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The PII protein-protein interaction network in Azospirillum brasilense reveals a novel role of PII in the control of c-di-GMP homeostasis

ABSTRACT: The PII family comprises a group of widely distributed signal transduction proteins ubiquitous present in prokaryotes and in the chloroplast of plants. The PII proteins sense the levels of key metabolites ATP, ADP and 2-oxoglutarate which affect the PII structure and the ability of PII to interact with a range of target proteins. Here we performed multiple PII ligand fishing assays to identify proteins that are likely to be part of the PII protein interaction network in plant-growth promoting nitrogen-fixing bacterium Azospirillum brasilense. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, co-enzyme synthesis, RNA catabolism and transcription. Direct binary PII-target complex was confirmed for xx protein complex using pull-down assays with recombinant proteins. Untargeted metabolome showed that PII is required to proper maintenance of Coenzyme A, panthotenate, PRPP and SAICAR levels. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII minus strain showed reduced c-di-GMP levels and swimming behavior. These data support that PII acts as a major metabolic hub controlling key metabolic enzymes and c-di-GMP homeostasis accordingly to the prevailing nutritional status.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Azospirillum Brasilense Sp245

SUBMITTER: Gustavo De Souza

LAB HEAD: Luciano Huergo

PROVIDER: PXD018530 | Pride | 2020-10-23

REPOSITORIES: Pride

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Project description:Bacterial lifestyles are dictated by the conditions encountered during colonization of a specific ecological niche or host. A wide range of environmental stimuli triggers sensory modules, that modify activity of proteins, or/and initiate a transfer of information in complex regulatory networks, which modulate gene expression, and therefore adaptation. The transition between planktonic and biofilm growth is dependent on the homeostasis of the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclase (DGC) activity favor biofilm while low levels maintained by phosphodiesterase (PDE) enzymes encourage planktonic style or biofilm dispersal. In Pseudomonas aeruginosa over 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual enzymes possessing both canonical DGC and PDE motifs, i.e. GGDEF and EAL, respectively. It has been previously reported that the deletion of the PDE/DGC dual enzyme PA0285, one of 5 c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms and causes elevated c-di-GMP levels in P. aeruginosa. Here we confirm that this role is conserved in various P. aeruginosa (PAO1, PA14 and PAK) and Pseudomonas putida strains. Deletion of PA0285 has the highest impact during the early stage of surface colonization, and RNA-seq analysis in biofilm context indicates that it may be associated with upregulation of cupA fimbrial genes. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds the respective GTP (GGDEF) and c-di-GMP (EAL) substrates but only exhibits PDE activity in vitro. Both GGDEF and EAL domains are important for PA0285 PDE activity in vivo as suggested by site-directed mutants in each of the key motifs. Complementation of the PA0285 mutant strain with the C-terminal GGDEF/EAL portion in trans is not as effective as with the full-length gene suggesting that the transmembrane region and PAS domains contained in PA0285’s N-terminal portion influence its PDE activity in vivo. We speculate that the transmembrane portion or/and PAS domains of PA0285 impede its PDE activity in vivo through modulating the conformation of the enzyme, as shown for P. aeruginosa RbdA, in response to an unknown stimulus.

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The PII protein-protein interaction network in Azospirillum brasilense reveals a novel role of PII in the control of c-di-GMP homeostasis

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