The First Proteomic Study of Nostoc sp. PCC 7120 Exposed to Cyanotoxin BMAA under Nitrogen Starvation

Abstract

The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid.

Proteomic Insights into Starvation of Nitrogen-Replete Cells of Nostoc sp. PCC 7120 under β-N-Methylamino-L-Alanine (BMAA) Treatment

“…In order to test BMAA effect on Nostoc under nitrogen-replete conditions, cyanobacterium was grown in three independent biological replicates in sodium nitrate containing BG11N medium for 48 h in two experimental settings: (1) control samples consisted of cells grown without the BMAA treatment and (2) the treated samples consisted of cells grown with addition of an aqueous solution of BMAA (20 µM), as it was performed earlier [ 22 , 23 ]. The analysis of cyanobacteria samples by using LC-MS/MS methods resulted in the identification of 1567 different proteins of Nostoc ( Supplementary Table S1 ).…”

“…The PII signal-transduction protein plays a key role in the control of various nitrogen-related transporters, enzymes and transcription factors ( Figure 1 and Figure 2 ) [ 28 ]. In our previous proteomic study [ 23 ], we found that BMAA downregulated the PII protein in nitrogen-starved Nostoc cells that should have formed heterocysts, however, this process is repressed by BMAA [ 21 ]. Remarkably, in nitrogen starvation conditions, the addition of BMAA alters the expression of the nitrate transporter nrtA ( alr0608 ) [ 23 ], a partner of PII protein [ 28 , 29 ].…”

“…For that reason, BMAA effects were studied, so far mostly in mammalian models, for review [ 15 ]. Knowledge on the synthesis mechanisms of this neurotoxic non-protein amino acid and on the functional role of BMAA in cyanobacteria cells is still scant [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. There are a few experimental studies dedicated to the notable and specific biological impact of BMAA on nitrogen-fixing filamentous cyanobacterium Nostoc (also known as Anabaena ) sp.…”

“…There are a few experimental studies dedicated to the notable and specific biological impact of BMAA on nitrogen-fixing filamentous cyanobacterium Nostoc (also known as Anabaena ) sp. PCC 7120 [ 20 , 21 , 22 , 23 ], which is known to be a favorable model organism for nitrogen fixation and heterocyst differentiation studies [ 24 ]. Its filaments contain only vegetative cells in the presence of combined nitrogen in the form of ammonium (NH 4 + ) or nitrate (NO 3− ) ions.…”

“…PCC 7120 [ 21 ]. Recently, we demonstrated experimentally, by using a proteomic approach, that under nitrogen starvation, the BMAA addition downregulates the key nitrogen regulatory protein PII (GlnB), glutamyl-tRNA synthetase ( gltX ) enzyme and different proteins involved in nitrogen metabolism and heterocyst formation, as well as proteins involved in carbon fixation and photosynthesis in cyanobacteria cells [ 23 ].…”