The Signal Transduction Protein PII Controls the Levels of the Cyanobacterial Protein PipX

Abstract

Cyanobacteria, microorganisms performing oxygenic photosynthesis, must adapt their metabolic processes to environmental challenges such as day and night changes. PipX, a unique regulatory protein from cyanobacteria, provides a mechanistic link between the signalling protein PII, a widely conserved (in bacteria and plants) transducer of carbon/nitrogen/energy richness, and the transcriptional regulator NtcA, which controls a large regulon involved in nitrogen assimilation. PipX is also involved in translational regulation through interaction with the ribosome-assembly GTPase EngA. However, increases in the PipX/PII ratio are toxic, presumably due to the abnormally increased binding of PipX to other partner(s). Here, we present mutational and structural analyses of reported PipX-PII and PipX-NtcA complexes, leading to the identification of single amino acid changes that decrease or abolish PipX toxicity. Notably, 4 out of 11 mutations decreasing toxicity did not decrease PipX levels, suggesting that the targeted residues (F12, D23, L36, and R54) provide toxicity determinants. In addition, one of those four mutations (D23A) argued against the over-activation of NtcA as the cause of PipX toxicity. Most mutations at residues contacting PII decreased PipX levels, indicating that PipX stability would depend on its ability to bind to PII, a conclusion supported by the light-induced decrease of PipX levels in Synechococcus elongatus PCC7942 (hereafter S. elongatus).

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions

“…Since L65 is outside the TLD/KOW domain providing the contacts with NtcA, the reason for the rather drastic effect of L65Q on interactions with NtcA in the BACTH system is unclear. However, L65Q was identified as a spontaneous mutation suppressing PipX toxicity and it has recently been shown to decrease PipX levels in S. elongatus [20,54], which suggests the reduced formation of in vivo complexes between PipX L65Q and PII. Thus, interaction or functional assays indicated that the L65Q mutation did have phenotypic consequences that differed for PipX-PII and PipX-NtcA complexes.…”

“…Because PipX mutations impairing binding to PII also impair PipX levels in S. elongatus [23,54,61], it was relevant to know whether GlnB and/or GlnK proteins can perform the same chaperon role in E. coli; in which case, the mutations affecting binding to GlnB and/or GlnK and PipX self-interaction should also impair protein levels.…”

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions

“…Since L65 is outside the TLD/KOW domain providing the contacts with NtcA, the reason for the rather drastic effect of L65Q on interactions with NtcA in the BACTH system is unclear. However, L65Q was identified as a spontaneous mutation suppressing PipX toxicity and has been recently shown to decrease PipX levels in S. elongatus [20,55], which suggests reduced formation of in vivo complexes between PipX L65Q and PII. Thus, interaction or functional assays indicated that the L65Q mutation did have phenotypic consequences that differed for PipX-PII and PipX-NtcA complexes.…”

“…Because PipX mutations impairing binding to PII also impair PipX in levels S. elongatus [24,55], it was relevant to know whether GlnB and/or GlnK proteins can perform the same chaperon role in E. coli, in which case the mutations affecting binding to GlnB and/or GlnK and PipX self-interaction should also impair protein levels.…”

Analysing the Cyanobacterial PipX Interaction Network Using NanoBiT Complementation in <em>Synechococcus elongatus</em> PCC7942

“…Since PipX levels in S. elongatus are impaired by mutations decreasing binding to PII or by environmental conditions disrupting PipX-PII complexes [22,25,48], the prediction is that in the absence of PII the levels of PipX would be significantly impaired. To test this idea while seeking evidence that our NanoBiT derivatives maintain regulatory features, we next asked whether the presence of either PII or PII-LgBiT have a positive effect on the levels of PipX-SmBiT.…”