title: Expanding the Cyanobacterial Nitrogen Regulatory Network: The GntR-Like Regulator PlmA Interacts with the PII-PipX Complex.
abstract: Cyanobacteria, phototrophic organisms that perform oxygenic photosynthesis, perceive nitrogen status by sensing 2-oxoglutarate levels. PII, a widespread signaling protein, senses and transduces nitrogen and energy status to target proteins, regulating metabolism and gene expression. In cyanobacteria, under conditions of low 2-oxoglutarate, PII forms complexes with the enzyme N-acetyl glutamate kinase, increasing arginine biosynthesis, and with PII-interacting protein X (PipX), making PipX unavailable for binding and co-activation of the nitrogen regulator NtcA. Both the PII-PipX complex structure andin vivofunctional data suggested that this complex, as such, could have regulatory functions in addition to PipX sequestration. To investigate this possibility we performed yeast three-hybrid screening of genomic libraries fromSynechococcus elongatusPCC7942, searching for proteins interacting simultaneously with PII and PipX. The only prey clone found in the search expressed PlmA, a member of the GntR family of transcriptional regulators proven here by gel filtration to be homodimeric. Interactions analyses further confirmed the simultaneous requirement of PII and PipX, and showed that the PlmA contacts involve PipX elements exposed in the PII-PipX complex, specifically the C-terminal helices and one residue of the tudor-like body. In contrast, PII appears not to interact directly with PlmA, possibly being needed indirectly, to induce an extended conformation of the C-terminal helices of PipX and for modulating the surface polarity at the PII-PipX boundary, two elements that appear crucial for PlmA binding. Attempts to inactiveplmAconfirmed that this gene is essential inS. elongatus. Western blot assays revealed thatS. elongatusPlmA, irrespective of the nitrogen regime, is a relatively abundant transcriptional regulator, suggesting the existence of a large PlmA regulon.In silicostudies showed that PlmA is universally and exclusively found in cyanobacteria. Based on interaction data, on the relative amounts of the proteins involved in PII-PipX-PlmA complexes, determined in western assays, and on the restrictions imposed by the symmetries of trimeric PII and dimeric PlmA molecules, a structural and regulatory model for PlmA function is discussed in the context of the cyanobacterial nitrogen interaction network.
authors.affiliation: Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante Alicante, Spain.
authors.forename: Jose I
authors.initials: JI
authors.lastname: Labella
authors.affiliation: Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante Alicante, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Obrebska
authors.affiliation: Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante Alicante, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Espinosa
authors.affiliation: Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante Alicante, Spain.
authors.forename: Paloma
authors.initials: P
authors.lastname: Salinas
authors.affiliation: Instituto de Biomedicina de Valencia of the CSIC Valencia, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Forcada-Nadal
authors.affiliation: Instituto de Biomedicina de Valencia of the CSIC Valencia, Spain.
authors.forename: Lorena
authors.initials: L
authors.lastname: Tremiño
authors.affiliation: Instituto de Biomedicina de Valencia of the CSICValencia, Spain; Group 739, CIBER de Enfermedades Raras (CIBERER-ISCIII)Valencia, Spain.
authors.forename: Vicente
authors.initials: V
authors.lastname: Rubio
authors.affiliation: Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante Alicante, Spain.
authors.forename: Asunción
authors.initials: A
authors.lastname: Contreras
keywords: GntR; PII; PipX; PlmA; cyanobacteria; nitrogen regulation; signaling; three hybrid interactions