mRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent in situ hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.

Nanoscale elemental and morphological imaging of nitrogen-fixing cyanobacteria.

Nitrogen-fixing cyanobacteria bind atmospheric nitrogen and carbon dioxide using sunlight. This experimental study focused on a laboratory-based model system, Anabaena sp., in nitrogen-depleted culture. When combined nitrogen is scarce, the filamentous prokaryotes reconcile photosynthesis and nitrogen fixation by cellular differentiation into heterocysts. To better understand the influence of micronutrients on cellular function, 2D and 3D synchrotron X-ray fluorescence mappings were acquired from whole biological cells in their frozen-hydrated state at the Bionanoprobe, Advanced Photon Source. To study elemental homeostasis within these chain-like organisms, biologically relevant elements were mapped using X-ray fluorescence spectroscopy and energy-dispersive X-ray microanalysis. Higher levels of cytosolic K+, Ca2+, and Fe2+ were measured in the heterocyst than in adjacent vegetative cells, supporting the notion of elevated micronutrient demand. P-rich clusters, identified as polyphosphate bodies involved in nutrient storage, metal detoxification, and osmotic regulation, were consistently co-localized with K+ and occasionally sequestered Mg2+, Ca2+, Fe2+, and Mn2+ ions. Machine-learning-based k-mean clustering revealed that P/K clusters were associated with either Fe or Ca, with Fe and Ca clusters also occurring individually. In accordance with XRF nanotomography, distinct P/K-containing clusters close to the cellular envelope were surrounded by larger Ca-rich clusters. The transition metal Fe, which is a part of nitrogenase enzyme, was detected as irregularly shaped clusters. The elemental composition and cellular morphology of diazotrophic Anabaena sp. was visualized by multimodal imaging using atomic force microscopy, scanning electron microscopy, and fluorescence microscopy. This paper discusses the first experimental results obtained with a combined in-line optical and X-ray fluorescence microscope at the Bionanoprobe.

Biosorption Ability of Pharmaceutically Active Compounds by Anabaena sp. and Chroococcidiopsis thermalis.

Drug overuse harms the biosphere, leading to disturbances in ecosystems' functioning. Consequently, more and more actions are being taken to minimise the harmful impact of xenopharmaceuticals on the environment. One of the innovative solutions is using biosorbents-natural materials such as cells or biopolymers-to remove environmental pollutants; however, this focuses mainly on the removal of metal ions and colourants. Therefore, this study investigated the biosorption ability of selected pharmaceuticals-paracetamol, diclofenac, and ibuprofen-by the biomass of the cyanobacteria Anabaena sp. and Chroococcidiopsis thermalis, using the LC-MS/MS technique. The viability of the cyanobacteria was assessed by determining photosynthetic pigments in cells using a UV-VIS spectrophotometer. The results indicate that both tested species can be effective biosorbents for paracetamol and diclofenac. At the same time, the tested compounds did not have a toxic effect on the tested cyanobacterial species and, in some cases, stimulated their cell growth. Furthermore, the Anabaena sp. can effectively biotransform DCF into its dimer.

Development of a base editor for convenient and multiplex genome editing in cyanobacteria.

Cyanobacteria are important primary producers, contributing to 25% of the global carbon fixation through photosynthesis. They serve as model organisms to study the photosynthesis, and are important cell factories for synthetic biology. To enable efficient genetic dissection and metabolic engineering in cyanobacteria, effective and accurate genetic manipulation tools are required. However, genetic manipulation in cyanobacteria by the conventional homologous recombination-based method and the recently developed CRISPR-Cas gene editing system require complicated cloning steps, especially during multi-site editing and single base mutation. This restricts the extensive research on cyanobacteria and reduces its application potential. In this study, a highly efficient and convenient cytosine base editing system was developed which allows rapid and precise C → T point mutation and gene inactivation in the genomes of Synechocystis and Anabaena. This base editing system also enables efficient multiplex editing and can be easily cured after editing by sucrose counter-selection. This work will expand the knowledge base regarding the engineering of cyanobacteria. The findings of this study will encourage the biotechnological applications of cyanobacteria.

Assessing thallium phycoremediation by applying Anabaena laxa and Nostoc muscorum and exploring its effect on cellular growth, antioxidant, and metabolic profile.

Thallium (Tl), a key element in high-tech industries, is recognized as a priority pollutant by the US EPA and EC. Tl accumulation threatens aquatic ecosystems. Despite its toxicity, little is known about its impact on cyanobacteria. This study explores the biochemical mechanisms of Tl(I) toxicity in cyanobacteria, focusing on physiology, metabolism, oxidative damage, and antioxidant responses. To this end, Anabaena and Nostoc were exposed to 400 µg/L, and 800 µg/L of Tl(I) over seven days. Anabaena showed superior Tl(I) accumulation with 7.8% removal at 400 µg/L and 9.5% at 800 µg/L, while Nostoc removed 2.2% and 7.4%, respectively. Tl(I) exposure significantly reduced the photosynthesis rate and function, more than in Nostoc. It also altered primary metabolism, increasing sugar levels and led to higher amino and fatty acids levels. While Tl(I) induced cellular damage in both species, Anabaena was less affected. Both species enhanced their antioxidant defense systems, with Anabaena showing a 175.6% increase in SOD levels under a high Tl(I) dose. This suggests that Anabaena's robust biosorption and antioxidant systems could be effective for Tl(I) removal. The study improves our understanding of Tl(I) toxicity, tolerance, and phycoremediation in cyanobacteria, aiding future bioremediation strategies.

This study presents novel insights into thallium (Tl) phycoremediation using Anabaena laxa and Nostoc muscorum, crucial for addressing the increasing contamination concerns stemming from high-tech industries. Elucidating the tolerance mechanisms and physiological responses of these cyanobacterial species to Tl(I) exposure. It highlights the potential of Anabaena laxa as an effective bio-remediator, offering a sustainable solution to mitigate Tl(I) environmental impact.

Unravelling the involvement of protein disorder in cyanobacterial stress responses.

This study has explored the involvement of Intrinsically Disordered Proteins (IDPs) in cyanobacterial stress response. IDPs possess distinct physicochemical properties, which allow them to execute diverse functions. Anabaena PCC 7120, the model photosynthetic, nitrogen-fixing cyanobacterium encodes 688 proteins (11 % of the total proteome) with at least one intrinsically disordered region (IDR). Of these, 130 proteins that showed >30 % overall disorder were designated as IDPs. Physico-chemical analysis, showed these IDPs to adopt shapes ranging from 'globular' to 'tadpole-like'. Upon exposure to NaCl, 41 IDP-encoding genes were found to be differentially expressed. Surprisingly, most of these were induced, indicating the importance of IDP-accumulation in overcoming salt stress. Subsequently, six IDPs were identified to be induced by multiple stresses (salt, ammonium and selenite). Interestingly, the presence of these 6-multiple stress-induced IDPs was conserved in filamentous cyanobacteria. Utilizing the experimental proteomic data of Anabaena, these 6 IDPs were found to interact with many proteins involved in diverse pathways, underscoring their physiological importance as protein hubs. This study lays the framework for IDP-related research in Anabaena by (a) identifying, as well as physiochemically characterizing, all the disordered proteins and (b) uncovering a subset of IDPs that are likely to be critical in adaptation to environmental stresses.

The cumulative impact of temperature and nitrogen availability on the potential nitrogen fixation and extracellular polymeric substances secretion by Dolichospermum.

Nitrogen-fixing cyanobacteria not only cause severe blooms but also play an important role in the nitrogen input processes of lakes. The production of extracellular polymeric substances (EPS) and the ability to fix nitrogen from the atmosphere provide nitrogen-fixing cyanobacteria with a competitive advantage over other organisms. Temperature and nitrogen availability are key environmental factors in regulating the growth of cyanobacteria. In this study, Dolichospermum (formerly known as Anabaena) was cultivated at three different temperatures (10 °C, 20 °C, and 30 °C) to examine the impact of temperature and nitrogen availability on nitrogen fixation capacity and the release of EPS. Initially, confocal laser scanning microscopy (CLSM) and the quantification of heterocysts at different temperatures revealed that lower temperatures (10 °C) hindered the differentiation of heterocysts under nitrogen-deprived conditions. Additionally, while heterocysts inhibited the photosynthetic activity of Dolichospermum, the secretion of EPS was notably affected by nitrogen limitation, particularly at 30 °C. Finally, real-time quantitative polymerase chain reaction (qPCR) was used to measure the expression of nitrogen-utilizing genes (ntcA and nifH) and EPS synthesis-related genes (wzb and wzc). The results indicated that under nitrogen-deprived conditions, the expression of each gene was upregulated, and there was a significant correlation between the upregulation of nitrogen-utilizing and EPS synthesis genes (P < 0.05). Our findings suggested that Dolichospermum responded to temperature variation by affecting the formation of heterocysts, impacting its potential nitrogen fixation capacity. Furthermore, the quantity of EPS released was more influenced by nitrogen availability than temperature. This research enhances our comprehension of interconnections between nitrogen deprivation and EPS production under the different temperatures.

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.

Insights into the growth and biochemical defense responses associated with fenitrothion toxicity and uptake by freshwater cyanobacteria.

The extensive use of fenitrothion (FNT) in agricultural practices induces its persistence in soil and waterways. Therefore, it is essential to implement effective management practices such as using cyanobacteria for FNT removal and accumulation, particularly under accidental contamination. To this end, we evaluated the responses of two freshwater cyanobacteria taxa, Nostoc muscorum and Anabaena laxa to mild (7.5 mg L-1) and high (15 mg L-1) levels of FNT over a period of 7 d. Compared to N. muscorum, A. laxa was more tolerant to FNT, exhibiting higher FNT uptake and removal efficiencies at mild (16.3%) and high (17.5%) levels. FNT induced a dose-dependent decrease in cell growth, Chl a, phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase activities, which were more pronounced in N. muscorum. Moreover, FNT significantly increased oxidative damage markers i.e., increased lipid peroxidation (MDA), protein oxidation, H2O2 levels and NADPH oxidase enzyme activity, to more extent in N. muscorum. Compared to N. muscorum, A. laxa had high antioxidant capacity (FRAP), glutathione and increased activities of glutathione-S-transferase, glutathione reductase, glutathione peroxidase and superoxide dismutase, suggesting a robust antioxidant defense mechanism to mitigate FNT toxicity. However, N. muscorum devoted the induction of ascorbate content and the activity of catalase, peroxidase, monodehydroascorbate reductase, ascorbate peroxidase, and dehydroascorbate reductase enzymes. Although A. laxa had greater intracellular FNT, it experienced less FNT-induced oxidative stress, likely due to over production of antioxidants. Consequently, A. laxa is considered as a promising candidate for FNT phycoremediation. Our findings provide fundamental information on species-specific toxicity of FNT among cyanobacteria and the environmental risk of FNT toxicity in aquatic environments.

Anabaena sp. A-1 mediated molybdenum oxide nanoparticles: A novel frontier in green synthesis, characterization and pharmaceutical properties.

Green-synthesized metal oxide nanoparticles have garnered considerable attention due to their simple, sustainable, and eco-friendly attributes, coupled with their diverse applications in biomedicine and environmental context. The current study shows a sustainable approach for synthesizing molybdenum oxide nanoparticles (MoONPs) utilizing an extract from Anabaena sp. A-1. This novel approach marks a significant milestone as various spectral approaches were employed for characterization of the green-synthesized MoONPs. Ultraviolet-visible (UV-Vis) spectroscopic analysis revealed a surface plasmon resonance (SPR) peak of MoONPs at 538 nm. Fourier transform infrared (FTIR) spectral analysis facilitated the identification of functional groups responsible for both the stability and production of MoONPs. Scanning electron microscopy (SEM) was utilized revealing a rod shape morphology of the MoONPs. X-ray diffraction (XRD) analysis yielded a calculated crystal size of 31 nm, indicating the crystalline nature of MoONPs. Subsequently, biological assays were employed to ascertain the potential of the bioengineered MoONPs. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to quantify free radical scavenging activity, revealing an antioxidant capacity of 68.1% at 200 µg/mL. To evaluate antibacterial and antifungal efficacy, the disc diffusion method was employed across varying concentrations of MoONPs (6.25, 12.5, 25, 50, 100, 150, 200 µg/mL). Quantification of cytotoxicity was performed via a brine shrimp assay, yielding an IC50 value of 552.3 µg/mL, a metric of moderate cytotoxicity. To assess the biocompatibility of MoONPs, an antihemolytic assay was conducted, confirming their safety profile. Additionally, MoONPs exhibited non-toxic attributes in an insecticidal assay. Notably, in anti-inflammatory assay MoONPs showed an inactive nature towards the reactive oxygen species. In conclusion, these findings highlight the potential versatility of MoONPs in various biological applications, extending beyond their recognized anti-inflammatory and insecticidal properties. RESEARCH HIGHLIGHTS: This study marks an advancement in nanotechnology, exploring ways for MoONPs fabrication, representing a unique and unexplored research domain. Green-synthesized MoONPs using Anabaena sp. A-1 extract offers a sustainable and eco-friendly approach. Characterized by UV-Vis, FTIR, SEM, and XRD, MoONPs demonstrate rod-shaped morphology and crystalline nature. Bioengineered MoONPs exhibit versatility in biological applications, demonstrating notable antioxidant, antibacterial and antifungal efficacy, moderate cytotoxicity, biocompatibility, and insecticidal properties, emphasizing their multifaceted utility. The research findings highlight the potential utilization of MoONPs across a spectrum of biological applications, thereby suggesting their promising role in the realm of biomedicine and environmental context.

Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120.

In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.

An ancient bacterial zinc acquisition system identified from a cyanobacterial exoproteome.

Bacteria have developed fine-tuned responses to cope with potential zinc limitation. The Zur protein is a key player in coordinating this response in most species. Comparative proteomics conducted on the cyanobacterium Anabaena highlighted the more abundant proteins in a zur mutant compared to the wild type. Experimental evidence showed that the exoprotein ZepA mediates zinc uptake. Genomic context of the zepA gene and protein structure prediction provided additional insights on the regulation and putative function of ZepA homologs. Phylogenetic analysis suggests that ZepA represents a primordial system for zinc acquisition that has been conserved for billions of years in a handful of species from distant bacterial lineages. Furthermore, these results show that Zur may have been one of the first regulators of the FUR family to evolve, consistent with the scarcity of zinc in the ecosystems of the Archean eon.

Synergistic regulation of hydrogen sulfide and nitric oxide on biochemical components, exopolysaccharides, and nitrogen metabolism in nickel stressed rice field cyanobacteria.

The present study examined the regulatory mechanism of hydrogen sulfide (H2S) and nitric oxide (NO) in nickel (Ni) stressed cyanobacteria viz., Nostoc muscorum and Anabaena sp. by analyzing growth, photosynthetic pigments, biochemical components (protein and carbohydrate), exopolysaccharides (EPS), inorganic nitrogen content, and activity of enzymes comprised in nitrogen metabolism and Ni accumulation. The 1 µM Ni substantially diminished growth by 18% and 22% in N. muscorum and Anabaena sp. respectively, along with declining the pigment contents (Chl a/Car ratio and phycobiliproteins), and biochemical components. It also exerted negative impacts on inorganic uptake of nitrate and nitrite contents; nitrate reductase and nitrite reductase; and ammonium assimilating enzymes (glutamine synthetase, glutamate synthase, and glutamate dehydrogenase exhibited a reverse trend) activities. Nonetheless, the adverse impact of Ni can be mitigated through the exogenous supplementation of NaHS [sodium hydrosulfide (8 µM); H2S donor] and SNP [sodium nitroprusside (10 µM); NO donor] which showed substantial improvement on growth, pigments, nitrogen metabolism, and EPS layer and noticeably occurred as a consequence of a substantial reduction in Ni accumulation content which minimized the toxicity effects. The accumulation of Ni on both the cyanobacterial cell surface (EPS layer) are confirmed by the SEM-EDX analysis. Further, the addition of NO scavenger (PTIO; 20 µM) and inhibitor of NO (L-NAME; 100 µM); and H2S scavenger (HT; 20 µM) and H2S inhibitor (PAG; 50 µM) reversed the positive responses of H2S and NO and damages were more prominent under Ni stress thereby, suggesting the downstream signaling of H2S on NO-mediated alleviation. Thus, this study concludes the crosstalk mechanism of H2S and NO in the mitigation of Ni-induced toxicity in rice field cyanobacteria.

Insights into energy quenching mechanisms and carotenoid uptake by orange carotenoid protein homologs: HCP4 and CTDH.

Photodamage to the photosynthetic apparatus by excessive light radiation has led to the evolution of a variety of energy dissipation mechanisms. A mechanism that exists in some cyanobacterial species, enables non-photochemical quenching of excitation energy within the phycobilisome (PBS) antenna complex by the Orange Carotenoid Protein (OCP). The OCP contains an active N-terminal domain (NTD) and a regulatory C-terminal domain (CTD). Some cyanobacteria also have genes encoding for homologs to both the CTD (CTDH) and the NTD (referred to as helical carotenoid proteins, HCP). The CTDH facilitates uptake of carotenoids from the thylakoid membranes to be transferred to the HCPs. Holo-HCPs exhibit diverse functionalities such as carotenoid carriers, singlet oxygen quenchers, and in the case of HCP4, constitutive OCP-like energy quenching. Here, we present the first crystal structure of the holo-HCP4 binding canthaxanthin molecule and an improved structure of the apo-CTDH from Anabaena sp. PCC 7120. We propose here models of the binding of the HCP4 to the PBS and the associated energy quenching mechanism. Our results show that the presence of the carotenoid is essential for fluorescence quenching. We also examined interactions within OCP-like species, including HCP4 and CTDH, providing the basis for mechanisms of carotenoid transfer from CTDH to HCPs.

Phytoremediation capability of Anabaena sp. for high organic and chromium-loaded wastewater in membrane bioreactors.

The efficiency of Anabaena sp. was analyzed for the phytoremediation of wastewater loaded with organic matter and heavy metals like chromium. Simulated wastewater was contaminated with chromium. A side-stream membrane bioreactor was used for the treatment of wastewater. A feed tank of 20 L capacity was used with a stirring arrangement. A ceramic microfiltration membrane composed of clay and alumina was obtained from Johnson & Johnson. The removal efficiency of chemical oxygen demand, biochemical oxygen demand, and chromium was evaluated. The process was used for algae harvesting and wastewater treatment. About 92% of chemical oxygen demand (COD), 98% chromium, and oil and grease were completely removed. Membrane fouling was explained by the pore blocking and cake resistance model. Stress in algal cells was determined from the superoxide dismutase (SOD) and catalase (CAT) analysis. The lipid content of algal cells was measured.

Structure of the intact tail machine of Anabaena myophage A-1(L).

The Myoviridae cyanophage A-1(L) specifically infects the model cyanobacteria Anabaena sp. PCC 7120. Following our recent report on the capsid structure of A-1(L), here we present the high-resolution cryo-EM structure of its intact tail machine including the neck, tail and attached fibers. Besides the dodecameric portal, the neck contains a canonical hexamer connected to a unique pentadecamer that anchors five extended bead-chain-like neck fibers. The 1045-Å-long contractile tail is composed of a helical bundle of tape measure proteins surrounded by a layer of tube proteins and a layer of sheath proteins, ended with a five-component baseplate. The six long and six short tail fibers are folded back pairwise, each with one end anchoring to the baseplate and the distal end pointing to the capsid. Structural analysis combined with biochemical assays further enable us to identify the dual hydrolytic activities of the baseplate hub, in addition to two host receptor binding domains in the tail fibers. Moreover, the structure of the intact A-1(L) also helps us to reannotate its genome. These findings will facilitate the application of A-1(L) as a chassis cyanophage in synthetic biology.

Chlorpyrifos enrichment enhances tolerance of Anabaena sp. PCC 7119 to dimethoate.

Organophosphorus (OP) insecticides are widely used for on-field pest control, constituting about 38% of global pesticide consumption. Insecticide tolerance has been recorded in microorganisms isolated from the contaminated soil. However, the cross-tolerance of laboratory-enriched cultures remains poorly understood. A chlorpyrifos tolerant (T) strain of Anabaena sp. PCC 7119 was developed through continuous enrichment of the wild strain (W). The cross-tolerance of the T strain to the OP insecticide dimethoate was assessed by measuring photosynthetic performance, key enzyme activities and degradation potential. The presence of dimethoate led to a significant reduction in the growth and pigment content of the W strain. In contrast, the T strain demonstrated improved growth and metabolic performance. Chl a and carotenoids were degraded faster than phycobiliproteins in both strains. The T strain exhibited superior photosynthetic performance, metabolic efficiency and photosystem functions, than of W strain, at both the tested dimethoate concentrations (100 and 200 µM). The treated T strain had more or less a normal OJIP fluorescence transient and bioenergetic functions, while the W strain showed a greater fluorescence rise at ≤ 300 µs indicating the inhibition of electron donation to PS II, and at 2 ms due to reduced electron release beyond QA. The T strain had significantly higher levels of esterase and phosphatases, further enhanced by insecticide treatment. Dimethoate degradation efficiency of the T strain was significantly higher than of the W strain. T strain also removed chlorpyrifos more efficiently than W strain at both the tested concentrations. The BCFs of both chlorpyrifos and dimethoate were lower in the T strain compared to the W strain. These findings suggest that the enriched strain exhibits promising results in withstanding dimethoate toxicity and could be explored for its potential as a bioremediating organism for OP degradation.

Unravelling HetC as a peptidase-based ABC exporter driving functional cell differentiation in the cyanobacterium Nostoc PCC 7120.

The export of peptides or proteins is essential for a variety of important functions in bacteria. Among the diverse protein-translocation systems, peptidase-containing ABC transporters (PCAT) are involved in the maturation and export of quorum-sensing or antimicrobial peptides in Gram-positive bacteria and of toxins in Gram-negative organisms. In the multicellular and diazotrophic cyanobacterium Nostoc PCC 7120, the protein HetC is essential for the differentiation of functional heterocysts, which are micro-oxic and non-dividing cells specialized in atmospheric nitrogen fixation. HetC shows similarities to PCAT systems, but whether it actually acts as a peptidase-based exporter remains to be established. In this study, we show that the N-terminal part of HetC, encompassing the peptidase domain, displays a cysteine-type protease activity. The conserved catalytic residues conserved in this family of proteases are essential for the proteolytic activity of HetC and the differentiation of heterocysts. Furthermore, we show that the catalytic residue of the ATPase domain of HetC is also essential for cell differentiation. Interestingly, HetC has a cyclic nucleotide-binding domain at its N-terminus which can bind ppGpp in vitro and which is required for its function in vivo. Our results indicate that HetC is a peculiar PCAT that might be regulated by ppGpp to potentially facilitate the export of a signaling peptide essential for cell differentiation, thereby broadening the scope of PCAT role in Gram-negative bacteria.IMPORTANCEBacteria have a great capacity to adapt to various environmental and physiological conditions; it is widely accepted that their ability to produce extracellular molecules contributes greatly to their fitness. Exported molecules are used for a variety of purposes ranging from communication to adjust cellular physiology, to the production of toxins that bacteria secrete to fight for their ecological niche. They use export machineries for this purpose, the most common of which energize transport by hydrolysis of adenosine triphosphate. Here, we demonstrate that such a mechanism is involved in cell differentiation in the filamentous cyanobacterium Nostoc PCC 7120. The HetC protein belongs to the ATP-binding cassette transporter superfamily and presumably ensures the maturation of a yet unknown substrate during export. These results open interesting perspectives on cellular signaling pathways involving the export of regulatory peptides, which will broaden our knowledge of how these bacteria use two cell types to conciliate photosynthesis and nitrogen fixation.

FurC (PerR) contributes to the regulation of peptidoglycan remodeling and intercellular molecular transfer in the cyanobacterium Anabaena sp. strain PCC 7120.

Microbial extracellular proteins and metabolites provide valuable information concerning how microbes adapt to changing environments. In cyanobacteria, dynamic acclimation strategies involve a variety of regulatory mechanisms, being ferric uptake regulator proteins as key players in this process. In the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120, FurC (PerR) is a global regulator that modulates the peroxide response and several genes involved in photosynthesis and nitrogen metabolism. To investigate the possible role of FurC in shaping the extracellular environment of Anabaena, the analysis of the extracellular metabolites and proteins of a furC-overexpressing variant was compared to that of the wild-type strain. There were 96 differentially abundant proteins, 78 of which were found for the first time in the extracellular fraction of Anabaena. While these proteins belong to different functional categories, most of them are predicted to be secreted or have a peripheral location. Several stress-related proteins, including PrxA, flavodoxin, and the Dps homolog All1173, accumulated in the exoproteome of furC-overexpressing cells, while decreased levels of FurA and a subset of membrane proteins, including several export proteins and amiC gene products, responsible for nanopore formation, were detected. Direct repression by FurC of some of those genes, including amiC1 and amiC2, could account for odd septal nanopore formation and impaired intercellular molecular transfer observed in the furC-overexpressing variant. Assessment of the exometabolome from both strains revealed the release of two peptidoglycan fragments in furC-overexpressing cells, namely 1,6-anhydro-N-acetyl-ß-D-muramic acid (anhydroMurNAc) and its associated disaccharide (ß-D-GlcNAc-(1-4)-anhydroMurNAc), suggesting alterations in peptidoglycan breakdown and recycling.IMPORTANCECyanobacteria are ubiquitous photosynthetic prokaryotes that can adapt to environmental stresses by modulating their extracellular contents. Measurements of the organization and composition of the extracellular milieu provide useful information about cyanobacterial adaptive processes, which can potentially lead to biomimetic approaches to stabilizing biological systems to adverse conditions. Anabaena sp. strain PCC 7120 is a multicellular, nitrogen-fixing cyanobacterium whose intercellular molecular exchange is mediated by septal junctions that traverse the septal peptidoglycan through nanopores. FurC (PerR) is an essential transcriptional regulator in Anabaena, which modulates the response to several stresses. Here, we show that furC-overexpressing cells result in a modified exoproteome and the release of peptidoglycan fragments. Phenotypically, important alterations in nanopore formation and cell-to-cell communication were observed. Our results expand the roles of FurC to the modulation of cell-wall biogenesis and recycling, as well as in intercellular molecular transfer.

Role of exopolysaccharides of Anabaena sp. in desiccation tolerance and biodeterioration of ancient terracotta monuments of Bishnupur.

Colonization of the cyanobacteria in the Bishnupur terracotta temples, one of the heritage sites of West Bengal, India is in an alarming state of deterioration now. Among the cyanobacteria Anabaena sp. (VBCCA 052002) has been isolated from most of the crust samples of terracotta monuments of Bishnupur. The identification was done using micromorphological characters and confirmed by 16S rRNA gene sequencing. The isolated strain produces enormous exopolysaccharides, which are extracted, hydrolyzed, and analyzed by HPLC. We have studied desiccation tolerance in this cyanobacterium and found biosynthesis of trehalose with an increase in durations of desiccation. The in vitro experiment shows that Chlorophyll-a and carotenoid content increase with fourteen days of desiccation, and cellular carbohydrates increase continuously. However, cellular protein decreases with desiccation. To gain insights into the survival strategies and biodeterioration mechanisms of Anabaena sp. in the desiccated conditions of the Bishnupur monuments, the present study focuses on the physiological aspects of the cyanobacteria under controlled in vitro conditions. Our study indicates that in desiccation conditions, trehalose biosynthesis takes place in Anabaena sp. As a result of the excessive sugar and polysaccharide produced, it adheres to the surface of the terracotta structure. The continuous contraction and expansion of these polysaccharides contribute to the biodeterioration of these monuments.