The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7.
Glutamine synthetase (GS), a key enzyme in biological nitrogen assimilation, is regulated in multiple ways in response to varying nitrogen sources and levels. Here we show a small regulatory RNA, NsiR4 (nitrogen stress-induced RNA 4), which plays an important role in the regulation of GS in cyanobacteria. NsiR4 expression in the unicellular Synechocystis sp. PCC 6803 and in the filamentous, nitrogen-fixing Anabaena sp. PCC 7120 is stimulated through nitrogen limitation via NtcA, the global transcriptional regulator of genes involved in nitrogen metabolism. NsiR4 is widely conserved throughout the cyanobacterial phylum, suggesting a conserved function. In silico target prediction, transcriptome profiling on pulse overexpression, and site-directed mutagenesis experiments using a heterologous reporter system showed that NsiR4 interacts with the 5'UTR of gifA mRNA, which encodes glutamine synthetase inactivating factor (IF)7. In Synechocystis, we observed an inverse relationship between the levels of NsiR4 and the accumulation of IF7 in vivo. This NsiR4-dependent modulation of gifA (IF7) mRNA accumulation influenced the glutamine pool and thus [Formula: see text] assimilation via GS. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between WT and an ΔnsiR4 KO mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis toward oscillating nitrogen levels. These results suggest a role for NsiR4 in the regulation of nitrogen metabolism in cyanobacteria, especially for the adaptation to rapid changes in available nitrogen sources and concentrations. NsiR4 is, to our knowledge, the first identified bacterial sRNA regulating the primary assimilation of a macronutrient.
Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany;
Stephan
S
Klähn
Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany;
Christoph
C
Schaal
Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany;
Jens
J
Georg
Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany;
Desirée
D
Baumgartner
Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany;
Gernot
G
Knippen
Plant Physiology Department, Institute of Biological Sciences, University of Rostock, D-18059 Rostock, Germany;
Martin
M
Hagemann
Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, E-41092 Sevilla, Spain.
Alicia M
AM
Muro-Pastor
Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany; wolfgang.hess@biologie.uni-freiburg.de.
Wolfgang R
WR
Hess
D001426:Bacterial Proteins / Q000378:metabolism*; D015152:Blotting, Northern; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors*; D015151:Immunoblotting; D046228:Microarray Analysis; D016297:Mutagenesis, Site-Directed; D009586:Nitrogen Fixation / Q000502:physiology*; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics / Q000502:physiology*; D046939:Synechocystis / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C120034:IF7 protein, Synechocystis sp.; D038621:Regulatory Sequences, Ribonucleic Acid; D014157:Transcription Factors; C075617:ntcA protein, Synechococcus; D005974:Glutamate-Ammonia Ligase
NtcA; Synechocystis; glutamine synthetase inactivating factors; nitrogen assimilation; regulatory RNA