Mutational Analysis of the Cyanobacterial Nitrogen Regulator PipX

PipX provides a functional link between the cyanobacterial global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as integrators of signals of the nitrogen and carbon balance. PipX, which is toxic in the absence of PII, can form alternative complexes with NtcA and PII and these interactions are respectively stimulated and inhibited by 2-oxoglutarate, providing a mechanism by which PII can modulate expression at the NtcA regulon. Structural information on PipX-NtcA complexes suggests that PipX coactivates NtcA controlled genes by stabilizing the active conformation of NtcA bound to 2-oxoglutarate and by possibly helping recruit RNA polymerase. To get insights into PipX functions, we perform here a mutational analysis of pipX informed by the structures of PipX-PII and PipX-NtcA complexes and evaluate the impact of point mutations on toxicity and gene expression. Two amino acid substitutions (Y32A and E4A) were of particular interest, since they increased PipX toxicity and activated NtcA dependent genes in vivo at lower 2-oxoglutarate levels than wild type PipX. While both mutations impaired complex formation with PII, only Y32A had a negative impact on PipX-NtcA interactions

Nitrogen interaction network in the cyanobacterium Synechococcus WH5701, a model organism with two PipX and two PII-like proteins

Resumen del póster presentado en ESF-EMBO Symposium, Molecular Bioenergetics of Cyanobacteria: From Cell to Community, Sant Feliu de Guixols, 10-15 April 2011

Mutations at pipX Suppress Lethality of PII-Deficient Mutants of Synechococcus elongatus PCC 7942▿

The PII proteins are found in all three domains of life as key integrators of signals reflecting the balance of nitrogen and carbon. Genetic inactivation of PII proteins is typically associated with severe growth defects or death. However, the molecular basis of these defects depends on the specific functions of the proteins with which PII proteins interact to regulate nitrogen metabolism in different organisms. In Synechococcus elongatus PCC 7942, where PII forms complexes with the NtcA coactivator PipX, attempts to engineer PII-deficient strains failed in a wild-type background but were successful in pipX null mutants. Consistent with the idea that PII is essential to counteract the activity of PipX, four different spontaneous mutations in the pipX gene were found in cultures in which glnB had been genetically inactivated

The nitrogen interaction network in Synechococcus WH5701, a cyanobacterium with two PipX and two PII-like proteins

Nitrogen regulation involves the formation of different types of protein complexes between signal transducers and their transcriptional or metabolic targets. In oxygenic phototrophs, the signal integrator PII activates the enzyme N-acetyl-l-glutamate kinase (NAGK) by complex formation. PII also interacts with PipX, a protein with a tudor-like domain that mediates contacts with PII and with the transcriptional regulator NtcA, to which it binds to increase its activity. Here, we use a combination of in silico, yeast two-hybrid and in vitro approaches to investigate the nitrogen regulation network of Synechococcus WH5701, a marine cyanobacterium with two PII (GlnB_A and GlnB_B) and two PipX (PipX_I and PipX_II) proteins. Our results indicate that GlnB_A is functionally equivalent to the canonical PII protein from Synechococcus elongatus. GlnB_A interacted with PipX and NAGK proteins and stimulated NAGK activity, counteracting arginine inhibition. GlnB_B had only a slight stimulatory effect on NAGK activity, but its potential to bind effectors and form heterotrimers in Synechococcus WH5701 indicates additional regulatory functions. PipX_II, and less evidently PipX_I, specifically interacted with GlnB_A and NtcA, supporting a role for both Synechococcus WH5701 PipX proteins in partner swapping with GlnB_A and NtcA.This work was supported by grants from Ministerio de Ciencia e Innovación (BFU2009-07371) and Ministerio de Educación y Ciencia (HA2007-0074)

Mutational analysis of PipX: effect of point mutations on NtcA activation and toxicity in the absence of PII

Resumen de la comunicación presentada en ESF-EMBO Symposium, Molecular Bioenergetics of Cyanobacteria: From Cell to Community, Sant Feliu de Guixols, 10-15 April 2011

Effect of point mutations on PipX levels.

<p>(A) Immunodetection of PipX from strains expressing wild type PipX (CS3X) and the indicated PipX point mutation derivatives (Y32A, E4A) alongside a <i>pipX</i> null strain (X^-) using an Anti-PipX antibody. Detection of endogenous PipX and recombinant H₆-PipX (10 ng loaded in a control lane) is indicated with black and grey arrowhead, respectively. 60 µg of protein extract were loaded per lane. A protein loading and transfer quality control is shown on the bottom panel. (B) Amplification of <i>pipX</i> and <i>rnpB</i> (used as a loading control) by RT-PCR in the CS3X and point mutant derivatives (E4A and Y32A) grown in the presence of nitrate. A representative experiment from two independent RNA extractions is shown.</p

Localization of PipX residues discussed in this work. Details of interactions between one PipX monomer and P_II

<p>(<b>A</b>) <b>or NtcA</b> (<b>B</b>) <b>are shown in the P_II-PipX (PDB file 2XG8) and NtcA-PipX complexes (PDB file 2XKO).</b> PipX structures are represented in blue ribbon while surface representation and pale colours are used for P_II (subunits in grey, yellow and pink) and NtcA (grey and cyan). In each case, the enlargement of PipX to the right shows relevant side chains of residues mutated to alanine (except Q34, mutated to glutamate). Mutated residues that increase, decrease or do not affect PipX toxicity are colored in red, green, and gold, respectively.</p

Effect of PipX substitutions in yeast two hybrid interaction signals.

<p>(A) An illustrative yeast two hybrid Interaction assay involving wild type PipX, P_II, NtcA and NAGK proteins fused to either GAL4AD and GAL4BD. Photographs show growths on diploid media (CONTROL), on histidine (<i>HIS3</i>) and adenine (<i>ADE2</i>) minus media, and x-gal test (<i>lacZ</i>) on a diploid plate. (B) Yeast two hybrid interaction pattern of PipX point mutants. Growth of diploids carrying GAL4AD:P_II or GAL4AD:NtcA and the wild type (PipX) or the BD:PipX substitutions indicated in bold type (representative for each group of proteins), on minus histidine medium. The table summarizes the level of interaction signals between fusions of GAL4AD and GAL4BD to PipX derivatives and their complementary domains fused to P_II, NtcA and NAGK (negative control), as indicated. Signs (from +++ to -) indicate levels of yeast two-hybrid expression from <i>HIS3</i>, <i>ADE2</i> and <i>lacZ</i> reporters according to previously described conventions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035845#pone.0035845-Burillo1" target="_blank">[8]</a>.</p

<i>S. elongatus</i> strains used in this work.

a<p>named as SA410 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035845#pone.0035845-Espinosa1" target="_blank">[11]</a>.</p>*<p>The asterisk represents, for CK1X strain derivatives, the following substitutions at PipX: Y6A, F12A, D23A, Q34E, R35A, L36A, F38A, R69A, Q82A and Q86A. For all CS3X derived strains it also represents substitutions E4A and Y32A.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p