Expanding the cyanobacterial nitrogen regulatory network: The GntR-like regulator PlmA interacts with the PII-PipX complex
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...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2016-10-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.01677/full |
| _version_ | 1819093363557662720 |
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| author | Jose Ignacio Labella Anna Obrebska Javier Espinosa Paloma Salinas Alicia Forcada-Nadal Lorena Tremiño Vicente Rubio Asunción Contreras |
| author_facet | Jose Ignacio Labella Anna Obrebska Javier Espinosa Paloma Salinas Alicia Forcada-Nadal Lorena Tremiño Vicente Rubio Asunción Contreras |
| author_sort | Jose Ignacio Labella |
| collection | DOAJ |
| description | 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 and in vivo functional 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 from Synechococcus elongatus PCC7942, 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 inactive plmA confirmed that this gene is essential in S. elongatus. Western blot assays revealed that S. elongatus PlmA, irrespective of the nitrogen regime, is a relatively abundant transcriptional regulator, suggesting the existence of a large PlmA regulon. In silico studies 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. |
| first_indexed | 2024-12-21T23:10:19Z |
| format | Article |
| id | doaj.art-6608eff73d6f4febba017fafa4b368b1 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-21T23:10:19Z |
| publishDate | 2016-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-6608eff73d6f4febba017fafa4b368b12022-12-21T18:47:02ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2016-10-01710.3389/fmicb.2016.01677223742Expanding the cyanobacterial nitrogen regulatory network: The GntR-like regulator PlmA interacts with the PII-PipX complexJose Ignacio Labella0Anna Obrebska1Javier Espinosa2Paloma Salinas3Alicia Forcada-Nadal4Lorena Tremiño5Vicente Rubio6Asunción Contreras7Universidad de AlicanteUniversidad de AlicanteUniversidad de AlicanteUniversidad de AlicanteInstituto de Biomedicina de Valencia (IBV-CSIC)Instituto de Biomedicina de Valencia (IBV-CSIC)Instituto de Biomedicina de Valencia (IBV-CSIC)Universidad de AlicanteCyanobacteria, 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 and in vivo functional 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 from Synechococcus elongatus PCC7942, 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 inactive plmA confirmed that this gene is essential in S. elongatus. Western blot assays revealed that S. elongatus PlmA, irrespective of the nitrogen regime, is a relatively abundant transcriptional regulator, suggesting the existence of a large PlmA regulon. In silico studies 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.http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.01677/fullCyanobacteriasignallingPIINitrogen regulationPipXPLMA |
| spellingShingle | Jose Ignacio Labella Anna Obrebska Javier Espinosa Paloma Salinas Alicia Forcada-Nadal Lorena Tremiño Vicente Rubio Asunción Contreras Expanding the cyanobacterial nitrogen regulatory network: The GntR-like regulator PlmA interacts with the PII-PipX complex Frontiers in Microbiology Cyanobacteria signalling PII Nitrogen regulation PipX PLMA |
| title | Expanding the cyanobacterial nitrogen regulatory network: The GntR-like regulator PlmA interacts with the PII-PipX complex |
| title_full | Expanding the cyanobacterial nitrogen regulatory network: The GntR-like regulator PlmA interacts with the PII-PipX complex |
| title_fullStr | Expanding the cyanobacterial nitrogen regulatory network: The GntR-like regulator PlmA interacts with the PII-PipX complex |
| title_full_unstemmed | Expanding the cyanobacterial nitrogen regulatory network: The GntR-like regulator PlmA interacts with the PII-PipX complex |
| title_short | Expanding the cyanobacterial nitrogen regulatory network: The GntR-like regulator PlmA interacts with the PII-PipX complex |
| title_sort | expanding the cyanobacterial nitrogen regulatory network the gntr like regulator plma interacts with the pii pipx complex |
| topic | Cyanobacteria signalling PII Nitrogen regulation PipX PLMA |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.01677/full |
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