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Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity.

Jovanovic M, James EH, Burrows PC, Rego FG, Buck M, Schumacher J - Nat Commun (2011)

Bottom Line: Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues.Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV.The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ(54)-RNA polymerase activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ(54)-dependent transcription at the hrpL promoter; triggering type-three secretion system-mediated pathogenicity. In contrast with singly acting EBPs, the evolution of the strictly co-operative HrpRS pair raises questions of potential benefits and mechanistic differences this transcription control system offers. Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues. Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV. The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ(54)-RNA polymerase activation.

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Related in: MedlinePlus

Regulation of the HrpRS complex in vitro.(a) In vitro full-length (FL) transcription assays (∼470 nucleotides) using the supercoiled hrpL promoter in the presence (+) or absence (−) of: σ54-RNAP (100 nM), dATP (4 mM), co-purified HrpRS (0.8 μM) and integration host factor (IHF) (20 nM). (b) HrpR alone (0.8 μM), HrpS alone (0.8 μM) and pre-mixed HrpR and HrpS (0.8 μM) (HrpR+HrpS) failed to activate transcription. (c) As in a, including dATP (4 mM), IHF (20 nM) with reconstituted σ54-RNAP using either purified σ54 from Ps (σ54-Ps) or from σ54 K. Pneumoniae (σ54-Kp). (d) HrpRS-dependent activation from the hrpL promoter as in a, in the presence of increasing HrpV concentrations. (e) Transcription from a test σ54 promoter (S. meliloti nifH), with the EBP AAA+ domain PspF1−275, or HrpRS, and in the presence (+) or absence (−) of HrpV (2 μM) and HrpG (2 μM). Gel images of transcripts for Figure 2a–e are shown in Supplementary Figure 4. (f) A bar graph showing electrophoretic mobility shifted hrpL DNA promoter probe comprising UAS sequences (in percentage of total hrpL DNA promoter signal) in the presence of HrpRS (2.5 μM) or HrpRS (2.5 μM and HrpV (4.2 μM). (g) A bar graph depicting the percentage of DNA bound in the closed promoter complex (CC) and transcriptionally proficient open promoter complex (OC; formed by PspF activation) in the presence of HrpRS–V or HrpV. (h) A bar graph depicting the relative amount of glutaraldehyde crosslinked HrpRS–HrpV species in either ATPase buffer (used to measure the ATPase activity of HrpRS+/−HrpV) or HGNED buffer. The relative amount of crosslinked HrpS–V complex was determined by fluorescence scanning. In f–h, estimated errors of measurements are shown as ±10%.
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f2: Regulation of the HrpRS complex in vitro.(a) In vitro full-length (FL) transcription assays (∼470 nucleotides) using the supercoiled hrpL promoter in the presence (+) or absence (−) of: σ54-RNAP (100 nM), dATP (4 mM), co-purified HrpRS (0.8 μM) and integration host factor (IHF) (20 nM). (b) HrpR alone (0.8 μM), HrpS alone (0.8 μM) and pre-mixed HrpR and HrpS (0.8 μM) (HrpR+HrpS) failed to activate transcription. (c) As in a, including dATP (4 mM), IHF (20 nM) with reconstituted σ54-RNAP using either purified σ54 from Ps (σ54-Ps) or from σ54 K. Pneumoniae (σ54-Kp). (d) HrpRS-dependent activation from the hrpL promoter as in a, in the presence of increasing HrpV concentrations. (e) Transcription from a test σ54 promoter (S. meliloti nifH), with the EBP AAA+ domain PspF1−275, or HrpRS, and in the presence (+) or absence (−) of HrpV (2 μM) and HrpG (2 μM). Gel images of transcripts for Figure 2a–e are shown in Supplementary Figure 4. (f) A bar graph showing electrophoretic mobility shifted hrpL DNA promoter probe comprising UAS sequences (in percentage of total hrpL DNA promoter signal) in the presence of HrpRS (2.5 μM) or HrpRS (2.5 μM and HrpV (4.2 μM). (g) A bar graph depicting the percentage of DNA bound in the closed promoter complex (CC) and transcriptionally proficient open promoter complex (OC; formed by PspF activation) in the presence of HrpRS–V or HrpV. (h) A bar graph depicting the relative amount of glutaraldehyde crosslinked HrpRS–HrpV species in either ATPase buffer (used to measure the ATPase activity of HrpRS+/−HrpV) or HGNED buffer. The relative amount of crosslinked HrpS–V complex was determined by fluorescence scanning. In f–h, estimated errors of measurements are shown as ±10%.

Mentions: The activity of the purified HrpRS complex was measured using an in vitro single-round transcription assay from the supercoiled hrpL promoter. As shown in Figure 2a, FL transcript formation required σ54-RNAP, dATP and HrpRS. Purified IHF greatly stimulated the amount of FL transcript obtained in vitro. Notably, the absence of FL transcripts with HrpR alone, HrpS alone or separately purified and mixed in vitro HrpR and HrpS (HrpR+HrpS), suggests that in vivo self-assembly of the HrpRS complex is important for forming the active HrpRS complex (Fig. 2b). HrpRS activated transcription when RNAP was reconstituted with either Klebsiella pneumoniae (Kp) or Ps σ54 (Fig. 2c), indicating that HrpRS can function independently of Ps-specific σ54.


Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity.

Jovanovic M, James EH, Burrows PC, Rego FG, Buck M, Schumacher J - Nat Commun (2011)

Regulation of the HrpRS complex in vitro.(a) In vitro full-length (FL) transcription assays (∼470 nucleotides) using the supercoiled hrpL promoter in the presence (+) or absence (−) of: σ54-RNAP (100 nM), dATP (4 mM), co-purified HrpRS (0.8 μM) and integration host factor (IHF) (20 nM). (b) HrpR alone (0.8 μM), HrpS alone (0.8 μM) and pre-mixed HrpR and HrpS (0.8 μM) (HrpR+HrpS) failed to activate transcription. (c) As in a, including dATP (4 mM), IHF (20 nM) with reconstituted σ54-RNAP using either purified σ54 from Ps (σ54-Ps) or from σ54 K. Pneumoniae (σ54-Kp). (d) HrpRS-dependent activation from the hrpL promoter as in a, in the presence of increasing HrpV concentrations. (e) Transcription from a test σ54 promoter (S. meliloti nifH), with the EBP AAA+ domain PspF1−275, or HrpRS, and in the presence (+) or absence (−) of HrpV (2 μM) and HrpG (2 μM). Gel images of transcripts for Figure 2a–e are shown in Supplementary Figure 4. (f) A bar graph showing electrophoretic mobility shifted hrpL DNA promoter probe comprising UAS sequences (in percentage of total hrpL DNA promoter signal) in the presence of HrpRS (2.5 μM) or HrpRS (2.5 μM and HrpV (4.2 μM). (g) A bar graph depicting the percentage of DNA bound in the closed promoter complex (CC) and transcriptionally proficient open promoter complex (OC; formed by PspF activation) in the presence of HrpRS–V or HrpV. (h) A bar graph depicting the relative amount of glutaraldehyde crosslinked HrpRS–HrpV species in either ATPase buffer (used to measure the ATPase activity of HrpRS+/−HrpV) or HGNED buffer. The relative amount of crosslinked HrpS–V complex was determined by fluorescence scanning. In f–h, estimated errors of measurements are shown as ±10%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f2: Regulation of the HrpRS complex in vitro.(a) In vitro full-length (FL) transcription assays (∼470 nucleotides) using the supercoiled hrpL promoter in the presence (+) or absence (−) of: σ54-RNAP (100 nM), dATP (4 mM), co-purified HrpRS (0.8 μM) and integration host factor (IHF) (20 nM). (b) HrpR alone (0.8 μM), HrpS alone (0.8 μM) and pre-mixed HrpR and HrpS (0.8 μM) (HrpR+HrpS) failed to activate transcription. (c) As in a, including dATP (4 mM), IHF (20 nM) with reconstituted σ54-RNAP using either purified σ54 from Ps (σ54-Ps) or from σ54 K. Pneumoniae (σ54-Kp). (d) HrpRS-dependent activation from the hrpL promoter as in a, in the presence of increasing HrpV concentrations. (e) Transcription from a test σ54 promoter (S. meliloti nifH), with the EBP AAA+ domain PspF1−275, or HrpRS, and in the presence (+) or absence (−) of HrpV (2 μM) and HrpG (2 μM). Gel images of transcripts for Figure 2a–e are shown in Supplementary Figure 4. (f) A bar graph showing electrophoretic mobility shifted hrpL DNA promoter probe comprising UAS sequences (in percentage of total hrpL DNA promoter signal) in the presence of HrpRS (2.5 μM) or HrpRS (2.5 μM and HrpV (4.2 μM). (g) A bar graph depicting the percentage of DNA bound in the closed promoter complex (CC) and transcriptionally proficient open promoter complex (OC; formed by PspF activation) in the presence of HrpRS–V or HrpV. (h) A bar graph depicting the relative amount of glutaraldehyde crosslinked HrpRS–HrpV species in either ATPase buffer (used to measure the ATPase activity of HrpRS+/−HrpV) or HGNED buffer. The relative amount of crosslinked HrpS–V complex was determined by fluorescence scanning. In f–h, estimated errors of measurements are shown as ±10%.
Mentions: The activity of the purified HrpRS complex was measured using an in vitro single-round transcription assay from the supercoiled hrpL promoter. As shown in Figure 2a, FL transcript formation required σ54-RNAP, dATP and HrpRS. Purified IHF greatly stimulated the amount of FL transcript obtained in vitro. Notably, the absence of FL transcripts with HrpR alone, HrpS alone or separately purified and mixed in vitro HrpR and HrpS (HrpR+HrpS), suggests that in vivo self-assembly of the HrpRS complex is important for forming the active HrpRS complex (Fig. 2b). HrpRS activated transcription when RNAP was reconstituted with either Klebsiella pneumoniae (Kp) or Ps σ54 (Fig. 2c), indicating that HrpRS can function independently of Ps-specific σ54.

Bottom Line: Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues.Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV.The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ(54)-RNA polymerase activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ(54)-dependent transcription at the hrpL promoter; triggering type-three secretion system-mediated pathogenicity. In contrast with singly acting EBPs, the evolution of the strictly co-operative HrpRS pair raises questions of potential benefits and mechanistic differences this transcription control system offers. Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues. Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV. The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ(54)-RNA polymerase activation.

Show MeSH
Related in: MedlinePlus