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Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.

ABSTRACT: Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

SUBMITTER: Whitney JC

PROVIDER: S-EPMC4432266 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.

Whitney John C JC Whitfield Gregory B GB Marmont Lindsey S LS Yip Patrick P Neculai A Mirela AM Lobsanov Yuri D YD Robinson Howard H Ohman Dennis E DE Howell P Lynne PL

The Journal of biological chemistry 20150327 20

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we repo ...[more]

PMID: 25817996

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Project description:In Pseudomonas aeruginosa PAO1, 41 genes encode proteins predicted to be involved in the production or degradation of c-di-GMP, a ubiquitous secondary messenger that regulates a variety of physiological behaviors closely related to biofilm and aggregate formation. Despite extensive effort, the entire picture of this important signaling network is still unclear, with one-third of these proteins remaining uncharacterized. Here, we show that the deletion of pipA, which produces a protein containing two PAS domains upstream of a GGDEF-EAL tandem, significantly increased the intracellular c-di-GMP level and promoted the formation of aggregates both on surfaces and in planktonic cultures. However, this regulatory effect was not contributed by either of the two classic pathways modulating biofilm formation, exopolysaccharide (EPS) overproduction or motility inhibition. Transcriptome sequencing (RNA-Seq) data revealed that the expression levels of 361 genes were significantly altered in a ΔpipA mutant strain compared to the wild type (WT), indicating the critical role of PipA in PAO1. The most remarkably downregulated genes were located on the Pf4 bacteriophage gene cluster, which corresponded to a 2-log reduction in the Pf4 phage production in the ΔpipA mutant. The sizes of aggregates in ΔpipA cultures were affected by exogenously added Pf4 phage in a concentration-dependent manner, suggesting the quantity of phage plays a part in regulating the formation of aggregates. Further analysis demonstrated that PipA is highly conserved across 83 P. aeruginosa strains. Our work therefore for the first time showed that a c-di-GMP phosphodiesterase can regulate bacteriophage production and provided new insights into the relationship between bacteriophage and bacterial aggregation. IMPORTANCE The c-di-GMP signaling pathways in P. aeruginosa are highly organized and well coordinated, with different diguanylate cyclases and phosphodiesterases playing distinct roles in a complex network. Understanding the function of each enzyme and the underlying regulatory mechanisms not only is crucial for revealing how bacteria decide the transition between motile and sessile lifestyles, but also greatly facilitates the development of new antibiofilm strategies. This work identified bacteriophage production as a novel phenotypic output controlled transcriptionally by a phosphodiesterase, PipA. Further analysis suggested that the quantity of phage may be important in regulating autoaggregation, as either a lack of phage or overproduction was associated with higher levels of aggregation. Our study therefore extended the scope of c-di-GMP-controlled phenotypes and discovered a potential signaling circuit that can be target for biofilm treatment.

Dataset Information

Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.

Publications

Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.

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