Modulation of oxidative stress machinery determines the contrasting ability of cyanobacteria to adapt to Se(VI) or Se(IV).

Excess of selenium (Se) in aquatic ecosystems has necessitated thorough investigations into the effects/consequences of this metalloid on the autochthonous organisms exposed to it. The molecular details of Se-mediated adaptive response remain unknown in cyanobacteria. This study aims to uncover the molecular mechanisms driving the divergent physiological responses of cyanobacteria on exposure to selenate [Se(VI)] or selenite [Se(IV)], the two major water-soluble oxyanions of Se. The cyanobacterium, Anabaena PCC 7120, withstood 0.4 mM of Se(VI), whereas even 0.1 mM of Se(IV) was detrimental, affecting photosynthesis and enhancing endogenous ROS. Surprisingly, Anabaena pre-treated with Se(VI), but not Se(IV), showed increased tolerance to oxidative stress mediated by H2O2/methyl viologen. RNA-Seq analysis showed Se(VI) to elevate transcription of genes encoding anti-oxidant proteins and Fe-S cluster biogenesis, whereas the photosynthesis-associated genes, which were mainly downregulated by Se(IV), remained unaffected. Specifically, the content of typical 2-Cys-Prx (Alr4641), a redox-maintaining protein in Anabaena, was elevated with Se(VI). In comparison to the wild-type, the Anabaena strain over-expressing the Alr4641 protein (An4641+) showed enhanced tolerance to Se(VI) stress, whereas the corresponding knockdown-strain (KD4641) was sensitive to this stressor. Incidentally, among these strains, only An4641+ was better protected from the ROS-mediated damage caused by high dose of Se(VI). These results suggest that altering the content of the antioxidant protein 2-Cys-Prx, could be a potential strategy for modulating resistance to selenate. Thus, involvement of oxidative stress machinery appears to be the major determinant, responsible for the contrasting physiological differences observed in response to selenate/selenite in cyanobacteria.

The atypical thioredoxin 'Alr2205', a newly identified partner of the typical 2-Cys-Peroxiredoxin, safeguards the cyanobacterium Anabaena from oxidative stress.

Thioredoxins (Trxs) are ubiquitous proteins that play vital roles in several physiological processes. Alr2205, a thioredoxin-like protein from Anabaena PCC 7120, was found to be evolutionarily closer to the Trx-domain of the NADPH-Thioredoxin Reductase C than the other thioredoxins. The Alr2205 protein showed disulfide reductase activity despite the presence a non-canonical active site motif 'CPSC'. Alr2205 not only physically interacted with, but also acted as a physiological reductant of Alr4641 (the typical 2-Cys-Peroxiredoxin from Anabaena), supporting its peroxidase function. Structurally, Alr2205 was a monomeric protein that formed an intramolecular disulfide bond between the two active site cysteines (Cys-38 and Cys-41). However, the Alr2205C41S protein, wherein the resolving cysteine was mutated to serine, was capable of forming intermolecular disulfide bond and exist as a dimer when treated with H2O2. Overproduction of Alr2205 in E. coli protected cells from heavy metals, but not oxidative stress. To delve into its physiological role, Alr2205/Alr2205C41S was overexpressed in Anabaena, and the ability of the corresponding strains (An2205+ or An2205C41S+) to withstand environmental stresses was assessed. An2205+ showed higher resistance to H2O2 than An2205C41S+, indicating that the disulfide reductase function of this protein was critical to protect cells from this peroxide. Although, An2205+ did not show increased capability to withstand cadmium stress, An2205C41S+ was more susceptible to this heavy metal. This is the first study that provides a vital understanding into the function of atypical thioredoxins in countering the toxic effects of heavy metals/H2O2 in prokaryotes.

Voyage of selenium from environment to life: Beneficial or toxic?

Selenium (Se), a naturally occurring metalloid, is an essential micronutrient for life as it is incorporated as selenocysteine in proteins. Although beneficial at low doses, Se is hazardous at high concentrations and poses a serious threat to various ecosystems. Due to this contrasting 'dual' nature, Se has garnered the attention of researchers wishing to unravel its puzzling properties. In this review, we describe the impact of selenium's journey from environment to diverse biological systems, with an emphasis on its chemical advantage. We describe the uneven distribution of Se and how this affects the bioavailability of this element, which, in turn, profoundly affects the habitat of a region. Once taken up, the subsequent incorporation of Se into proteins as selenocysteine and its antioxidant functions are detailed here. The causes of improved protein function due to the incorporation of redox-active Se atom (instead of S) are examined. Subsequently, the reasons for the deleterious effects of Se, which depend on its chemical form (organo-selenium or the inorganic forms) in different organisms are elaborated. Although Se is vital for the function of many antioxidant enzymes, how the pro-oxidant nature of Se can be potentially exploited in different therapies is highlighted. Furthermore, we succinctly explain how the presence of Se in biological systems offsets the toxic effects of heavy metal mercury. Finally, the different avenues of research that are fundamental to expand our understanding of selenium biology are suggested.

Loss of 2-Cys-Prx affects cellular ultrastructure, disturbs redox poise and impairs photosynthesis in cyanobacteria.

In a striking similarity to plant chloroplasts, the cyanobacterium Anabaena displays very low catalase activity, but expresses several peroxiredoxins (Prxs), including the typical 2-Cys-Prx (annotated as Alr4641), that detoxify H2 O2 . Due to the presence of multiple Prxs, the precise contribution of Alr4641 to the oxidative stress response of Anabaena is not well-defined. To unambiguously assess its in vivo function, the Alr4641 protein was knocked down using the CRISPRi approach in Anabaena PCC 7120. The knockdown strain (An-KD4641), which showed over 85% decrease in the content of Alr4641, was viable, but grew slower than the control strain (An-dCas9). An-KD4641 showed elevated levels of reactive oxygen species and the expression of several redox-responsive genes was analogous to that of An-dCas9 subjected to oxidative stress. The knockdown strain displayed reduced filament size, altered thylakoid ultrastructure, a marked drop in the ratio of phycocyanin to chlorophyll a and decreased photosynthetic parameters compared to An-dCas9. In comparison to the control strain, exposure to H2 O2 had a more severe effect on the photosynthetic parameters or survival of An-KD4641. Thus, in the absence of adequate catalase activity, 2-Cys-Prx appears to be the principal Prx responsible for maintaining redox homoeostasis in diverse photosynthetic systems ranging from chloroplasts to cyanobacteria.

Unique functional insights into the antioxidant response of the cyanobacterial Mn-catalase (KatB).

KatB, a hexameric Mn-catalase, plays a vital role in overcoming oxidative and salinity stress in the ecologically important, N2-fixing cyanobacterium, Anabaena. The 5 N-terminal residues of KatB, which show a high degree of conservation in cyanobacteria, form an antiparallel ß-strand at the subunit interface of the KatB hexamer. In this study, the contribution of these N-terminal non-active site residues, towards the maintenance of the structure, biochemical properties, and redox balance was evaluated. Each N-terminal amino acid residue from the 2nd to the 7th position of KatB was individually mutated to Ala (to express KatBF2A/KatBF3A/KatBH4A/KatBK5E/KatBK6A/KatBE7A) or this entire 6 amino acid stretch was deleted (to yield KatBTrunc). All the above-mentioned KatB variants, along with the wild-type KatB protein (KatBWT), were overproduced in E. coli and purified. In comparison to KatBWT, the KatBF2A/KatBH4A/KatBTrunc proteins were less compact, more prone to chemical/thermal denaturation, and were unexpectedly inactive. KatBF3A/KatBK5E/KatBK6A showed biophysical/biochemical properties that were in between that of KatBWT and KatBF2A/KatBH4A/KatBTrunc. Surprisingly, KatBE7A was more thermostable with higher activity than KatBWT. On exposure to H2O2, E. coli expressing KatBWT/KatBE7A showed considerably reduced formation of ROS and increased survival than the other KatB variants. Utilizing the KatB structure, the molecular basis responsible for the altered stability/activity of the KatB mutants was delineated. This study demonstrates the physiological importance of the N-terminal ß-strand of Mn-catalases in combating H2O2 stress and shows that the non-active site residues can be used for rational protein engineering to develop Mn-catalases with improved characteristics.

Functional and mechanistic insights into the differential effect of the toxicant 'Se(IV)' in the cyanobacterium Anabaena PCC 7120.

Selenium, an essential trace element for animals, poses a threat to all forms of life above a threshold concentration. The ubiquitously present cyanobacteria, a major photosynthetic biotic component of aquatic and other ecosystems, are excellent systems to study the effects of environmental toxicants. The molecular changes that led to beneficial or detrimental effects in response to different doses of selenium oxyanion Se(IV) were analyzed in the filamentous cyanobacterium Anabaena PCC 7120. This organism showed no inhibition in growth up to 15 mg/L sodium selenite, but above this dose i.e. 20-100 mg/L of Se(IV), both growth and photosynthesis were substantially inhibited. Along with the increased accumulation of non-protein thiols, a consistent reduction in levels of ROS was observed at 10 mg/mL dose of Se(IV). High dose of Se(IV) (above 20 mg/L) enhanced endogenous reactive oxygen species (ROS)/lipid peroxidation, and decreased photosynthetic capability. Treatment with 100 mg/L Se(IV) downregulated transcription of several photosynthesis pathways-related genes such as those encoding photosystem I and II proteins, phycobilisome rod-core linker protein, phycocyanobilin, phycoerythrocyanin-associated proteins etc. Interestingly, at a dose range of 10-15 mg/L Se(IV), Anabaena showed an increase in PSII photosynthetic yield and electron transport rate (at PSII), suggesting improved photosynthesis. Se was incorporated into the Anabaena cells, and Se-enriched thylakoid membranes showed higher redox conductivity than the thylakoid membranes from untreated cells. Overall, the data supports that modulation of photosynthetic machinery is one of the crucial mechanisms responsible for the dose-dependent contrasting effect of Se(IV) observed in Anabaena.

Novel molecular aspects of the CRISPR backbone protein 'Cas7' from cyanobacteria.

The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.