The Escherichia coli uropathogenic-specific-protein-associated immunity protein 3 (Imu3) has nucleic acid -binding activity
© Črnigoj et al.; licensee BioMed Central Ltd. 2014
Received: 11 September 2013
Accepted: 20 January 2014
Published: 28 January 2014
The Escherichia coli uropathogenic-specific protein (Usp) is a bacteriocin-like genotoxin, active against mammalian cells and associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia. Usp is encoded by a small pathogenicity island with three downstream small open reading frames (Imu1-3) that are believed to provide immunity to the producer. To prevent host suicide, colicins, bacteriocins of E. coli, form tight complexes with their cognate immunity proteins. Colicin – immunity protein complexes are among the strongest protein complexes known. Here, the Usp associated immunity protein 3 (Imu3) was partially characterized to gain insight into its role and mechanism of activity.
Isolation and partial characterisation of the Usp-associated immunity protein-3 (Imu3) revealed that, while Usp and Imu3 do not form a high affinity complex, Imu3 exhibits DNA and RNA binding activity. Imu3 was also shown to protect DNA against degradation by colicin E7.
Our data infer that nonspecific DNA binding of the Imu3 immunity protein, prevents suicide of E. coli producing the genotoxin Usp.
The Escherichia coli uropathogenic-specific protein (Usp) has been shown to be associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia, and with increased virulence and fitness of pathogenic strains of E. coli[1–4]. Nucleotide sequence analysis has shown approximately 45% sequence identity of the Usp C-terminal region with that of the E. coli bacteriocin colicin E7, which has nuclease activity, while the Usp N-terminal region is similar to the Type VI protein secretion system component (Hcp like)[5–7]. It has been proposed that Usp acts as a bacteriocin against competing E. coli strains and that it also enhances infectivity in the urinary tract. Recently, we demonstrated the genotoxic activity of Usp against mammalian cells[5, 8]. To protect the colicin-producing cell from its own toxin, colicin-encoding operons generally harbour one cognate immunity gene. Colicins and their immunity proteins have some of the strongest protein-protein affinities, which result in the formation of stable colicin–immunity protein complexes[10, 11]. In contrast, downstream of the usp gene, there are three short open reading frames designated orfU1-3 (288, 294, and 291 bp long, respectively), that are believed to be involved in the protection of the Usp-producing cell from its own nuclease activity. The immunity proteins coded by the usp gene operon have a characteristic two-histidine region which appears to enable the inactivation of the Usp DNase activity. However, Usp-encoding strains that do not have all three orfU immunity protein genes have been described. All three immunity proteins are thus not essential for the protection of the Usp producers, although Usp is lethal when it is expressed alone in E. coli. It has been postulated that none of the three proteins is exclusively required for Usp protein synthesis. As protection of the Usp-producing bacterial cell might be provided by a mechanism that is different from that of the colicins, we have investigated the E. coli Usp-associated immunity protein Imu3, previously designated OrfU3. Our study indicates that Imu3 has protective non specific DNA-binding abilities that could have possible biotechnological potential.
Results and discussion
Protection of Usp producing E. coli by the individual Imu proteins
% of transformants relative to control (usp + imu1-3)
1.7 ± 1.2
usp + imu1
2.4 ± 1.2
usp + imu2
4.1 ± 2.0
usp + imu3
10.6 ± 4.0
Imu3 dimerisation and USP binding
Imu3 has fairly high sequence similarity to the colicin E7 immunity protein Cei, approximately 66% sequence identity as established with the MEGA program package, which was previously reported to form monomers. We investigated potential dimer formation by Imu3, using the cross-linking glutaraldehyde assay, native PAGE electrophoresis and size exclusion chromatography (HPLC). Native PAGE as well as HPLC experiments clearly showed that, Imu3 does not form dimers or multimers since a single peak of size between 11 and 13 kDa was observed regardless of the presence or absence of DNA (Figure 1B). Cross-linking studies of equimolar mixtures of Imu3 and Usp also showed no complex formation (Additional file2: Figure S2).
Minimal DNA length for Imu3 binding
EMSA tests with short double stranded DNA fragments (re-annealed oligonucleotides) were also performed however, the results were inconclusive since we repeatedly observed the recurring effect of unbound Imu3 that re-/dis-appeared every 3-5 nucleotides of the oligonucleotide length; however, the underlying basis of this phenomena is unclear.
Separation of Imu3 from DNA and subsequent DNA integrity analysis
Separation of the DNA-Imu3 complex, was examined under different conditions. Exposure of the DNA–Imu3 complex to pH values between pH 3 and pH 13 showed separation at values between pH 11 and pH 12. However, at pH values higher than pH 12.5, DNA degradation was also observed. When the DNA–Imu3 complex was heated to 100°C for 5 min in the presence of different NaCl concentrations, separation of Imu3 from DNA was observed at 0.5 M NaCl or higher (Additional file3: Figure S3). Incubation of Imu3-DNA complexes with proteinase K resulted in unbound DNA due to degradation of Imu3.
To the best of our knowledge, no known functions have been described yet for the protein products of orfU1, orfU2 and orfU3 (here referred to as Imu1, Imu2 and Imu3). Although all three immunity proteins of the Usp cluster share a high degree of sequence similarity, the present study shows that Imu3 has a distinct RNA- and DNA-binding ability. Nevertheless, additional as of yet unresolved mechanisms could be involved in protection of Usp producing cells by its cognate immunity proteins. Interestingly, protein-mediated DNA precipitation has been reported in studies describing eukaryotic histones and the E. coli global regulator, protein HU, a known DNA-binding protein[13, 14]. Operons, such as those of colicins, that encode proteins that can be detrimental to the producing cell are regulated precisely to ensure appropriate timing of synthesis and avoid untimely death of the producer[15–17]. We can thus speculate that synthesis of Usp and its associated Imu1-3 proteins could also be tightly regulated, limiting their production to avoid overt degradation and masking of the producers’ genome. Indeed high expression levels of imu3 (IPTG induced for protein isolation) are toxic for producing cells. DNA-binding (basic) proteins usually have an overall positive charge that facilitates their binding to DNA. The Imu3 protein, has a theoretical isoelectric point of ca. 4.4, which implies that the DNA-binding region must be localised only on part of the tertiary structure of the molecule. Different online DNA-binding motif search tools were used to identify a potential Imu3 DNA binding motif[18, 19]. The results imply that the DNA-binding ability of Imu3 probably originates from the helix-turn-helix motif.
In conclusion, our study shows that Imu3 like the colicin E7 immunity protein Cei, does not form dimers and in addition, does not form a tight complex with the Usp protein. However, in contrast to the two other small proteins of the Usp pathogenicity island, Imu1 and 2, Imu3 does bind DNA and RNA. We propose that Usp producing cells are protected from genome fragmentation by Imu3 DNA masking. Further, as Imu3 precipitates but does not damage DNA we believe that could have biotechnological potential.
Plasmid construction and protein expression
The nucleotide sequences that encode Imu3 (USP-associated immunity protein 3 from E. coli) were amplified from the genomic DNA of the uropathogenic E. coli strain TA211 using standard PCR reactions. The Imu3n-F 5′-TTTCTCGAG CTATAATTTTAAAGATGAAATAG-3′ and Imu3n.R 5′-TTTACGCGT TATTTAGAGTCTTTAAACAAG-3′ primers were used, with Xho I and Mlu I restriction sites, respectively (in italics). The PCR product was cloned into the blunted pJET1.2 plasmid (Fermentas), and Usp-coding sequences were subsequently excised and re-cloned into the Xho I and Mlu I sites of the pET8c expression plasmid, with an N-terminal His tag (Novagen). Subsequently, Imu3 was expressed in the E. coli strain BL21(DE3) pLysE as described by the manufacturer (QIAgen). Briefly, an overnight culture of E. coli BL21(DE3) pLysE was diluted in liquid Luria Bertani medium supplemented with 120 mg/L ampicillin (LBAp medium) to an OD600 of 0.05 at 37°C, and grown to an OD600 of 0.6. Imu3 production was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to 0.8 mM final concentration. The culture was grown for an additional 4 h, and then the biomass was collected by 10 min centrifugation at 4,000× g.
All of the isolation steps were carried out at 4°C. The collected biomass was treated with DNase, RNase and lysozyme on ice for 1 h, as described by the manufacturer (QIAgen), and complete EDTA-free protease inhibitor cocktail (Roche) was added. The cells were ruptured with 12 consecutive ultrasonication bursts (alternating 30 s pulse, 30 s pause) at the 55 setting (Sonics Vibra Cell). The cell lysates were cleared by three 20 min centrifugations at 20,000× g. All of the other protein isolation steps were carried out. When needed, Imu3 was further purified with size-exclusion FPLC chromatography (Superdex 75 HR 10/30, Amersham Biosciences) equilibrated with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl. Buffer exchanges were carried out using Amicon MWCO 3 kDa microconcentrators (Millipore). The his-tag was removed with the Thrombin Cleavage Capture Kit (Novagen) as described by the manufacturer. Actual mass of Imu3 protein was determined via mass spectrometry ESI + and Q-Tof (Waters-Micromass, United Kingdom).
The degree of Usp-producing cell protection provided by each of the three individual immunity proteins (Imu1-3) was examined in E. coli BL21(DE3) pLysE cells that were transformed with the plasmid pET8c carrying the combination of Usp and either Imu1, Imu2 or Imu3. The transformants were isolated on LB Ap plates with IPTG (0.8 mM final concentration) after being grown overnight at 37°C.
Imu3 and Usp binding
Formation of a Imu3 dimer was checked using the cross-linking glutaraldehyde assay as previously described, native PAGE and size exclusion chromatography (HPLC).
Imu3 samples (2 mg/mL) with or without the addition of 2.7 kbp double-stranded linear DNA (pUC19/Eco RI) were initially incubated at 37°C for 30 min, to allow for potential multimerization. Samples were then subjected to either native PAGE resolution or to the glutaraldehyde cross-linking procedure and SDS-PAGE resolution, with the LexA protein as a dimerisation-positive control. Aditionally, Imu3 was checked for dimerisation with size exclusion chromatography (HPLC, Phenomenex Biosep SEC-S2000 column, flow rate: 1 mL/min, 50 mM NaH2PO4, 300 mM NaCl, pH8), self-cleaved LexA protein was used as a standard (11 kDa, 13 kDa and 26 kDa).
Formation of the Imu3–USP complex was also investigated using the glutaraldehyde assay, after Imu3 and Usp had been mixed in equimolar ratios.
Various concentrations of either Eco RI linearised pUC19 DNA or total RNA (isolated from E. coli) and the Imu3 protein were used to establish the nucleic-acid-binding ability of Imu3. The Imu3 was incubated with either the DNA or RNA in TE buffer (10 mM Tris, 1 mM EDTA, pH 8) at 37°C for 30 min, prior to the electromobility shift assays (EMSAs) with 0.8% agarose gels. The effects of temperature (10 min incubation at 70°C, 80°C, 90°C and 100°C), pH (6, 7, 8, 9), NaCl (0-500 mM) and Mg2+ ions concentrations (0-50 mM) on the DNA binding ability of Imu3 were studied.
Thermal denaturation curves of linearised pUC19 DNA and the Imu3 protein were carried out in 5 mM cacodylic buffer (pH 6.5) using a UV-vis spectrophotometer (Cary Varian Cary 100 Bio, Australia) equipped with a thermoelectrically controlled cell holder. UV absorption was measured as a function of temperature (UV melting curves) for different ratios of linear DNA and Imu3 (0, 0.3 and 1.0 μg per 100 ng DNA), at 260 nm. The UV melting temperature ranged from 25°C to 99°C, with a heating rate of 1°C•min-1 and an equilibration time of 1 min. The melting curves of buffer and of the Imu3 protein alone were subtracted from the melting curves of the DNA–Imu3 protein complex, providing curves that show only the changes in the thermal stability of the DNA.
Further, the influences of pH, temperature and ionic strength on the separation of the DNA–Imu3 complex were examined. The effects of pH, were examined in the range from pH 3 to pH 13. Buffers used for these pH values were the following: pH 3-5, citric buffer; pH 6, MES buffer; pH 7-9, TRIS buffer; pH 10-12, glycine/NaOH buffer; pH 13, NaOH. The impacts of various ions on the separation of the DNA–Imu3 complex were studied as 0-1 M NaCl, 350 mM KCl, 350 mM NaSCN, 70 mM MgCl2, 0.7% SDS, 1-3 M (NH4)2SO4 and 2.3 M guanidinium thiocyanate. The effects of temperature were studied 80°C and 95°C, with a 10 min incubation of the complex, and at 100°C, with a 5 min incubation.
To examine whether Imu3 binding to DNA triggers any DNA damage, religation experiments were performed. Initially, the linear plasmid DNA (pUC19) was incubated with the Imu3 protein at 37°C for 30 min, to allow for the DNA–Imu3 complex to form. The samples were subsequently purified using the QIAprep Spin Miniprep kits (QIAgen). To check DNA integrity, the linearised DNA was used for a (self) ligation reaction (Fermentas); half of the ligation mixture was transformed into E. coli DH5α, while the other half was subjected to a second restriction (Eco RI).
The structural integrity of the Imu3 precipitated plasmid DNA was also investigated on the basis of detection of potential mutations within a non-selected marker, the ampicillin resistance gene. For this purpose, plasmid pBR322 carrying both tetracycline and ampicillin resistance genes was employed. Plasmid DNA was digested with Pst I, with a single restriction site within the ampicillin resistance gene to yield one linear DNA fragment. Following gel electrophoresis the linear plasmid DNA was precipitated with Imu3 and centrifuged for 10 minutes at 4°C, followed by washing with 0.5 ml of TE buffer. The pellet was subsequently treated with the PCR Cleaning Kit (Thermo Scientific) and several μl of the isolate were employed for re-ligation. In control experiments, ligase was omitted. Transformants were isolated upon selection for tetracycline resistance. Five hundred transformants were further screened and all found to express ampicillin resistance.
Minimum DNA length for Imu3 binding
Oligonucleotide sequences used to determine the minimal length of single-stranded and double-stranded DNA for DNA–Imu3 complex formation
15-TATA (poly AT)
15-gCgC (poly GC)
This work was financed by the Slovene Research Agency (ARRS). We would like to thank Dušan Žigon for help with mass spectroscopy, Nataša Poklar Ulrih with DNA melting experiments and Luka Ausec for assistance with bioinformatics issues.
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