Analysing the Cyanobacterial PipX Interaction Network Using NanoBiT Complementation in Synechococcus elongatus PCC7942

Abstract

The conserved cyanobacterial protein PipX is part of a complex interaction network with regulators involved in essential processes that include metabolic homeostasis and ribosome assembly. Because PipX interactions depend on the relative levels of their different partners and of the effector molecules binding to them, in vivo studies are required to understand the physiological significance and contribution of environmental factors to the regulation of PipX complexes. Here, we have used the NanoBiT complementation system to analyse the regulation of complex formation in Synechococcus elongatus PCC 7942 between PipX and each of its two best-characterized partners, PII and NtcA. Our results confirm previous in vitro analyses on the regulation of PipX-PII and PipX-NtcA complexes by 2-oxoglutarate and on the regulation of PipX-PII by the ATP/ADP ratio, showing the disruption of PipX-NtcA complexes due to increased levels of ADP-bound PII in Synechococcus elongatus. The demonstration of a positive role of PII on PipX-NtcA complexes during their initial response to nitrogen starvation or the impact of a PipX point mutation on the activity of PipX-PII and PipX-NtcA reporters are further indications of the sensitivity of the system. This study reveals additional regulatory complexities in the PipX interaction network, opening a path for future research on cyanobacteria.

Keywords: 2-oxoglutarate; NanoLuc; NtcA; PCAs; PII; complementation reporter; energy regulation; environmental factors; nitrogen regulation; protein-fragment complementation assays.

Figure 1

NanoBiT constructs and strategy used to analyse the PipX-PII and PipX-NtcA interactions in S. elongatus. (A) The NSI region and derivatives containing the C.S3 selection marker and the corresponding gene fusions are schematically illustrated, with the relevant products depicted to the right. * refers to PipX or PipX^Y6A. (B) Schematic representation of the pipX and glnB alleles. (C) Left panel: PCR analysis indicating the primers, depicted as black arrows, in (A,B) and the size of bands at the left and right, respectively. M: λ EcoRI/HindIII size marker. Right panel: strains analysed. See text for additional details.

Figure 2

Levels of PipX and PII derivatives in S. elongatus. Representative immunodetection of PipX and PII of S. elongatus strains differing in their NSI constructs or genetic background (in brackets) as indicated. Relative PipX and PII levels were normalized by the PlmA signal and referred to the WT. Data are presented as means and error bars (standard deviation) from three biological replicates.

Figure 3

Real-time responses of PipX-PII and PipX-NtcA reporters to changes in energy levels and nitrogen sources. Reporter strains are indicated, and relevant proteins illustrated on top of the corresponding results. (A) Bioluminescence signals (black scale and curves) and normalized ATP levels (red scale and curves) from cultures grown with BG11 in the presence or absence of 200 µM DCCD. (B) Bioluminescence signals after transfer to the indicated nitrogen regimens. Data in (A,B) are presented as means with error bars (standard deviation) due to the indicated number of biological replicates (top rectangles) performed in each case. Wilcoxon rank-sum tests between the indicated comparisons produced p-values < 0.05 (*). (a–c) refer to the indicated nitrogen conditions.

Figure 4

Regulation of PipX levels and PipX-NtcA interactions by PII in response to nitrogen deprivation. (A) Representative immunodetection and relative levels of PipX (PipX) from S. elongatus cultures after two centrifuge/washing steps with BG11₀ (-N), BG11 (Fresh) or the same BG11 supernatant (Used), normalised to the intensity shown in the same blot by endogenous PlmA, and respective to the “Used” values. Data are presented as means and error bars (standard deviation) from five biological replicates of two independent experiments. Wilcoxon rank-sum test produced p-values < 0.05 (*) (B) Real-time comparison of bioluminescence signals under the indicated nitrogen regimens at different times between the pipX (Δ) and pipXglnB (○) strains. Data are presented as means and error bars (standard deviation) from two biological replicates. Other details as in Figure 3.

Figure 5

Impact of the mutation Y6A on PipX-PII and PipX-NtcA interactions. Bioluminescence signal of the indicated strains under different nitrogen regimen conditions at timepoints 0 and 60′. Black, grey, and red lines correspond to the WT or Y6A versions of the reporter and to the PipX control, respectively. Data are presented as means and error bars (standard deviation) from the indicated biological replicates (squares inside the graphics). Inset covers enlarge their corresponding regions.