The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense

Abstract

The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status. IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense. The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.

Arabidopsis PII Proteins Form Characteristic Foci in Chloroplasts Indicating Novel Properties in Protein Interaction and Degradation

“…In addition to well characterized interactors like AtNAGK, AtBCCP1 and 2, and AtBADC1-3 [12] the number of proteins as well as their functional range seems to be growing. This resembles the situation found for bacterial PII homologs and their interaction partners: For the PII homolog GlnZ from Azospirillum brasilense 37 interaction partners could be identified in ligand fishing assays [4]. In the cyanobacterium Synechocystis sp.…”

“…The T-loop can adopt various conformations, depending on the effector molecules ATP, ADP or Mg-ATP plus 2-oxoglutarate, bound in the three effector binding sites, which are located in the clefts between the subunits. Thereby the levels of the carbon/nitrogen status-reporter 2-oxoglutarate as well as the energy state, sensed by the ATP to ADP ratio, are integrated by PII and allow PII to control a multitude of cellular functions, mainly related to nitrogen assimilation but also to central carbon flux and other core features of metabolism such as NAD-synthesis [3,4].…”

“…The signalling principles are highly conserved as compared to heterotrophic bacteria, however, cyanobacteria appear to have evolved some specific PII regulatory targets. The controlling enzyme of the arginine pathway, N-acetyl-L-glutamate kinase (NAGK), appears to be a dominant target in these organisms, although recent studies revealed PII-NAGK interaction also in non-photosynthetic bacteria [4]. Moreover, in cyanobacteria PII controls the flux of newly fixed carbon by controlling a protein that acts as inhibitor of phosphoglycerate mutase (PGAM) [6].…”

CdgC, a Cyclic-di-GMP Diguanylate Cyclase of Azospirillum baldaniorum Is Involved in Internalization to Wheat Roots

“…Accordingly, we observed that the cdgC gene is involved in the formation of biofilms and exopolysaccharide production under nitrogen fixation ( Figures 2 , 3B ), as previously outlined in A. brasilense Sp7 and Pseudomonas stutzeri ( Bible et al, 2015 ; Wang et al, 2017 ; Shang et al, 2021 ). The role of the second messenger c-di-GMP levels in cells grown under nitrogen-fixing conditions has been described in A. brasilense , where the PII protein, which is a global regulator of nitrogen fixation, additionally mediates the control of homeostasis at the cell level of c-di-GMP in vivo , modulating cell motility, aerotaxis, and adherence behaviors, which are regulated through binding with two enzymes involved in the biosynthesis of c-di-GMP, albeit the genetic and biochemical mechanisms involved in this regulation have not yet been described ( Gerhardt et al, 2020 ). Biofilm formation in cells grown in NFB ∗ medium supplemented with KNO 3 as a nitrogen source ( Figure 3A ), was not affected by the absence of CdgC.…”

Split NanoLuc technology allows quantitation of interactions between PII protein and its receptors with unprecedented sensitivity and reveals transient interactions

“…This is achieved through transient interactions with PII, which alter the activities of the PII-receptor proteins. Although initially thought to be confined to the regulation of nitrogen metabolism, research in the past years has unveiled a plethora of PII regulated proteins 2 , 3 .…”