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A cyclic-di-GMP receptor required for bacterial exopolysaccharide production.

Lee VT, Matewish JM, Kessler JL, Hyodo M, Hayakawa Y, Lory S - Mol. Microbiol. (2007)

Bottom Line: Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide.Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP.The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been shown to be a global regulatory molecule that modulates the reciprocal responses of bacteria to activate either virulence pathways or biofilm formation. The mechanism of c-di-GMP signal transduction, including recognition of c-di-GMP and subsequent phenotypic regulation, remain largely uncharacterized. The key components of these regulatory pathways are the various adaptor proteins (c-di-GMP receptors). There is compelling evidence suggesting that, in addition to PilZ domains, there are other unidentified c-di-GMP receptors. Here we show that the PelD protein of Pseudomonas aeruginosa is a novel c-di-GMP receptor that mediates c-di-GMP regulation of PEL polysaccharide biosynthesis. Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide. Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP. The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.

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Role of conserved residues in PelD in c-di-GMP binding. A. Sequence alignment of PelD homologues. Similar residues are shaded in light grey and conserved residues (> 75%) are indicated by bold lettering. Residues conserved in all homologues are indicated by blue asterisks. Locations of point mutations introduced into PelD are indicate by red asterisks. B. Cartoon of the locations of introduced point mutations in the PelD sequence. C. Coomassie-stained SDS-PAGE of purified MBP-PelD with indicated point mutations. D. Binding of [32P]-c-di-GMP to each of the purified MBP-PelD fusion proteins.
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fig03: Role of conserved residues in PelD in c-di-GMP binding. A. Sequence alignment of PelD homologues. Similar residues are shaded in light grey and conserved residues (> 75%) are indicated by bold lettering. Residues conserved in all homologues are indicated by blue asterisks. Locations of point mutations introduced into PelD are indicate by red asterisks. B. Cartoon of the locations of introduced point mutations in the PelD sequence. C. Coomassie-stained SDS-PAGE of purified MBP-PelD with indicated point mutations. D. Binding of [32P]-c-di-GMP to each of the purified MBP-PelD fusion proteins.

Mentions: Analysis of the PelD sequence using blast algorithms identified gene clusters in different bacterial species that encode PelD orthologues (Fig. 3A). Sequence alignment of PelD orthologues revealed conservation of the four transmembrane domains and an additional number of highly conserved residues (Fig. 3A). To avoid disrupting folding of the protein, we chose only hydrophilic and charged amino acids in the region C-terminal to the transmembrane segments as indicated in the schematic in Fig. 3B. The wild-type PelD and each of the point mutations were expressed as fusions to 6-histidine-MBP and were purified on Ni-NTA agarose (Fig. 3C). The purified proteins, immobilized on Ni-NTA agarose, were then tested for their ability to retain [32P]-c-di-GMP (Fig. 3D). PelD with substitutions R131A, R161A, D305A and a mutant with two substitutions, S145A/H146A, retained the ability to bind [32P]-c-di-GMP, whereas proteins containing R367A, D370A and R402A were unable to bind [32P]-c-di-GMP. These results identify three critical charged residues in the C-terminus of PelD that are required for the protein to bind c-di-GMP.


A cyclic-di-GMP receptor required for bacterial exopolysaccharide production.

Lee VT, Matewish JM, Kessler JL, Hyodo M, Hayakawa Y, Lory S - Mol. Microbiol. (2007)

Role of conserved residues in PelD in c-di-GMP binding. A. Sequence alignment of PelD homologues. Similar residues are shaded in light grey and conserved residues (> 75%) are indicated by bold lettering. Residues conserved in all homologues are indicated by blue asterisks. Locations of point mutations introduced into PelD are indicate by red asterisks. B. Cartoon of the locations of introduced point mutations in the PelD sequence. C. Coomassie-stained SDS-PAGE of purified MBP-PelD with indicated point mutations. D. Binding of [32P]-c-di-GMP to each of the purified MBP-PelD fusion proteins.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2170427&req=5

fig03: Role of conserved residues in PelD in c-di-GMP binding. A. Sequence alignment of PelD homologues. Similar residues are shaded in light grey and conserved residues (> 75%) are indicated by bold lettering. Residues conserved in all homologues are indicated by blue asterisks. Locations of point mutations introduced into PelD are indicate by red asterisks. B. Cartoon of the locations of introduced point mutations in the PelD sequence. C. Coomassie-stained SDS-PAGE of purified MBP-PelD with indicated point mutations. D. Binding of [32P]-c-di-GMP to each of the purified MBP-PelD fusion proteins.
Mentions: Analysis of the PelD sequence using blast algorithms identified gene clusters in different bacterial species that encode PelD orthologues (Fig. 3A). Sequence alignment of PelD orthologues revealed conservation of the four transmembrane domains and an additional number of highly conserved residues (Fig. 3A). To avoid disrupting folding of the protein, we chose only hydrophilic and charged amino acids in the region C-terminal to the transmembrane segments as indicated in the schematic in Fig. 3B. The wild-type PelD and each of the point mutations were expressed as fusions to 6-histidine-MBP and were purified on Ni-NTA agarose (Fig. 3C). The purified proteins, immobilized on Ni-NTA agarose, were then tested for their ability to retain [32P]-c-di-GMP (Fig. 3D). PelD with substitutions R131A, R161A, D305A and a mutant with two substitutions, S145A/H146A, retained the ability to bind [32P]-c-di-GMP, whereas proteins containing R367A, D370A and R402A were unable to bind [32P]-c-di-GMP. These results identify three critical charged residues in the C-terminus of PelD that are required for the protein to bind c-di-GMP.

Bottom Line: Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide.Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP.The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been shown to be a global regulatory molecule that modulates the reciprocal responses of bacteria to activate either virulence pathways or biofilm formation. The mechanism of c-di-GMP signal transduction, including recognition of c-di-GMP and subsequent phenotypic regulation, remain largely uncharacterized. The key components of these regulatory pathways are the various adaptor proteins (c-di-GMP receptors). There is compelling evidence suggesting that, in addition to PilZ domains, there are other unidentified c-di-GMP receptors. Here we show that the PelD protein of Pseudomonas aeruginosa is a novel c-di-GMP receptor that mediates c-di-GMP regulation of PEL polysaccharide biosynthesis. Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide. Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP. The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.

Show MeSH