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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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Phenotypic comparison of ΔretS mutation or overexpression of diguanylate cyclase on pellicle production and pel mRNA levels. A and B. Pellicle formation in static culture as revealed by crystal violet staining for (A) PA14, PA14ΔpelA, PA14ΔpelD and PA14ΔpelE harbouring vector control or induced for expression of diguanylate cyclases PA1107, PA1120 or PA3702; (B) PA14ΔretS, PA14ΔretSΔpelA, PA14ΔretSΔpelD and PA14ΔretSΔpelE. C. Expression of pel was determined by measuring β-galactosidase levels using a pelA–lacZ reporter construct in strains PA14, PA14ΔpelA, PA14ΔpelD and PA14ΔpelE harbouring vector control or induced for expression of diguanylate cyclases PA1107, PA1120 or PA3702. D. Quantification of PEL polysaccharide by binding to Congo red was normalized to total protein for strains PA14 pMMB-PA3702, PA14ΔpelD pMMB-PA3702, PA14ΔretS and PA14ΔretSΔpelD.
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fig01: Phenotypic comparison of ΔretS mutation or overexpression of diguanylate cyclase on pellicle production and pel mRNA levels. A and B. Pellicle formation in static culture as revealed by crystal violet staining for (A) PA14, PA14ΔpelA, PA14ΔpelD and PA14ΔpelE harbouring vector control or induced for expression of diguanylate cyclases PA1107, PA1120 or PA3702; (B) PA14ΔretS, PA14ΔretSΔpelA, PA14ΔretSΔpelD and PA14ΔretSΔpelE. C. Expression of pel was determined by measuring β-galactosidase levels using a pelA–lacZ reporter construct in strains PA14, PA14ΔpelA, PA14ΔpelD and PA14ΔpelE harbouring vector control or induced for expression of diguanylate cyclases PA1107, PA1120 or PA3702. D. Quantification of PEL polysaccharide by binding to Congo red was normalized to total protein for strains PA14 pMMB-PA3702, PA14ΔpelD pMMB-PA3702, PA14ΔretS and PA14ΔretSΔpelD.

Mentions: In-frame deletions of the pelA, pelD and pelE genes abrogated the ability of P. aeruginosa PA14 to produce biofilms in static culture in either the wild-type or ΔretS genetic background (Fig. 1A and B) (Vasseur et al., 2005). These results suggest that genes encoded by the pel operon are essential for pellicle formation. Using a lacZ transcriptional reporter fused to the pel promoter, we examined whether manipulating the levels of c-di-GMP by overexpressing proteins that have been previously shown to possess diguanylate cyclase activity would also effect the transcription of the pel operon (Kulasakara et al., 2006). In the wild-type PA14 parent carrying the pMMB vector alone, approximately 200 Miller units of β-galactosidase activity was detected and comparable expression of the pel–lacZ fusion was observed in the ΔpelA, ΔpelD or ΔpelE mutants (Fig. 1C). Induction of three independent diguanylate cyclases, PA1107, PA1120 and PA3702 (WspR), resulted in an increase in pel transcription in wild-type PA14 by five- to seven-fold, while in the ΔpelA, ΔpelD or ΔpelE backgrounds, transcription also increased by three-fold. These results indicate that high levels of cellular c-di-GMP enhance the level of pel mRNA. In each case, we consistently observed a higher induction of β-galactosidase in wild-type P. aeruginosa PA14 compared with ΔpelA, ΔpelD or ΔpelE mutants, suggesting a role for the functional PEL biosynthetic complex in regulating its own expression.


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)

Phenotypic comparison of ΔretS mutation or overexpression of diguanylate cyclase on pellicle production and pel mRNA levels. A and B. Pellicle formation in static culture as revealed by crystal violet staining for (A) PA14, PA14ΔpelA, PA14ΔpelD and PA14ΔpelE harbouring vector control or induced for expression of diguanylate cyclases PA1107, PA1120 or PA3702; (B) PA14ΔretS, PA14ΔretSΔpelA, PA14ΔretSΔpelD and PA14ΔretSΔpelE. C. Expression of pel was determined by measuring β-galactosidase levels using a pelA–lacZ reporter construct in strains PA14, PA14ΔpelA, PA14ΔpelD and PA14ΔpelE harbouring vector control or induced for expression of diguanylate cyclases PA1107, PA1120 or PA3702. D. Quantification of PEL polysaccharide by binding to Congo red was normalized to total protein for strains PA14 pMMB-PA3702, PA14ΔpelD pMMB-PA3702, PA14ΔretS and PA14ΔretSΔpelD.
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Related In: Results  -  Collection

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fig01: Phenotypic comparison of ΔretS mutation or overexpression of diguanylate cyclase on pellicle production and pel mRNA levels. A and B. Pellicle formation in static culture as revealed by crystal violet staining for (A) PA14, PA14ΔpelA, PA14ΔpelD and PA14ΔpelE harbouring vector control or induced for expression of diguanylate cyclases PA1107, PA1120 or PA3702; (B) PA14ΔretS, PA14ΔretSΔpelA, PA14ΔretSΔpelD and PA14ΔretSΔpelE. C. Expression of pel was determined by measuring β-galactosidase levels using a pelA–lacZ reporter construct in strains PA14, PA14ΔpelA, PA14ΔpelD and PA14ΔpelE harbouring vector control or induced for expression of diguanylate cyclases PA1107, PA1120 or PA3702. D. Quantification of PEL polysaccharide by binding to Congo red was normalized to total protein for strains PA14 pMMB-PA3702, PA14ΔpelD pMMB-PA3702, PA14ΔretS and PA14ΔretSΔpelD.
Mentions: In-frame deletions of the pelA, pelD and pelE genes abrogated the ability of P. aeruginosa PA14 to produce biofilms in static culture in either the wild-type or ΔretS genetic background (Fig. 1A and B) (Vasseur et al., 2005). These results suggest that genes encoded by the pel operon are essential for pellicle formation. Using a lacZ transcriptional reporter fused to the pel promoter, we examined whether manipulating the levels of c-di-GMP by overexpressing proteins that have been previously shown to possess diguanylate cyclase activity would also effect the transcription of the pel operon (Kulasakara et al., 2006). In the wild-type PA14 parent carrying the pMMB vector alone, approximately 200 Miller units of β-galactosidase activity was detected and comparable expression of the pel–lacZ fusion was observed in the ΔpelA, ΔpelD or ΔpelE mutants (Fig. 1C). Induction of three independent diguanylate cyclases, PA1107, PA1120 and PA3702 (WspR), resulted in an increase in pel transcription in wild-type PA14 by five- to seven-fold, while in the ΔpelA, ΔpelD or ΔpelE backgrounds, transcription also increased by three-fold. These results indicate that high levels of cellular c-di-GMP enhance the level of pel mRNA. In each case, we consistently observed a higher induction of β-galactosidase in wild-type P. aeruginosa PA14 compared with ΔpelA, ΔpelD or ΔpelE mutants, suggesting a role for the functional PEL biosynthetic complex in regulating its own expression.

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