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

Abstract

All cyanobacteria produce a neurotoxic non-protein amino acid β-N-methylamino-L-alanine (BMAA). However, the biological function of BMAA in the regulation of cyanobacteria metabolism still remains undetermined. It is known that BMAA suppresses the formation of heterocysts in diazotrophic cyanobacteria under nitrogen starvation conditions, and BMAA induces the formation of heterocyst-like cells under nitrogen excess conditions, by causing the expression of heterocyst-specific genes that are usually “silent” under nitrogen-replete conditions, as if these bacteria receive a nitrogen deficiency intracellular molecular signal. In order to find out the molecular mechanisms underlying this unexpected BMAA effect, we studied the proteome of cyanobacterium Nostoc sp. PCC 7120 grown under BMAA treatment in nitrogen-replete medium. Experiments were performed in two experimental settings: (1) in 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). In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by LC-MS/MS spectrometry. Among them, 80 proteins belonging to different functional categories were chosen for further functional analysis and interpretation of obtained proteomic data. Here, we provide the evidence that a pleiotropic regulatory effect of BMAA on the proteome of cyanobacterium was largely different under conditions of nitrogen-excess compared to its effect under nitrogen starvation conditions (that was studied in our previous work). The most significant difference in proteome expression between the BMAA-treated and untreated samples under different growth conditions was detected in key regulatory protein PII (GlnB). BMAA downregulates protein PII in nitrogen-starved cells and upregulates this protein in nitrogen-replete conditions. PII protein is a key signal transduction protein and the change in its regulation leads to the change of many other regulatory proteins, including different transcriptional factors, enzymes and transporters. Complex changes in key metabolic and regulatory proteins (RbcL, RbcS, Rca, CmpA, GltS, NodM, thioredoxin 1, RpbD, ClpP, MinD, RecA, etc.), detected in this experimental study, could be a reason for the appearance of the “starvation” state in nitrogen-replete conditions in the presence of BMAA. In addition, 15 proteins identified in this study are encoded by genes, which are under the control of NtcA—a global transcriptional regulator—one of the main protein partners and transcriptional regulators of PII protein. Thereby, this proteomic study gives a possible explanation of cyanobacterium starvation under nitrogen-replete conditions and BMAA treatment. It allows to take a closer look at the regulation of cyanobacteria metabolism affected by this cyanotoxin.

β-N-Methylamino-L-Alanine (BMAA) Causes Severe Stress in Nostoc sp. PCC 7120 Cells under Diazotrophic Conditions: A Proteomic Study

“…Both described mechanisms can explain BMAA’s impact on nitrogen fixation. It was demonstrated in two previous proteomic studies [ 23 , 24 ] and in the present study ( Table 2 , Section 2.6 ) that BMAA affects enzymes involved in glutamate metabolism. At the same time, the discussed above reaction of BMAA with pyridoxal-5′-phosphate can occur, leading to oxidative stress, which can trigger diverse defense mechanisms.…”

“…To elucidate the effect of BMAA on Nostoc PCC 7120 under diazotrophic conditions cyanobacteria cells were grown in three independent biological replicates in nitrogen-free BG11 0 medium for 48 h in two experimental settings, in which: (1) control samples consisted of cells grown without BMAA addition and (2) treated samples contained cells grown with BMAA (20 µmol), as it was performed before [ 23 , 24 ]. The analysis of cyanobacteria samples was performed by using the LC-MS/MS method and resulted in identifying 1567 proteins of Nostoc PCC 7120 ( Supplementary Table S1 ).…”

“…Urease subunit α ( alr3670 ) [EC:3.5.1.5] is downregulated in the way it changes in nitrogen starvation growth conditions [ 23 ]. However, in nitrogen-replete conditions, this subunit is upregulated under BMAA treatment [ 24 ]. Urease subunit α is the central functional part of the nickel-containing metalloenzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia [ 61 ].…”

“…In our previous works we have demonstrated by using different experimental approaches that the BMAA’s impact on diazotrophic filamentous cyanobacteria is connected with nitrogen-carbon balance regulation, and is different under nitrogen starvation and in the nitrogen-replete growth medium [ 17 , 18 , 23 , 24 ] ( Figure 1 ). The most significant difference in proteome expression consists of regulating a key nitrogen regulatory protein PII under BMAA treatment.…”

“…The most significant difference in proteome expression consists of regulating a key nitrogen regulatory protein PII under BMAA treatment. This protein is downregulated during nitrogen-starvation, and it is upregulated in nitrogen-rich growth conditions [ 23 , 24 ]. This fact is consistent with the specific regulatory effect of BMAA on heterocyst differentiation and heterocyst- and nitrogenase-related gene expression in Nostoc sp.…”