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. 2011 Apr 18;12:16.
doi: 10.1186/1471-2199-12-16.

Functional Characterization of an Alkaline Exonuclease and Single Strand Annealing Protein From the SXT Genetic Element of Vibrio Cholerae

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Free PMC article

Functional Characterization of an Alkaline Exonuclease and Single Strand Annealing Protein From the SXT Genetic Element of Vibrio Cholerae

Wen-yang Chen et al. BMC Mol Biol. .
Free PMC article

Abstract

Background: SXT is an integrating conjugative element (ICE) originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively.

Results: SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn2+ or Mg2+ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo.

Conclusions: The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V. cholerae cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.

Figures

Figure 1
Figure 1
Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography. Panel A: Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B: Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C: 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/E. coli BL21 (DE3) pLysS Rosetta whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.
Figure 2
Figure 2
Qualitative analysis of the metal ion dependence, DNA substrate preferences and mode of digestion of the SXT-Exo alkaline exonuclease. Panel A: Agarose gel showing ability of SXT-Exo to digest linear dsDNA (NdeI-linerized pET28a; lanes 2-5), circularized dsDNA (undigested pET28a; lanes 6 and 7), circularized ssDNA (M13 phage DNA; lanes 8 and 9) in Tris-HCl pH7.4, 50 mM NaCl with/without 10 mM MgCl2; λ-HindIII (NEB) DNA ladder (lane1). Panel B: Agarose gel showing the ability of SXT-Exo and lambda-Exo to digest 5'-phosphorylated linear dsDNA substrates ('unmodified'; lanes 2, 3, 6 and 7), compared with analogous 5'-phosphorylated linear dsDNA substrates containing 3 consecutive phosphorothioate linkages at the 5'-termini of each strand (PT-modified; lanes 4, 5, 8 and 9). The 712 bp 'unmodified' or 'PT-modified' dsDNA substrates (0.1 mg) were incubated at 37°C with lambda-Exo (3 μg) or SXT-Exo (30 μg) in Tris-HCl, (25 mM, pH7.4), 50 mM NaCl, 10 mM MgCl2 (total volume 40 μl). Aliquots (20 μl) were quenched (20 mM EDTA + 1% SDS) immediately, and after 30 mins, and analyzed on 1% agarose TAE gels. 1 Kb Plus DNA Ladder (Invitrogen; lane 1). Panel C: Agarose gel showing time-course of digestion of 5'-phosphorylated linear dsDNA (NdeI-linearized pET28a, 0.56 pmol) by SXT-Exo (50 pmol of trimers) in Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl2; at 37°C, with aliquots removed at times indicated (0-160 minutes; lanes 2-11); 1 Kb Plus DNA Ladder (lane 1).
Figure 3
Figure 3
Optimal pH, temperature and Mg(II) and Mn(II) ion concentrations for the dsDNA exonuclease activities of SXT-Exo, as determined by quenched PicoGreen Assays. Panel A: Optimum Mg2+ ion concentrations. SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl containing MnCl2 (0-10 mM); was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 30 mins. Panel B: Optimum Mn2+ ion concentrations. SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl containing MgCl2 (0-50 mM); was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 30 mins. Panel C: Optimum pH. SXT-Exo (2 pmol of trimers) in Tris-HCl (50 mM, adjusted to pH 7.0-9.0), 50 mM NaCl, 0.5 mM MnCl2; was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 30 mins. Panel D: Optimum temperature. SXT-Exo (6 pmol of trimers) in Tris-HCl (25 mM, pH 7.4), 50 mM NaCl, 0.5 mM MnCl2; was incubated with PstI-linearized pUC18 (5 ng, 0.003 pmol) at 37°C for 1 min. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.
Figure 4
Figure 4
Effects of addition of various monovalent and divalent salts on the double strand DNA activities of SXT-Exo, as determined by quenched PicoGreen assays. Panel A: Inhibition of the dsDNA exonuclease activities of SXT-Exo with sodium chloride (black squares), sodium phosphate (buffered to pH7.4; red circles) and sodium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl2; as well as the salt indicated in the figure (NaCl, Na2HPO4 (pH7.4), or Na2SO4; 0-500 mM); were incubated at 37°C for 30 mins. Panel B: Inhibition of the dsDNA exonuclease activities of SXT-Exo with potassium chloride (black squares), calcium chloride (red squares) and potassium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl2; as well as the salt indicated in the figure (KCl, CaCl2 or K2SO4; 0-500 mM); were incubated at 37°C for 30 mins. Relative dsDNA exonuclease activities were calculated (as a percentage) by comparison with results from analogous assays that contained: SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl2, 50 mM NaCl. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.
Figure 5
Figure 5
Double stranded DNA end preferences of SXT-Exo. SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl2, 50 mM NaCl; was incubated at 37°C for 30 mins with: i) 5'-phosphorylated linear dsDNA with 4 nt 3'-overhangs (PstI-linearized pUC18, black shaded circles); ii) 5'-hydroxylated linear dsDNA with 4 nt 3'-overhangs (dephosphorylated PstI-linerarized pUC18, un-shaded circles); iii) 5'-phosphorylated blunt-ended dsDNA (SspI-linerized pUC18, shaded inverted triangles); or iv) 5'-phosphorylated linear dsDNA with 4 nt 5'-overhangs (BamHI-linearized pUC18, un-shaded green triangles). Aliquots were removed at 1, 2, 5, 10, 20 and 40 minutes; quenched, then dsDNA levels were quantified using the PicoGreen reagent. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.
Figure 6
Figure 6
Digestion of 5'-phosphorylated and non-phosphorylated oligonucleotides by SXT-Exo, monitored by size exclusion chromatography. Panel A: Overlay of three size exclusion chromatograms of the time-wise digestion of a non-phosphorylated synthetic 75 mer of oligothymidine (dT75, 400 pmol) by SXT-Exo (100 pmol of trimers) in 25 mM Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl2; incubated at 37°C. Black-coloured trace: amounts of oligo-dT75 present immediately after addition of SXT-Exo; red and blue-coloured traces: oligo-dT75 present 3 minutes, and 25 minutes after the addition of SXT-Exo, respectively. Panel B: Overlay of three size exclusion chromatograms of the time-wise digestion of a 5'-phosphorylated 75-mer of oligothymidine (5'-P-dT75, 400 pmol) by SXT-Exo (100 pmol of trimers) in 25 mM Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl2; incubated at 37°C. Black-coloured trace: 5'-phosphorylated oligo-dT75 present immediately after the addition of SXT-Exo; red and blue-coloured traces: 5'-phosphorylated oligo-dT75 present 3 minutes, and 25 minutes after the addition of SXT-Exo, respectively.
Figure 7
Figure 7
Digestion of fluorescently-labeled annealed oligonucleotide substrates by SXT-Exo. The ability of SXT-Exo to digest three different (partially) dsDNA substrates (prepared from two annealed oligonucleotides, see Additional File 4) was monitored by quantifying the digestion of the 5'-phosphorylated-3'-Cy3-labeled strand using fluorescence gel scanning. In each assay, SXT-Exo (50 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 0.5 mM MnCl2. Aliquots were removed and quenched at 0, 1, 2, 4, 10 and 20 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A: Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate (annealed 5'-PO4-70Cy3 + 50blunt oligonucleotides) by SXT-Exo. Panel B: Representative gel image showing digestion of the Blunt ended DNA substrate (annealed 5'-PO4-50Cy3 + 50blunt oligonucleotides) by SXT-Exo. Panel C: Representative gel image showing digestion of the 3'-overhang DNA substrate (annealed 5'-PO4-50Cy3 + 70overhang oligonucleotides) by SXT-Exo. Panel D: Plot showing the digestion of the three DNA substrates by SXT-Exo over a 20 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.
Figure 8
Figure 8
Digestion of fluorescently-labeled annealed oligonucleotide substrates by lambda-Exo. In experiments analogous to those described for SXT-Exo (see Figure 7), the ability of lambda-Exo to digest three different (partially) dsDNA substrates was investigated. In each assay, lambda-Exo (3 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 5 mM MgCl2. Aliquots were removed and quenched at 0, 0.5, 1, 2, 4 and 10 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A: Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate by SXT-Exo. Panel B: Representative gel image showing digestion of the Blunt ended DNA substrate by lambda-Exo. Panel C: Representative gel image showing digestion of the 3'-overhang DNA substrate by lambda-Exo. Panel D: Plot showing the digestion of the three DNA substrates by lambda-Exo over a 10 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.
Figure 9
Figure 9
Processivity of SXT-Exo digestion of double stranded DNA. Heparin-trap experiments were used to calculate the average number of nucleotides hydrolyzed by an SXT-Exo trimer during a single binding event. SXT-Exo (41 nmol of trimers) and PstI-linearized pUC18 DNA (2686 bp in length; 18.1 pmol) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl2; were incubated at 25°C for 30 s, before trapping unbound protein by addition of a large excess of heparin. Aliquots were removed at 0, 1, 2, 5, 10, 20 and 30 minutes; quenched, then dsDNA levels immediately quantified using PicoGreen assays to determine the number of nucleotides digested from each end (red circles) at each time point. Analogous control experiments without heparin were performed (black circles). Four independent replicates of each experiment were conducted, and graphs show the mean values ± standard deviation. See materials section for details.
Figure 10
Figure 10
Stimulation of double strand DNA exonuclease activities of SXT-Exo and lambda-Exo by SSAP and Ssb proteins. Panel A. SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of the protein indicated in the text (BSA, lambda-Bet, SXT-Bet or SXT-Ssb) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl2; were incubated at 25°C for 30 mins before EDTA quenching. dsDNA levels were immediately quantified using PicoGreen reagent. The level of DNA digestion by SXT-Exo in the absence of added protein (-) was normalized to a value of 100%. Panel B. In analogous sets of experiments, lambda-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of BSA, lambda-Bet, SXT-Bet or SXT-Ssb; in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 5 mM MgCl2; were incubated at 25°C for 10 mins. Digestion levels were normalized to those of lambda-Exo in the absence of added protein (-). See methods section for detailed experimental procedure. Six independent replicates were performed for each experiment, and error bars indicate standard deviation from the mean values. Analysis using ANOVA indicated all results were statistically significant (P < 0.05) when compared to the no-protein control (-), with respective P values indicated above each bar.
Figure 11
Figure 11
Efficiency of SXT-Bet + SXT-Exo mediated recombination between a PCR-generated DNA fragment and its homologous target on the E. coli chromosome. Panel A: Schematic overview of the chromosomal targeting assay use to score exonuclease + SSAP-mediate recombination efficiency. A dsDNA 'targeting' molecule (galK<>Cmr) was synthesized by PCR using two primers sharing (ca. 20 nt) sequence homology to a chloramphenicol resistance cassette at one end; and 50 nt of sequence homology to the 5'-end (ECgalKF1) or 3'-end (ECgalKR1) of the non-essential E. coli galK gene at the other. The purified galK<>Cmr targeting cassette was electroporated into DH10B cells expressing pairs of Exo and SSAP proteins from established arabinose-inducible plasmids. The Exo and SSAP proteins mediate homologous recombination between the galK<>Cmr dsDNA cassette and the galK locus of the E. coli chromosome via 50 bp regions of flanking sequence homology. This creates a mutant E. coli strain that has its galK gene replaced with a chloramphencol resistance cassette. Panel B. Comparison of dsDNA recombination efficiencies of SXT-Bet-Exo with those of RecET and lambda-Bet-Exo. The RecET; lambda-Bet-Exo; SXT-Bet-Exo and SXT-Ssb-Bet-Exo sets of homologous recombination-promoting proteins were expressed from arabinose-inducible (PBAD/araC) plasmids established in E. coli DH10B cells (see Additional File 3). pBAD-ETγ: RecE + RecT + lambda-Gam [35]; pB1E4A: lambda-Bet + lambda Exo; pBex4b1: SXT-Bet + SXT-Exo; pBX2B: SXT-Ssb + SXT-Bet + SXT-Exo; pBAD-28MCS (negative control). Homologous recombination events were scored by plating galK<>Cmr-electroporated cells onto LB-agar with/without chloramphenicol. The dsDNA recombination efficiency was calculated by dividing the number of Cmr colonies by the total number of cells (CFUs) that survived electroporation. 8 replicates were performed; the mean recombination efficiency ± standard deviation is reported.

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