1 Nitrogen interaction network in Synechococcus WH 5701 , a cyanobacterium 2 with two PipX and two PII - like proteins 3

2 Nitrogen regulation involves formation of different types of proteins complexes 3 between signal transducers and their transcriptional or metabolic targets. In oxygenic 4 phototrophs, the signal integrator PII activates the enzyme N-acetyl-L-glutamate kinase 5 (NAGK) by complex formation. PII also interacts with PipX, a protein with a tudor-like 6 domain that mediates contacts with PII and with the transcriptional regulator NtcA, to 7 which it binds to increase its activity. Here we use a combination of in silico, yeast two8 hybrid and in vitro approaches to investigate the nitrogen regulation network of 9 Synechococcus WH5701, a marine cyanobacterium with two PII (GlnB_A and GlnB_B) 10 and two PipX (PipX_I and PipX_II) proteins. Our results indicate that GlnB_A is 11 functionally equivalent to the canonical PII protein from S. elongatus. GlnB_A 12 interacted with PipX and NAGK proteins and stimulated NAGK activity counteracting 13 arginine inhibition. GlnB_B had only a slight stimulatory effect on NAGK activity, but 14 its potential to bind effectors and form heterotrimers in Synechococcus WH5701 15 indicates additional regulatory functions. PipX_II, and less evidently PipX_I, 16 specifically interacted with GlnB_A and NtcA, supporting a role for both 17 Synechococcus WH5701 PipX proteins in partner swapping with GlnB_A and NtcA. 18