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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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In vivo transcription and protein–protein interactions of Hrp proteins.(a) Domain organization of EBPs showing the seven conserved regions (C1–C7) and their associated functions. HrpR and HrpS key motif sequences are given with the sites of amino-acid substitutions (used in c, d, e below) indicated (black typeface numbering refers to amino-acid position in either HrpR or HrpS). (b) In vivo transcription activation activities measured from the chromosomal promoter hrpL∷lacZ reporter construct—given as a percentage of WT co-expressed (single operon) HrpRS activity (where WT=100%)—of singly expressed HrpR or HrpS, and co-expressed HrpR+HrpS (HrpR+S) (from different plasmids). HrpV expression abolishes HrpRS in vivo activity (HrpRS+HrpV), which is partly relieved when co-expressing HrpG (HrpRS+HrpV+HrpG). (c) As in b transcription activation by HrpRS, where HrpR carries mutations as indicated. (d) As in b transcription activation by HrpRS, where HrpS carries mutations as indicated. (e) As in b transcription activation by HrpRS, where HrpS carries the gain of function mutation Y85F. (f) Bar graph depicting results of the BACTH protein–protein binding interactions between HrpR (black), HrpS (light grey), HrpS1−275 (dark grey), HrpV (white), HrpG (grey) inferred by β-galactosidase production and displayed in Miller Units (MU)32. B/ground represents the background level of β-galactosidase activity detected for the negative control (30 MU—obtained with the two-hybrid vectors in the absence of protein fusions). (g) Scheme of the expression construct for the BACTH three component system. (h) Bar graph depicting the binding interactions between T25-fused proteins HrpR, HrpS, HrpV and HrpG with T18C-fused HrpS in the presence of co-expressed HrpV in a three-component two-hybrid system35 inferred by β-galactosidase production and displayed in Miller Units. In b–e, all assays were minimally performed in triplicate and standard errors of the mean are shown. Protein expression was verified by western blotting (see Supplementary Fig. S3).
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f1: In vivo transcription and protein–protein interactions of Hrp proteins.(a) Domain organization of EBPs showing the seven conserved regions (C1–C7) and their associated functions. HrpR and HrpS key motif sequences are given with the sites of amino-acid substitutions (used in c, d, e below) indicated (black typeface numbering refers to amino-acid position in either HrpR or HrpS). (b) In vivo transcription activation activities measured from the chromosomal promoter hrpL∷lacZ reporter construct—given as a percentage of WT co-expressed (single operon) HrpRS activity (where WT=100%)—of singly expressed HrpR or HrpS, and co-expressed HrpR+HrpS (HrpR+S) (from different plasmids). HrpV expression abolishes HrpRS in vivo activity (HrpRS+HrpV), which is partly relieved when co-expressing HrpG (HrpRS+HrpV+HrpG). (c) As in b transcription activation by HrpRS, where HrpR carries mutations as indicated. (d) As in b transcription activation by HrpRS, where HrpS carries mutations as indicated. (e) As in b transcription activation by HrpRS, where HrpS carries the gain of function mutation Y85F. (f) Bar graph depicting results of the BACTH protein–protein binding interactions between HrpR (black), HrpS (light grey), HrpS1−275 (dark grey), HrpV (white), HrpG (grey) inferred by β-galactosidase production and displayed in Miller Units (MU)32. B/ground represents the background level of β-galactosidase activity detected for the negative control (30 MU—obtained with the two-hybrid vectors in the absence of protein fusions). (g) Scheme of the expression construct for the BACTH three component system. (h) Bar graph depicting the binding interactions between T25-fused proteins HrpR, HrpS, HrpV and HrpG with T18C-fused HrpS in the presence of co-expressed HrpV in a three-component two-hybrid system35 inferred by β-galactosidase production and displayed in Miller Units. In b–e, all assays were minimally performed in triplicate and standard errors of the mean are shown. Protein expression was verified by western blotting (see Supplementary Fig. S3).

Mentions: In Ps (including DC3000), hrpR and hrpS are arranged in tandem (hrpRS), transcribed as a single operon8 and exhibit high sequence similarity, suggesting that they have evolved from a single ancestral gene duplication event. A search for the highest similar sequences for group I-type pathogenicity islands (PAIs) identified ten non-redundant hrpRS tandem arrangements (in Ps pathovars) and ten single-hrpS sequences (in Erwinia-type pathovars as well as the hrpS homologue rspR from P. fluorescens). All 20 inspected PAIs also harbour the regulatory genes hrpV and hrpG, including P. mendocina (a P. aeruginosa-related human pathogen20), suggesting that the appearance of hrpRS is more recent than that of the presumed hrpS regulatory genes (hrpV and hrpG). Although regulatory roles for HrpV and HrpG remain to be established for non-syringae strains they have been clearly annotated as hrpV and hrpG across genera and the synteny of the intra-operon loci are strictly conserved. A phylogenetic tree based on protein sequences derived from the 30 EBPs obtained is shown in Supplementary Figure S1. Where present HrpR and HrpS have 55–65% sequence identity and 70–79% sequence similarity to each other (Supplementary Table S1—compared with other well-studied bacterial EBPs, such as ZraR, PspF, NtrC1, which have 38–41% identity; Supplementary Table S2). Strikingly, hrpRS was found in all Ps pathovars, making the dual hrpRS EBP system a distinguishing feature in the hrp/hrc gene cluster of group I PAI in Ps pathovars. Further, no polymorphism (hrpRS or hrpS) has been reported for Ps. We did not find polymorphism among identified PAI (including among redundant sequences); strongly suggesting that hrpRS in Ps provides a selective advantage compared with singly acting hrpS. The exact branch pattern symmetry between the HrpSRS and HrpR groups (Supplementary Fig. S1) suggests that following gene duplication, hrpR and hrpS may not have diverged freely as is commonly assumed in gene duplication due to functional redundancy2122. Inspection of the HrpR and HrpS sequences of Ps DC3000 and their predicted structures indicate no obvious basis of their co-dependency. They show clear congruence with other EBPs (Supplementary Fig. S2), in that the seven conserved regions identified for all EBPs and the conserved AAA+ motifs are present (Fig. 1a)5.


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)

In vivo transcription and protein–protein interactions of Hrp proteins.(a) Domain organization of EBPs showing the seven conserved regions (C1–C7) and their associated functions. HrpR and HrpS key motif sequences are given with the sites of amino-acid substitutions (used in c, d, e below) indicated (black typeface numbering refers to amino-acid position in either HrpR or HrpS). (b) In vivo transcription activation activities measured from the chromosomal promoter hrpL∷lacZ reporter construct—given as a percentage of WT co-expressed (single operon) HrpRS activity (where WT=100%)—of singly expressed HrpR or HrpS, and co-expressed HrpR+HrpS (HrpR+S) (from different plasmids). HrpV expression abolishes HrpRS in vivo activity (HrpRS+HrpV), which is partly relieved when co-expressing HrpG (HrpRS+HrpV+HrpG). (c) As in b transcription activation by HrpRS, where HrpR carries mutations as indicated. (d) As in b transcription activation by HrpRS, where HrpS carries mutations as indicated. (e) As in b transcription activation by HrpRS, where HrpS carries the gain of function mutation Y85F. (f) Bar graph depicting results of the BACTH protein–protein binding interactions between HrpR (black), HrpS (light grey), HrpS1−275 (dark grey), HrpV (white), HrpG (grey) inferred by β-galactosidase production and displayed in Miller Units (MU)32. B/ground represents the background level of β-galactosidase activity detected for the negative control (30 MU—obtained with the two-hybrid vectors in the absence of protein fusions). (g) Scheme of the expression construct for the BACTH three component system. (h) Bar graph depicting the binding interactions between T25-fused proteins HrpR, HrpS, HrpV and HrpG with T18C-fused HrpS in the presence of co-expressed HrpV in a three-component two-hybrid system35 inferred by β-galactosidase production and displayed in Miller Units. In b–e, all assays were minimally performed in triplicate and standard errors of the mean are shown. Protein expression was verified by western blotting (see Supplementary Fig. S3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: In vivo transcription and protein–protein interactions of Hrp proteins.(a) Domain organization of EBPs showing the seven conserved regions (C1–C7) and their associated functions. HrpR and HrpS key motif sequences are given with the sites of amino-acid substitutions (used in c, d, e below) indicated (black typeface numbering refers to amino-acid position in either HrpR or HrpS). (b) In vivo transcription activation activities measured from the chromosomal promoter hrpL∷lacZ reporter construct—given as a percentage of WT co-expressed (single operon) HrpRS activity (where WT=100%)—of singly expressed HrpR or HrpS, and co-expressed HrpR+HrpS (HrpR+S) (from different plasmids). HrpV expression abolishes HrpRS in vivo activity (HrpRS+HrpV), which is partly relieved when co-expressing HrpG (HrpRS+HrpV+HrpG). (c) As in b transcription activation by HrpRS, where HrpR carries mutations as indicated. (d) As in b transcription activation by HrpRS, where HrpS carries mutations as indicated. (e) As in b transcription activation by HrpRS, where HrpS carries the gain of function mutation Y85F. (f) Bar graph depicting results of the BACTH protein–protein binding interactions between HrpR (black), HrpS (light grey), HrpS1−275 (dark grey), HrpV (white), HrpG (grey) inferred by β-galactosidase production and displayed in Miller Units (MU)32. B/ground represents the background level of β-galactosidase activity detected for the negative control (30 MU—obtained with the two-hybrid vectors in the absence of protein fusions). (g) Scheme of the expression construct for the BACTH three component system. (h) Bar graph depicting the binding interactions between T25-fused proteins HrpR, HrpS, HrpV and HrpG with T18C-fused HrpS in the presence of co-expressed HrpV in a three-component two-hybrid system35 inferred by β-galactosidase production and displayed in Miller Units. In b–e, all assays were minimally performed in triplicate and standard errors of the mean are shown. Protein expression was verified by western blotting (see Supplementary Fig. S3).
Mentions: In Ps (including DC3000), hrpR and hrpS are arranged in tandem (hrpRS), transcribed as a single operon8 and exhibit high sequence similarity, suggesting that they have evolved from a single ancestral gene duplication event. A search for the highest similar sequences for group I-type pathogenicity islands (PAIs) identified ten non-redundant hrpRS tandem arrangements (in Ps pathovars) and ten single-hrpS sequences (in Erwinia-type pathovars as well as the hrpS homologue rspR from P. fluorescens). All 20 inspected PAIs also harbour the regulatory genes hrpV and hrpG, including P. mendocina (a P. aeruginosa-related human pathogen20), suggesting that the appearance of hrpRS is more recent than that of the presumed hrpS regulatory genes (hrpV and hrpG). Although regulatory roles for HrpV and HrpG remain to be established for non-syringae strains they have been clearly annotated as hrpV and hrpG across genera and the synteny of the intra-operon loci are strictly conserved. A phylogenetic tree based on protein sequences derived from the 30 EBPs obtained is shown in Supplementary Figure S1. Where present HrpR and HrpS have 55–65% sequence identity and 70–79% sequence similarity to each other (Supplementary Table S1—compared with other well-studied bacterial EBPs, such as ZraR, PspF, NtrC1, which have 38–41% identity; Supplementary Table S2). Strikingly, hrpRS was found in all Ps pathovars, making the dual hrpRS EBP system a distinguishing feature in the hrp/hrc gene cluster of group I PAI in Ps pathovars. Further, no polymorphism (hrpRS or hrpS) has been reported for Ps. We did not find polymorphism among identified PAI (including among redundant sequences); strongly suggesting that hrpRS in Ps provides a selective advantage compared with singly acting hrpS. The exact branch pattern symmetry between the HrpSRS and HrpR groups (Supplementary Fig. S1) suggests that following gene duplication, hrpR and hrpS may not have diverged freely as is commonly assumed in gene duplication due to functional redundancy2122. Inspection of the HrpR and HrpS sequences of Ps DC3000 and their predicted structures indicate no obvious basis of their co-dependency. They show clear congruence with other EBPs (Supplementary Fig. S2), in that the seven conserved regions identified for all EBPs and the conserved AAA+ motifs are present (Fig. 1a)5.

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