 μg ml⁻¹); and containing a trace amount of histidine (1 μg ml⁻¹). Plates were incubated at 37 °C for three days. The results presented in Figs. 2, 3 and S1 represent the average number of His⁺ mutants from nine plates per cultured transformant (± standard error of the mean [SEM]).

2.3. Visualization of cellular levels of RumA

A 1:100 dilution in fresh LB medium, plus appropriate antibiotics, of the overnight cultures used in the spontaneous reversion of hisG4(oc) assay (see above) were incubated at 37 °C until they reached early exponential phase. A 1.0 ml aliquot of each culture was centrifuged, and the resulting cell pellet was suspended in 4 x SDS sample buffer [40% glycerol (v/v), 9.2% SDS (w/v), 4 mM DTT, 250 mM Tris–HCl (pH 6.8), and 0.2% (w/v) bromophenol blue]. Equal protein concentrations of the aliquots were determined using the Pierce BCA Protein Assay (ThermoFisher Scientific) and electrophoresed on 12% SDS-polyacrylamide gels. Next, proteins were transferred to an Immobilon P membrane (Millipore) and subsequently incubated with polyclonal antibodies directed against the RumA protein (Covance). Visualization of the transferred RumA protein was performed on Kodak BioMaxMR film using the BioRad Immun-Star AP substrate.

2.4. Phylogenetic analysis of transcriptional repressors

A multiple sequence alignment of the core region of the SetR protein from ICE391 and various other homologous proteins were performed using the ClustalW algorithm in MacVector (version 14.0.6). The alignment was exported from MacVector as a Phylip file. This Phylip file was imported into the SplitsTree4 (version 4.14.4) and an unrooted phylogenetic tree was generated by setting the distance method to BioNJ.

2.5. Expression and purification of native SetR_ICE391

The ICE391 SetR protein was overproduced using the T7 protein expression system (EMD Millipore). Overproduced SetR_ICE391 was purified using protocols previously developed for the purification of LexA and DinR proteins [45,46]. Strain RW1290 harboring pDH9 was grown overnight at 37 °C in LB broth containing 100 μg ml⁻¹ ampicillin. Cells were diluted 100-fold in LB broth containing ampicillin and grown at 37 °C. At an OD_{600 nm} of approximately 0.5, IPTG was added to a final concentration of 1 mM, and growth continued at 37 °C for an additional 2 h, after which time cells were harvested by centrifugation at 2600 x g for 10 min at 4 °C. The cell pellet was resuspended in RS buffer [50 mM Tris (pH 7.8) and 10% w/v sucrose] in 1/25 the original culture volume and stored at −80 °C. Cells were slowly thawed on ice and lysed via sonication. The resulting material was centrifuged at 185,500 x g for 1 h. The supernatant was collected, and ammonium sulfate added to a final concentration of 35% (w/v). The precipitate was centrifuged at 185,500 x g for 30 min and the supernatant was collected. Ammonium sulfate was then added to the supernatant to a final concentration of 50% (w/v). The precipitate was centrifuged, and the pellet resuspended in buffer B [20 mM potassium phosphate (pH 7.0), 0.1 mM EDTA, 10% glycerol (v/v), and 1 mM DTT] containing 250 mM KCl and dialyzed against the same buffer overnight. Immediately before loading on a phosphocellulose column (Whatman, Clifton, NJ), the dialysate was diluted in buffer B to a final concentration of 100 mM KCl. The phosphocellulose column was washed with two column volumes of buffer B containing 100 mM KCl and SetRICE391 was eluted with a linear gradient of 100–500 mM KCl in buffer B. Fractions containing SetR_ICE391 (as judged by visual inspection of Coomassie-stained gels) were pooled and applied to a hydroxyapatite column (Bio-Rad, Hercules, CA). The column was washed with two column volumes of buffer C [0.1 mM EDTA, 10% glycerol (v/v), and 1 mM DTT] containing 50 mM KHPO₄ (pH 7.0). SetR_ICE391 was eluted with a linear gradient of 50–300 mM KHPO₄ in buffer C. Fractions containing highly purified SetRICE391 were pooled and stored at −80 °C after increasing the glycerol concentration to 20% (v/v).

2.6. SetR_ICE391 in vitro cleavage reactions

Autodigestion reactions of purified SetR_ICE391 occurred at 37 °C in 50 mM CAPS-NaOH (pH 10.0). Each reaction mixture (50 μl) contained 3 μg of SetR_ICE391. Reactions were terminated at the appropriate times by the addition of 4 x SDS sample buffer and rapidly freezing the samples on dry ice.

RecA-mediated cleavage of SetR_ICE391 was based upon a previously described procedure for the RecA-mediated cleavage of UmuD [21]. Standard reaction mixtures (50 μl) contained 10 mM Tris–HCl (pH 8.0), 10 mM MgCl₂, 0.1 μM UTT48 DNA oligo (5′-TCG ATA CTG GTA CTA ATG ATT AAC GAA TTA AGC ACG TCC GTA CCA TCG-3′), 1 mM ATPγS, 2 μg of purified E. coli RecA (New England Biolabs), and 3 μg of SetRICE391. Reactions were incubated at 37 °C for the appropriate times and terminated by adding 4 x SDS sample buffer and freezing on dry ice.

The products of the autodigestion and RecA-mediated cleavage reactions were subsequently subjected to electrophoresis in SDS-polyacrylamide gel electrophoresis (PAGE) gels containing 4–12% polyacrylamide. Proteins were visualized after staining gels with Coomassie brilliant blue R-250.

2.7. Electrophoretic mobility shift gel assays

The 123 bp region upstream of SetR_ICE391 was generated by annealing two complementary synthetic oligonucleotides, corresponding to the intergenic regions between the croS_ICE391 and setR_ICE391 genes (Fig. 6A). A 194 bp PCR amplicon containing the SetR_ICE391 binding site (s) upstream of the rumAB operon was generated using primers RumF; 5′-GCG AGC TAT CCC CAC ATC TA-3′ and RumR; 5′-CTA CGG CCT ATC AGC GAG AC-3′ (Genscript, Piscatway, NJ) and the rumAB operon–containing plasmid, pRW290, as a template. Similarly, the SetR_ICE391 binding site mutant used in Fig. 7, was initially synthesized as a 205 bp EcoRI-HindIII fragment (Genscript) that was cloned into pUC57 and subsequently amplified using the RumF and RumR PCR primers. DNAs were 5′ end labeled with [γ-³²P] ATP (Perkin Elmer, 3000 Ci/mmol) using T4 polynucleotide kinase (Roche). The purity of the probes was analyzed on a 20% polyacrylamide gel. Labeled probes had an average specific activity of 2000 cpm/fmol of DNA.

DNA-binding assay and DNA band-shifts were modifications of the method used in Winterling et al., 1996 [46]. Reaction mixtures containing labeled probe (~10,000–30,000 cpm μl⁻¹) and purified SetR_ICE391, or LexA protein, ranging from 0 to 200 nM were incubated at room temperature for 25 min in binding buffer (150 mM NaCl, 20 mM Tris–HCl [pH 7.5], 0.2 mM EDTA, 1.0 mM MgCl₂, 5% glycerol [v/v], 50 μg ml⁻¹ bovine serum albumin [BSA], 1 mM DTT) with a final reaction volume of 20 μl each. Binding assays were performed multiple times with and without polydIdC as a competitor of nonspecific binding. However, we found that the presence or absence of polydIdC did not influence the outcome of the results. These results were expected, since the EMSAs were performed with purified proteins, rather than cell extracts [47]. Protein-DNA complexes were separated in native polyacrylamide gels (6% acrylamide). Gels were dried and subsequently exposed to Kodak Biomax XAR film for appropriate periods of time.

3. Results

3.1. Phylogenetic relationship of SetR_ICE391 to λcI-like repressors

Characterization of the ICE391 rumAB operon identified a DNA polV ortholog that belongs to an evolutionary distinct subfamily of the polV-like Y-family polymerases [29]. Relative to the other cloned polV orthologs characterized, polV_R391 demonstrates a higher frequency of spontaneous mutagenesis and transversion mutations [31]. polV_ICE391 also promotes much higher induced mutation frequencies than that found for MucA′₂B (polR1), another member of the polV-like family, that has historically played a major role in increasing the sensitivity of the “Ames Test” used to detect carcinogens [48]. Interestingly, the high mutation frequency of polV_ICE391 is only evident with the sub-cloned operon and is not seen with the native ICE391 [31,34,35].

ICE391 is a mobile genetic element capable of transfer between different species of bacteria; therefore, it is plausible that ICE391 carries factors necessary to minimize aberrant polV_ICE391-mediated mutagenesis. We chose to address the potential role of SetR_ICE391 in limiting expression of the rumAB operon since SetR_ICE391 is identical at the amino acid level to the repressor of SXT excision and transfer, SetR_SXT [38].

The cI-like family of transcriptional repressors are represented by chromosomally encoded members, as well as by those carried on mobile genetic elements. Phylogenetic analysis generated from multiple alignments of the core amino acid sequences of the cI-like repressor sequences separated the chromosomal E. coli LexA and B. subtilis DinR proteins from the bacteriophage cI-like proteins and SetR_ICE391 (Fig. 1A). Indeed, SetRICE391 was more closely aligned with the bacteriophage λimm434 cI and P22 c2 repressors (Fig. 1A). All transcriptional repressors subject to RecA-mediated self-cleavage, including SetR_ICE391, possess a conserved alanine-glycine cleavage site and a serine-lysine dyad responsible for carrying out the activated RecA-mediated self-cleavage reaction (Fig. 1B). As with the identical SXT SetR protein, SetR_ICE391 is predicted to possess the amino-terminal helix turn helix DNA binding domain of the cI-like repressors [40].

Fig. 1.

Phylogenetic analysis of SetR_ICE391 related transcriptional repressors. A. An unrooted phylogenetic tree indicating the evolutionary relatedness of the SetR transcriptional repressor from ICE391 and other various related transcriptional repressors. As can be seen, SetR_ICE391is more closely related to cI-like repressors than LexA-like repressors. B. Alignment of core residues of repressors. A ClustalW multiple sequence alignment of the core binding region of the SetR protein and other related transcriptional repressors. Protein sequences were obtained from the following Genbank sequence files: NP_418467 (Escherichia coli LexA); WP_019714520 (Bacillus subtilis DinR); NP_040628 (λ phage cI); WP_000028394 (λ P434 cI); WP_001095982 (P22 c2); AY090559 (ICE391 orf96 SetR). Residues that are identical or highly conserved are shaded and blocked together. A consensus sequence is shown below the core repressor protein sequences. The Ala-Gly cleavage site is indicated with an arrow and the asterisks identify the conserved Ser and Lysine residues required for cleavage.

3.2. Expression of rumAB-dependent mutagenesis is LexA regulated

Ideally, the role of SetRICE391 in rumAB-dependent mutagenesis would be best studied in the context of the entire ICE391. However, previous work on SXT revealed manipulation of setR_SXT in a cell harboring SXT was deleterious to the host cell [39]. In order to address the role of SetRICE391 in regulating rumAB-dependent mutagenic activity, we utilized pRLH421, a plasmid construct containing an ~21.5 kb fragment of ICE391 cloned into the low-copy number plasmid, pGB2 [36]. The ~88.5 kb ICE391 normally integrates into the 5′ end of the prfC gene in the E.coli genome [49]. setR_ICE391 is located at the very 3′end of the linear ICE391 (bp 87561–88208). However, it is evident that pRLH421 was generated by a partial EcoRI digest of episomal ICE391, since the ~21.5 kb ICE391 insert contains sequences from both the 5′ (bp 1–20241) and 3′ (bp 87297–88532) ends of the linear ICE391 and includes numerous open reading frames including the rumAB operon and setR_ICE391 (Fig. 2 A).

Fig. 2.

LexA and SetR regulation of polV_ICE391-dependent spontaneous mutagenesis. A: Location of the 29 open reading frames encoded by the ~21.5 kb ICE391 fragment cloned into the low-copy number plasmid, pRLH421. B: Arrangement of setR_ICE391 and rumAB_ICE391 in the low copy number plasmid, pJM1317. C: Spontaneous mutagenesis promoted by pRLH421 (wild-type setR_ICE391) (Red), or pJM1300 (as pRLH421, but expressing setR_ICE391(G94R) (yellow) in lexA⁺, lexA(Ind⁻) and lexA(Def) strains. D: Spontaneous mutagenesis promoted by pRLH421 (wild-type SetR_ICE391) (Red), or pJM1300 (as pRLH421 but expressing setR_ICE391(G94R) (yellow) in a recA718 lexA(Def) strain. E: Spontaneous mutagenesis promoted by pRW290 (rumAB without setR_ICE391) (Red), pJM1317 (as pRW290, but expressing wild-type setR_ICE391) (yellow), or pJM1318 (as pJM1317, but expressing setR_ICE391(G94R) (green) in lexA⁺, lexA(Ind⁻) and lexA(Def) strains. F: Spontaneous mutagenesis promoted by pRW290 (rumAB without setR) (Red), pJM1317 (wild-type setR_ICE391) (yellow), or pJM1318 (as pJM1317, but setR_ICE391(G94R) (green) in a recA718 lexA(Def) strain.

To assess the mutagenic capacity of the rumAB operon in pRLH421, the levels of spontaneous mutagenesis were measured via a qualitative mutagenic assay that measures the reversion of the hisG4 ochre allele [43] The rumAB-mediated mutagenic activity in the strains expressing different lexA alleles was consistent with LexA-regulated rumAB expression. The number of histidine revertants per plate was greatest in the strain lacking a functional LexA [lexA51(Def)] while the strain expressing a non-cleavable LexA protein [lexA3(Ind⁻)] produced the fewest histidine revertants per plate (Fig. 2C). Because the wild-type lexA⁺ strain maintains a low basal rate of cleavage, the number of histidine revertants was intermediate of that seen for the lexA51(Def) strain and the non-cleavable lexA3(Ind⁻) strain (Fig. 2C). LexA regulation of the rumAB operon is therefore consistent with the identification of a potential LexA-binding site in the promoter region of the rumAB operon [29].

3.3. A non-cleavable G94R mutant of SetR_ICE391 abrogates rumAB-dependent mutagenesis in a recA718 lexA(Def) strain

Interaction of RecA with single stranded DNA formed following a DNA damaging event activates RecA (RecA*) thus facilitating the self-cleavage of repressors, such as the E. coli LexA repressor and B. subtilis DinR repressor, thereby leading to the induction of the SOS response [45,50]. RecA*-mediated self-cleavage has also been described for the prophage repressors of numerous temperate bacteriophage [7,51,52]. Although non-cleavable mutants of these repressors span the entire gene sequence, a substantial number of these mutants impact the cleavage site, or the catalytic serine-lysine dyad [53,54]. To determine if SetRICE391 cleavage is required for derepression of rumAB, we constructed a plasmid similar to pRLH421, designated pJM1300, expressing a mutant of SetR_ICE391 at the highly conserved cleavage site of the transcriptional repressors that we predicted would render the protein non-cleavable. Based on the inducible minus mutations previously characterized in the bacteriophage λcI repressor [53], we changed the Gly94 residue to Arg94 (G94R). The SetR_ICE391(G94R) expressing plasmid was then transformed into strains expressing the different lexA alleles described above. As expected, the pattern of mutagenic activity for the SetR_ICE391(G94R) expressing plasmid mimics that of the wild-type SetR_ICE391 (Fig. 2C).

Inactivation of SetR_ICE391 was demonstrated in the E. coli strain MVG114 [recA718 lexA51(Def) ΔumuDC596::ermGT hisG4]. The recA718 allele produces an activated RecA* protein when expressed in a LexA defective background, or following DNA damage to the cell [55]. Due to the proficient co-protease activity of RecA718 and possible inactivation of the SetR_ICE391 protein, plasmid encoded SetR_ICE391 is incapable of reducing RumAB activity in the recA718 strain (Fig. 2D). This was in dramatic contrast to the non-cleavable SetR_ICE391(G94R) mutant, which is largely insensitive to the activated RecA718 protein, and exhibited reduced levels of rumAB-dependent spontaneous mutagenesis (Fig. 2D). These observations support the hypothesis that SetR_ICE391 cleavage is required for maximal rumAB expression and polV_ICE391 mutagenesis-promoting activity.

Given the phylogenetic relationship of SetRICE391 to λcI-like transcriptional repressors, we considered the possibility that SetR_ICE391 might help attenuate expression of the RumAB proteins. To address this issue, we examined the intracellular levels of RumA expressed in the presence and absence of SetR_ICE391 (Supplementary Fig. 1A, B). Indeed, intracellular levels of RumA were substantially lower in the presence of SetR_ICE391 than in its absence (Supplementary Fig. 1A, B). Therefore, in the presence of SetR_ICE391 the intracellular levels of RumA (and most likely RumB as well) are reduced, which in turn, impacts the extent of rumAB-mediated spontaneous mutagenesis.

The ~21.5 kb DNA fragment from ICE391 cloned into plasmids pRLH421 and pJM1300 include numerous open reading frames, in addition to the rumAB operon and setR_ICE391 gene, that may influence polV_ICE391 mutagenesis-promoting activity. In order to definitively assess the role of setR_ICE391 in rumAB expression, we constructed low-copy plasmids that were devoid of these multiple open reading frames (Fig. 2B). Expression of either setR_ICE391 (pJM1317), or setR_ICE391(G94R) (pJM1318) in the presence of the rumAB operon, as well as expression of rumAB in the absence of setRICE391 (pRW290, see [43]) leads to comparable numbers of histidine revertants in both recA⁺ lexA⁺ and recA⁺ lexA3(Ind⁻) strains (Fig. 2E). However, the impact of SetR_ICE391 on polV_ICE391 mutagenesis-promoting activity is fully evident in the recA⁺ lexA51(Def) strain. The presence of setR_ICE391 or setR_ICE391(G94R) leads to a greater than four-fold reduction in histidine revertants compared to the plasmid expressing just rumAB (Fig. 2E). Moreover, expression of these plasmids in a recA718 lexA51(Def) strain further reinforces the idea that cleavage of SetR_ICE391 is required for maximal rumAB expression (Fig. 2F).

3.4. SetR_ICE391 down-regulates ICE391-encoded rumAB, but not related polV-orthologs

Characterization of SetR_SXT involvement in the regulation of SXT excision and transfer previously identified four comparable 14-bp SetR_SXT binding sites with partial dyad symmetry, each containing an 8-bp AT-rich spacer region. Indeed, SetR_SXT binding at the four operators inhibits expression from the P_L promoter responsible for the transcription of setCD, which are activators of SXT excision and transfer, and from the P_R promoter for setR_SXT [40,41]. Therefore, the reduction in spontaneous mutagenesis evident in the presence of SetR_ICE391 (Fig.2D) is most likely the result of SetR_ICE391 repressor activity upon the regulatory region upstream of rumAB. We addressed the specificity of SetR_ICE391 for the plasmid encoded rumAB operon (pRW290) by measuring the level of spontaneous mutagenesis of the E.coli umuDC (pRW154) and mucAB (pRW144) operons in the recA⁺ lexA51(Def) Δ(umuDC)596::ermGT hisG4 background (RW570). Analysis of the levels of spontaneous mutagenesis promoted by polV_ICE391, E.coli polV and R46 encoded polRI, reveals that SetR_ICE391 specifically down-regulates only ICE391-encoded rumAB activity and has minimal effect on the ability of either polV or polRI to promote spontaneous mutagenesis in vivo (Fig. 3).

Fig. 3.

Specificity of SetR_ICE391. Mutagenesis promoted by rumAB, mucAB or umuDC in the presence or absence of SetR_ICE391 were assayed in RW570 (hisG4(Oc) lexA51(Def) ΔumuDC596::ermGT) harboring either pAMG1 (SetR_ICE391, basal level expression from Ptac) or pMal-p2X (vector) and the individually transformed compatible plasmid pRW290 (rumAB), pRW144 (mucAB) or pRW154 (umuDC). All three polV operons were under control of their natural LexA-regulated promoter. Fresh transformants were cultured overnight in LB and processed to measure reversion to histidine prototrophy. The results presented represent the average number of His⁺ mutants from nine plates per strain (± standard error of the mean [SEM]).

3.5. SetR_ICE391 undergoes self-cleavage in the presence of RecA or under alkaline conditions

SetR_ICE391 possesses the necessary amino acid residues to undergo a RecA*-mediated self-cleavage reaction (Fig. 1B). The fact that plasmid encoded SetR_ICE391 could only suppress rumAB-mediated mutagenesis in a recA⁺ strain, and not in a recA718 strain, is a strong indicator that like LexA and cI-like proteins, SetR_ICE391 is also subject to a RecA-mediated cleavage reaction. We wanted to directly test this hypothesis and to do so we first overproduced and purified the native (untagged) SetR_ICE391 protein in E.coli, using the methodology and protocols (see Materials and methods) that were previously used to purify E.coli LexA and B.subtilis DinR proteins [2,45]. Using this strategy, SetR_ICE391 was purified to greater then 95% homogeneity (Fig. 4).

Fig. 4.

Purification of native SetR_ICE391. SetR_ICE391 was purified to greater than 95% homogeneity as described in Materials and methods. Each lane of the gel represents fractions taken at each step of the purification process. (U) RW1290/pDH9 in the absence of inducing treatment; (I) RW1290/pDH9 induced with 1 mM IPTG for 2 h; (Am) Soluble cell extract precipitated with 35–50% ammonium sulfate; (PC) Pooled SetRICE391 fractions after elution from the phosphocellulose column; (HA) Pooled SetR_ICE391 fractions after elution from the hydroxyapatite column. The position of SetR_ICE391 is marked on the right of the gel and the positions of molecular weight markers (in kDa) are indicated on the left.

The highly purified SetR_ICE391 was then incubated with RecA, single-stranded DNA as a polynucleotide cofactor, and ATPγS as a mononucleotide cofactor in an in vitro cleavage reaction (Fig. 5). Reactions were terminated at different time points and samples separated by SDS-PAGE and visualized after staining the gel with Coomassie Blue dye. As early as two hours into the reaction the SetR_ICE391 C-terminal fragment generated by the RecA-mediated self-cleavage at the Ala-Gly cleavage site begin to appear and by 24 h almost the entire SetR_ICE391 sample had been cleaved (Fig. 5). We can therefore conclude that SetR_ICE391 does indeed undergo a RecA*-mediated cleavage reaction.

Fig. 5.

Self-cleavage of SetR_ICE391. Left hand panel, RecA-mediated cleavage of SetR_ICE391. SetR_ICE391 (3 μg) was incubated with activated RecA (2 μg) at 37 °C for the number of hours indicated at the top of the lanes. The products of the reaction were separated by SDS-PAGE (4–12%) and the proteins were visualized by staining with Coomassie blue. Migration positions are indicated for RecA, intact SetR_ICE391, and the C-terminal (SetR-C) cleavage fragment. Right hand panel, Autodigestion of SetR_ICE391. SetR_ICE391 (3 μg) was incubated in 50 mM CAPS-NaOH (pH 10.0) at 37 °C for the number of hours indicated at the top of each lane. The products of the reaction were separated by SDS-PAGE (4–12%) and the proteins visualized by staining with Coomassie blue. Migration positions are indicated for intact SetR_ICE391 and C-terminal (SetR-C) cleavage fragment.

The LexA and cI-like repressors also possess the ability to undergo RecA-independent auto-catalytic cleavage when incubated at alkaline conditions [7]. To determine if the SetRICE391 cleavage is mechanistically similar to LexA and cI-like proteins, self-cleavage of the purified SetRICE391 protein was assayed in the absence of RecA* at pH 10.0. Under these conditions, LexA undergoes rapid self-cleavage, whilst that of λcI is much slower [7]. Considering the closer phylogenetic relationship of SetR_ICE391 to the cI-like proteins than to the LexA-like repressors (Fig. 1A), it was not surprising that we observed minimal pH-dependent self–cleavage of SetRICE391 even after 24 h incubation at pH 10.0 (Fig. 5). The limited pH dependent auto-cleavage therefore firmly places SetR_ICE391 in the cI-like family of transcriptional repressors, rather than the LexA-like repressors.

3.6. Binding of SetR_ICE391 to sites upstream of setR_ICE391 and the ICE391 rumAB operon

Reduced levels of rumAB-mediated spontaneous mutagenesis in the presence of SetR_ICE391 (Fig. 2), coupled with the RecA-mediated self-cleavage of SetR_ICE391 (Fig. 5) strongly indicate the involvement of SetR_ICE391 in the transcriptional repression of the mutagenic SOS response of the ICE391. The discovery that the primary amino acid sequence of the SetR_SXT protein is identical to that of SetR_ICE391, prompted us to examine the DNA region upstream of the rumAB operon for possible SetR_ICE391 binding sites. Based on the previously described SetR_SXT binding sites (Fig. 6A) [41], we identified an operator sequence partially overlapping the putative-35 promoter region of the rumAB operon (Fig. 6B). We were therefore interested in assaying whether highly purified SetR_ICE391 physically interacts with the region upstream of rumAB (Fig. 6C). First, we confirmed that the purified SetR_ICE391 protein would bind to sites upstream of the setR_ICE391 gene, similar to that previously reported for setR_SXT [40]. To do so, we synthesized two 123bp oligonucleotides which when annealed to each other would be identical to the intragenic region between croS_ICE391 and setR_ICE391 of ICE391 (Fig. 6A). This intragenic region is identical between ICE391 and SXT and was previously identified in SXT to contain four SetR_SXT binding sites [40,41]. As clearly seen, based upon differential retardation of the ³²P-labeled DNA probe, SetR_ICE391 appears to bind to a minimum of three discrete sites on the probe (Fig. 6C), and this observation is consistent with the previous results of SetR_SXT binding to the same DNA region on SXT [40].

Fig. 6.

SetR_ICE391 binding upstream of the rumAB operon. A. Nucleotide sequence of the 123 bp DNA probe used to study binding of SetR_ICE391 to regulatory elements upstream of setR_ICE391. B. Nucleotide sequence of the 194 bp DNA probe used to study binding of SetR_ICE391 to regulatory elements upstream of rumAB_ICE391. C. Electrophoretic gel mobility shift assays with E.coli LexA and SetR_ICE391. The DNA probes described above were radiolabeled and incubated with increasing concentrations of either highly purified SetR_ICE391 or LexA as specified at the bottom of each lane. DNA probe/protein complexes were incubated at 25 °C for 25 min, separated in a native 6% polyacrylamide gel and visualized on Kodak BioMax XAR film.

To determine if SetR_ICE391 physically interacts with the upstream regulatory region of rumAB, we used two primers to PCR amplify a 194bp amplicon spanning the upstream promoter region of rumAB including the very 5′ region of rumA (Fig. 6B) and the ³²P-labelled 194-bp substrate was incubated with increasing concentrations of SetR_ICE391 protein. Weak binding of SetR_ICE391 was observed at a protein concentration of 10 nM (Fig. 6C). As the SetRICE391 protein concentration increases beyond 10 nM, the initial band intensifies, and additional gel retardation occurs suggesting that there may be an additional SetR binding site located within the 194 bp probe (Fig. 6C)

Only one LexA binding site has been identified upstream of the rumAB operon [29] (Fig. 6B), and as expected, only one retarded band is observed when the rumAB probe was incubated with purified E.coli LexA protein (Fig. 6C).

3.7. Identification of the specific SetR_ICE391 binding site upstream of the rumAB operon

In order to unequivocally determine the SetR_ICE391 binding site(s) upstream of the rumAB operon, DNase I footprint analysis was under-taken as a custom contract service by scientists at Profacgen (Shirley, NY). DNAse I footprint analysis in the presence and absence of highly purified SetR_ICE391 with the rumAB promoter/operator sequence identified two distinct protected areas that coincide with the proposed SetR_ICE391 binding site that overlaps the −35 promoter region (Figs. 6C and Fig. 7A). No additional sites that are protected by SetR_ICE391 were identified by DNase I footprinting, suggesting that the “super-shift” of the probe observed at high concentrations of SetR_ICE391 in Fig. 6C, is likely due to oligomerization of the SetR_ICE391 on the single SetR_ICE391 binding site located in the −35 region of the rumAB promoter. We then created mutations within the two half-sites of the SetR_ICE391 binding site and utilized EMSA to determine SetR_ICE391 specificity for the binding-site mutant (Fig. 7A). Alteration of the protected nucleotides of the rumAB -35 promoter region equivalent to the most conserved nucleotides of the SetR_SXT consensus sequence resulted in a loss of affinity of SetR_ICE391 even at concentrations of the protein which gave a substantial shift of the wild-type DNA sequence (Fig. 7B). SetR_ICE391 binding within the rumAB promoter region, combined with the data presented above (Figs. 2 & 3), is therefore consistent with SetR_ICE391 acting as a transcriptional repressor of the rumAB operon.

Fig. 7.

Identification of the SetR_ICE391 box located upstream of rumAB. A. Nucleotide sequence of the putative SetR_ICE391 operator and surrounding sequence. The SetR_ICE391 operator is identified by the boxed region with areas protected during DNase I footprinting and the subsequent mutations generated within these protected areas also noted. B. Electrophoretic mobility shift assays with SetR_ICE391 and both the wild-type and mutated SetR_ICE391 operator DNA probes. The DNA probes were radiolabeled and incubated with increasing concentrations of either highly purified SetR_ICE391 as specified at the bottom of each lane. DNA probe/protein complexes were incubated at 25 °C for 25 min, separated in a native 6% polyacrylamide gel and visualized on Kodak BioMax XAR film.

4. Discussion

The mutagenic SOS response in E.coli is regulated at multiple levels [56]. The first key step relies on the RecA*-mediated self-cleavage and inactivation of the LexA transcriptional repressor to derepress expression of LexA-regulated proteins. The E.coli umuDC operon has a LexA-binding site with high homology to the consensus-binding site [2] and as a consequence, the UmuD and UmuC proteins are induced late in the SOS response. The rumAB operon, encoding polV_ICE391, also possesses an upstream LexA-binding site, but it differs from the consensus LexA-binding site at two additional positions, suggesting that LexA-dependent transcriptional regulation of rumAB might not be as vigorous as that observed for E.coli umuDC. We therefore considered the possibility that expression of rumAB might be subject to alternate levels of regulation, thereby keeping the mutagenesis promoting activity the RumA′B-encoded polV_ICE391 to a minimum. We present evidence here that SetR_ICE391 provides one such level of regulation.

Unlike E.coli polV, or R46-encoded polRI, SetR_ICE391 specifically functions as an additional transcriptional repressor of the polV_ICE391 polymerase. Indeed, alignment and phylogenetic analyses of the core amino acid sequences of SetR_ICE391 and its homologs identified SetR_ICE391 as a member of the cI-like family of transcriptional repressors, which includes LexA (Fig. 1). Expression of SetR_ICE391 in an E.coli recA⁺ lexA51(Def) strain results in reduced levels of rumAB-directed spontaneous mutagenesis (Fig. 2). In contrast, there was little negative regulation in a recA718 strain expressing RecA* in the lex-A51(Def) strain (Fig. 2D & F). This observation can be explained by the fact that purified SetR_ICE391 was shown to undergo RecA*-mediated cleavage in vitro (Fig. 5). We assume that such processing occurs rapidly in a recA718 strain in vivo thereby abrogating the ability of SetR_ICE391 to serve as a transcriptional repressor of the rumAB operon. This hypothesis is supported by the observation that rumAB-dependent mutagenesis was greatly reduced in a recA718 strain in the presence of a non-cleavable SetR_ICE391 variant (G94R) (Fig. 2D & F). It is important to note that the recA718 lexA51(Def) background more closely mimics the physiological state of a recA⁺ cell following extensive DNA damage suggesting that the ability of SetR_ICE391 to down-regulate the rumAB operon after DNA damage may also be limited.

The level of genetic diversity seen within a species of bacteria is absolutely dependent on the process of horizontal gene transfer [57]. The movement of genetic material between organisms can provide an advantage to the recipient cell; one of the most clinically relevant examples is the acquisition of antibiotic resistance genes. Another mechanism capable of imparting resistance to antibiotics is the error-prone nature of the members of the Y-family of DNA polymerases. Because of its mobility, and with the identification and characterization of the error-prone polV_R391, it has become apparent that ICE391 not only factors in the establishment of antibiotic resistance but possibly influences the overall genetic diversity of the integrated ICE391 and the host organism.

ICEs are found integrated within the genome of many species of the γ-Proteobacteria. It was in an isolate of Providencia rettgeri, formally known as Proteus rettgeri, that ICE391 (originally identified as the IncJ plasmid, R391) was first identified [58]. Although SXT and ICE391 both carry the genes necessary for a mutagenic SOS response, an SXT-mediated mutagenic SOS response is lacking due to an ISCR2 element insertion in rumB [38]. The broad range transmissibility of ICE391 poses a compelling question: how is the rumAB-mediated aberrant mutagenesis minimized in these varied bacterial genetic backgrounds? The identification of a LexA box within the promoter region of the rumAB operon linked regulation of the mutagenic capacity of ICE391 with the host LexA repressor. While LexA-like regulation is widely distributed amongst bacteria, it is not universally present in all bacteria [59,60]. Furthermore, not all host LexA homologs are expected to bind the rumAB LexA-box with sufficient affinity, or at all, to adequately repress the ICE391 mutagenic SOS response. For instance, employing a broad host range plasmid encoding an E. coli recA-lacZ fusion, a wide variety of gram-negative microorganisms were found to be capable of repression and induction of the lacZ fusion based on the DNA damage state of the cell [61]; however, it was also demonstrated that the more distantly related the organism, the less affinity the LexA-like repressors possess for the E. coli recA LexA box.

Identification of SetR_ICE391 as an SOS-induced repressor of rumAB reveals that ICE391 harbors a self-contained regulatory mechanism to curtail the mutagenic SOS response. Induction of the SetR regulon (setCD, setR and rumAB), probably functions to maximize the continued proliferation of ICE391 following exposure to a stressor by activating conjugal transfer, which requires DNA synthesis, and conceivably TLS, if the stressor is a DNA damaging agent.

Regulation of the ICE391 mutagenic SOS response can also be influenced by the rate at which SetR_ICE391 undergoes RecA*mediated self-cleavage. In this work, SetR_ICE391 has been shown to undergo in vitro self-cleavage at both alkaline pH and in the presence of RecA*, which is common to the cI-like repressors. However, the in vitro rate of self-cleavage of SetR_ICE391 is limited in relation to that of LexA. Under similar conditions, the in vitro RecA-mediated cleavage of the E. coli LexA has an estimated half-life of less than 30 min [45] compared to a half-life of > 6 h for SetRICE391 (Fig. 5). Excision and transfer of an ICE, along with the induction of an error-prone DNA polymerase, are events with serious consequences that require fine control and timing to favor the best evolutionary outcome. Expression of LexA-repressed genes is affected by the degree of DNA damage present, the affinity of LexA for the respective LexA box, the number of LexA boxes upstream of a gene and the efficiency at which it undergoes RecA*-mediated self-cleavage. In SXT the expression of setCD from the P_L promoter is subject to negative regulation by SetR_SXT. However, following mitomycin C induction of the SOS response, expression from P_L is not fully induced suggesting that a portion of the cellular SetR_SXT remains intact and functional [40]. This information, together with the data presented here, showing the reduced in vitro rate of self-cleavage of SetR_ICE391, support the idea that the rate of SetR_ICE391 self-cleavage could have evolved to safeguard the genetic integrity of ICE391 by minimizing SOS mutagenesis and conjugal transfer, while the error-free systems act first to address any DNA damage.

Although much work remains to be done to fully understand the extent of SetR_ICE391 activity in the mutagenic SOS response of ICE391, our findings strongly suggest that SetR_ICE391 is a repressor of the rumAB operon. Mobile genetic elements such as Integrative Conjugative Elements are agents of rapid bacterial evolution. One trait of medical concern passed on by many ICEs is the acquisition of antibiotic resistance genes by horizontal gene transfer. For example, ICESXT is capable of conferring resistance to sulfamethoxazole, trimethoprim and streptomycin [62], while ICE391 integration into a host genome imparts resistance to kanamycin, as well as the heavy metal, mercury [63]. In addition, ICE391 also encodes a very potent error-prone DNA polymerase, polV_ICE391, which is capable of contributing to the de novo generation of antibiotic resistance-conferring mutations. The fact that polV_ICE391 is so potent when sub-cloned from its native ICE391 environment [29,31,36] suggests that its mutagenic-promoting activity is normally regulated by trans-activating factors that are located on the intact ICE391. Here, we have identified SetR_ICE391 as one such trans-activating factor, but suspect others remain to be discovered.

Abbreviations:

TLS

translesion DNA synthesis

ICE

integrating conjugative element

polV

DNA polymerase V

RecA*

activated RecA-nucleoprotein filament