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prlF and yhaV encode a new toxin-antitoxin system in Escherichia coli - PubMed

  • ️Mon Jan 01 2007

prlF and yhaV encode a new toxin-antitoxin system in Escherichia coli

Oliver Schmidt et al. J Mol Biol. 2007.

Abstract

Toxin-antitoxin systems consist of a stable toxin, frequently with endonuclease activity, and a small, labile antitoxin, which sequesters the toxin into an inactive complex. Under unfavorable conditions, the antitoxin is degraded, leading to activation of the toxin and resulting in growth arrest, possibly also in bacterial programmed cell death. Correspondingly, these systems are generally viewed as agents of the stress response in prokaryotes. Here we show that prlF and yhaV encode a novel toxin-antitoxin system in Escherichia coli. YhaV, a ribonuclease of the RelE superfamily, causes reversible bacteriostasis that is counteracted by PrlF, a swapped-hairpin transcription factor homologous to MazE. The two proteins form a tight, hexameric complex, which binds with high specificity to a conserved sequence in the promoter region of the prlF-yhaV operon. As homologs of MazE and RelE, respectively, PrlF and YhaV provide an evolutionary connection between the two best-characterized toxin-antitoxin systems in E. coli, mazEF and relEB.

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Figures

Figure 1
Figure 1

The prlF-yhaV operon in E. coli. (a) Schematic depiction of the gene structure; the stop codon of prlF (TAA) overlaps by one base with the start codon of yhaV (ATG) and both genes are controlled by a shared promoter. (b) Alignment of the N-terminal domain of PrlF with AbrB and three antitoxins of the MazEF family (PemK, MazE, ChpBI). All proteins belong to a superfamily of prokaryotic transcription factors with a swapped-hairpin barrel fold; the secondary structure of the fold (S = β-strand, H = α-helix) and the location of two characteristic sequence motifs are shown above the alignment. For the proteins of known structure, the Protein Data Bank identifiers are given. The organisms are: Ec Escherichia coli, Bs Bacillus subtilis, Ph Pyrococcus horikoshii. (c) Alignment of YhaV with toxins of the RelE superfamily. Conserved residues of the superfamily are shown bold and the catalytically important residues Arg85 and Arg94 are highlighted in red. Other annotations are as in panel (b). (d) Effect of the prlF1 mutation on PrlF. The duplication of 7 nt at position 257 – 263 of prlF introduces a frame-shift that results in a slightly shorter gene product rich in basic (blue) instead of acidic (red) residues. Moreover, an intergenic gap of 24 nt is formed between the prlF and yhaV open reading frames.

Figure 2
Figure 2

Comparison of protein expression from the prlF-yhaV operon with prlF1-yhaV, both placed under control of a lactose-inducible T7 promotor, indicating that translation of YhaV is abolished by the prlF1 mutation. For detection in western blot, PrlF and PrlF1 carry a His6-tag at the N-terminus and YhaV at the C-terminus. A strain expressing the untagged proteins from a similar plasmid (PY/pET30) is used as a control. The cross reaction with an E. coli protein of 33 kD serves as a loading control. MW: molecular markers (GE Healthcare LMW-SDS-Marker kit).

Figure 3
Figure 3

Co-expressed PrlF and His6-YhaV form a complex and co-elute on both preparative gel size exclusion (a) and Ni-NTA metal affinity chromatography (b). (c) Migration of natively purified PrlF-His6-YhaV complex and of His6-YhaV on analytical Superose 6 gel size exclusion columns. A second peak of approximately twice the molecular weight overlaps the main peak of the PrlF-His6-YhaV hexamer. Lower panel: Coomassie Blue-stained SDS-gel of the peak fractions from the PrlF-His6-YhaV run. Size calibration of the column: GroEL (800 kD), bovine serum albumin (BSA; 66 kD), carbonic anhydrase (CA; 29 kD), cytochrome c (Cyt c; 12.4 kD), aprotinin (Aprot.; 6.5 kD).

Figure 3
Figure 3

Co-expressed PrlF and His6-YhaV form a complex and co-elute on both preparative gel size exclusion (a) and Ni-NTA metal affinity chromatography (b). (c) Migration of natively purified PrlF-His6-YhaV complex and of His6-YhaV on analytical Superose 6 gel size exclusion columns. A second peak of approximately twice the molecular weight overlaps the main peak of the PrlF-His6-YhaV hexamer. Lower panel: Coomassie Blue-stained SDS-gel of the peak fractions from the PrlF-His6-YhaV run. Size calibration of the column: GroEL (800 kD), bovine serum albumin (BSA; 66 kD), carbonic anhydrase (CA; 29 kD), cytochrome c (Cyt c; 12.4 kD), aprotinin (Aprot.; 6.5 kD).

Figure 4
Figure 4

(a) Growth phenotype of strain NJH134 expressing the plasmids PrlF/pBAD, YhaV/pBAD, PrlF/YhaV/pBADtwin, PrlF1/YhaV/pBADtwin and the pBAD vector as a control. The curves are averaged from four independent cultures and are normalized by setting cell density of each culture at induction time (t = 0 min) to 0 % and average density of the vector control at t = 300 min to 100%. Error bars show standard deviation. (b) Expression of YhaV from YhaV/pBAD in the wild type strain E. coli W3110 induces growth arrest, from which the cells recover when the inducing agent is removed. No loss of colony forming units is observed in a drop dilution test on LB agar in cells expressing the toxin YhaV, compared to cells coexpressing PrlF and YhaV. Each consecutive drop is a ten-fold dilution of the previous, starting from OD600 = 6×10−3. c) High-level expression of His6-YhaV from the IPTG-induced YhaV/pET28b plasmid in the C41 (DE3) strain reduces the number of viable cells by three orders of magnitude within 2 hrs with respect to cells expressing simultaneously the PrlF/pET31b and YhaV/pET28b plasmids. The YhaV-expressing strain harbored an additional empty pET15b plasmid to provide ampicillin resistance.

Figure 5
Figure 5

(a) Time course for proteolysis of purified PrlF-His6-YhaV with proteinase K (PK) (10 μg/ml). A 18 kD protease-resistant fragment of YhaV is formed, while PrlF is almost completely degraded. (b) Differential proteolysis pattern of the PrlF-His6-YhaV complex and of His6-YhaV. Left panel: Coomassie Blue-stained SDS-gel; right panel: corresponding western blot probed for His6-tagged proteins.

Figure 6
Figure 6

RNase activity of YhaV towards a preparation of cellular RNA of E. coli W3110 (10 μg/lane), showing (a) His6-YhaV, (b) His6-YhaV preincubated for 30 min with a three-fold molar excess of His6-PrlF, (c) refolded PrlF-His6-YhaV, (d) PrlF-His6-YhaV and refolded His6-YhaV pretreated with proteinase K. RNA alone (control) and RNA with just proteinase K (PK) are shown for comparison; the positions of 23S and 16S ribosomal RNA are indicated.

Figure 7
Figure 7

(a) Growth curve of E. coli NJH134 expressing pBAD plasmids of YhaV and of the mutants YhaV R85A, YhaV R94A and YhaV R85A,R94A. The experimental setup and data processing are as in Fig. 4a. (b) RNase activity of the natively purified double-mutant His6-YhaV R85A,R94A (0.33 μM and 16.5 μM). Control is RNA alone. (c) RNase activity of His6-YhaV R85A,R94A preincubated with His6-PrlF.

Figure 8
Figure 8

Electrophoretic mobility shift assays of a 250 bp PCR product containing the operator region of prlF (1.33 pmol/lane), titrated with increasing amounts of (a) PrlF-His6-YhaV, (b) His6-PrlF, (c) His6-PrlF1 and (d) His6-PrlF-N. The molar ratios of DNA to hexameric complex (PrlF-YhaV), and dimer (PrlF, PrlF1, PrlF-N) are indicated for each lane. The dimer was chosen as the basic unit, since it foms one putative DNA-binding domain. (e) A consensus DNA sequence motif in the operator region of prlF in various organisms. The positions are counted from the first nucleotide of the annotated start codon of prlF (+1). E. coli harbors a second, less conserved motif at position +199, while in Pseudomonas putida a second motif overlaps the predicted start codon.

Figure 9
Figure 9

Electrophoretic mobility shift assays of PrlF constructs. (a) Protein binding to the operator region of prlF in competition with a two-fold molar excess of the operator region of B. subtilis spoVT. (b) PrlF-His6-YhaV binding to overlapping fragments of the prlF operator region. (c) Comparison of PrlF-His6-YhaV binding to the native operator (wt), an unrelated DNA carrying the 14 nt consensus motif (chimera), the same DNA carrying only the first eight palindromic nucleotides of the motif (half chimera), and two additional native operator sequences where either the first (1st half) or last (2nd half) eight nucleotides are retained while the rest of the motif is mutated. Two shifted bands are detected, labelled ‘binary complex’ and ‘ternary complex’, which presumably correspond to one and two PrlF-His6-YhaV complexes bound to DNA, respectively. (d) Schematic model of the PrlF-YhaV complex binding to its recognition site.

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