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Interaction of the cyclic-di-GMP binding protein FimX and the Type 4 pilus assembly ATPase promotes pilus assembly.

ABSTRACT: Type IVa pili (T4P) are bacterial surface structures that enable motility, adhesion, biofilm formation and virulence. T4P are assembled by nanomachines that span the bacterial cell envelope. Cycles of T4P assembly and retraction, powered by the ATPases PilB and PilT, allow bacteria to attach to and pull themselves along surfaces, so-called "twitching motility". These opposing ATPase activities must be coordinated and T4P assembly limited to one pole for bacteria to show directional movement. How this occurs is still incompletely understood. Herein, we show that the c-di-GMP binding protein FimX, which is required for T4P assembly in Pseudomonas aeruginosa, localizes to the leading pole of twitching bacteria. Polar FimX localization requires both the presence of T4P assembly machine proteins and the assembly ATPase PilB. PilB itself loses its polar localization pattern when FimX is absent. We use two different approaches to confirm that FimX and PilB interact in vivo and in vitro, and further show that point mutant alleles of FimX that do not bind c-di-GMP also do not interact with PilB. Lastly, we demonstrate that FimX positively regulates T4P assembly and twitching motility by promoting the activity of the PilB ATPase, and not by stabilizing assembled pili or by preventing PilT-mediated retraction. Mutated alleles of FimX that no longer bind c-di-GMP do not allow rapid T4P assembly in these assays. We propose that by virtue of its high-affinity for c-di-GMP, FimX can promote T4P assembly when intracellular levels of this cyclic nucleotide are low. As P. aeruginosa PilB is not itself a high-affinity c-di-GMP receptor, unlike many other assembly ATPases, FimX may play a key role in coupling T4P mediated motility and adhesion to levels of this second messenger.

SUBMITTER: Jain R

PROVIDER: S-EPMC5595344 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Interaction of the cyclic-di-GMP binding protein FimX and the Type 4 pilus assembly ATPase promotes pilus assembly.

Jain Ruchi R Sliusarenko Oleksii O Kazmierczak Barbara I BI

PLoS pathogens 20170830 8

Type IVa pili (T4P) are bacterial surface structures that enable motility, adhesion, biofilm formation and virulence. T4P are assembled by nanomachines that span the bacterial cell envelope. Cycles of T4P assembly and retraction, powered by the ATPases PilB and PilT, allow bacteria to attach to and pull themselves along surfaces, so-called "twitching motility". These opposing ATPase activities must be coordinated and T4P assembly limited to one pole for bacteria to show directional movement. How ...[more]

PMID: 28854278

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Project description:UnlabelledHigh levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein.ImportanceIn many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.

Dataset Information

Interaction of the cyclic-di-GMP binding protein FimX and the Type 4 pilus assembly ATPase promotes pilus assembly.

Publications

Interaction of the cyclic-di-GMP binding protein FimX and the Type 4 pilus assembly ATPase promotes pilus assembly.

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