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1.
Nat Commun ; 12(1): 5497, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535665

RESUMO

Phycobilisomes (PBS) are the major light-harvesting machineries for photosynthesis in cyanobacteria and red algae and they have a hierarchical structure of a core and peripheral rods, with both consisting of phycobiliproteins and linker proteins. Here we report the cryo-EM structures of PBS from two cyanobacterial species, Anabaena 7120 and Synechococcus 7002. Both PBS are hemidiscoidal in shape and share a common triangular core structure. While the Anabaena PBS has two additional hexamers in the core linked by the 4th linker domain of ApcE (LCM). The PBS structures predict that, compared with the PBS from red algae, the cyanobacterial PBS could have more direct routes for energy transfer to ApcD. Structure-based systematic mutagenesis analysis of the chromophore environment of ApcD and ApcF subunits reveals that aromatic residues are critical to excitation energy transfer (EET). The structures also suggest that the linker protein could actively participate in the process of EET in both rods and the cores. These results provide insights into the organization of chromophores and the mechanisms of EET within cyanobacterial PBS.


Assuntos
Cianobactérias/metabolismo , Transferência de Energia , Ficobilissomas/metabolismo , Anabaena/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Pigmentos Biliares/metabolismo , Cianobactérias/ultraestrutura , Modelos Moleculares , Ficobilissomas/química , Ficobilissomas/ultraestrutura , Multimerização Proteica , Rodófitas/metabolismo , Homologia Estrutural de Proteína
2.
J Proteome Res ; 20(8): 3963-3976, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34270261

RESUMO

Anabaena sp. PCC 7120 (Anabaena 7120) is a photoautotrophic filamentous cyanobacterium capable of fixing atmospheric nitrogen. It is a model organism used for studying cell differentiation and nitrogen fixation. Under nitrogen deficiency, Anabaena 7120 forms specialized heterocysts capable of nitrogen fixation. However, the molecular mechanisms involved in the cyanobacterial adaptation to nitrogen deficiency are not well understood. Here, we employed a label-free quantitative proteomic strategy to systematically investigate the nitrogen deficiency response of Anabaena 7120 at different time points. In total, 363, 603, and 669 proteins showed significant changes in protein abundance under nitrogen deficiency for 3, 12, and 24 h, respectively. With mapping onto metabolic pathways, we revealed proteomic perturbation and regulation of carbon and nitrogen metabolism in response to nitrogen deficiency. Functional analysis confirmed the involvement of nitrogen stress-responsive proteins in biological processes, including nitrogen fixation, photosynthesis, energy and carbon metabolism, and heterocyst development. The expression of 10 proteins at different time points was further validated by using multiple reaction monitoring assays. In particular, many dysregulated proteins were found to be time-specific and involved in heterocyst development, providing new candidates for future functional studies in this model cyanobacterium. These results provide novel insights into the molecular mechanisms of nitrogen stress responses and heterocyst development in Anabaena 7120.


Assuntos
Anabaena , Proteômica , Anabaena/genética , Anabaena/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Nitrogênio/metabolismo , Fixação de Nitrogênio
3.
Arch Microbiol ; 203(7): 4367-4383, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34120198

RESUMO

The present study was undertaken to investigate the arsenite (As III)-induced changes in the diazotrophic cyanobacterium Anabaena PCC 7120. It was observed that the growth of cyanobacterial decreased with increase in As (III) concentration. The cells exposed to As (III) showed morphological variation (deformity) due to the formation of deeper constrictions in vegetative cells. Strain showed increased heterocyst differentiation (1.6-fold higher) whereas decreased nitrogenase activity at the concentration of 40 ppm As (III). The activities of NR, NiR, urease and GS decreased with increase in As (III) concentrations and attained their minimum levels at 40 ppm of As (III). The Ca2+-dependent ATPase activity increased with increase in As (III) concentration and attained its about 2.72-fold higher level at 40 ppm of As (III). In contrast, sharp decline in Mg2+-dependent ATPase activity (28%) was recorded at 1 ppm of As (III) over untreated control. The rates of photosynthetic O2 evolution and respiration decreased with increase in As (III) concentration and attained its minimal level at 40 ppm of As (III). Therefore, this study highlighted arsenite regimes efficiently correlated with behavioral changes in consort with strain.


Assuntos
Anabaena , Arsenitos , Anabaena/efeitos dos fármacos , Anabaena/metabolismo , Arsenitos/farmacologia , Proteínas de Bactérias/metabolismo , Poluentes Ambientais/farmacologia , Ativação Enzimática/efeitos dos fármacos , Nitrogênio/metabolismo , Fixação de Nitrogênio/efeitos dos fármacos , Nitrogenase/metabolismo
4.
Environ Sci Pollut Res Int ; 28(37): 51088-51104, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33974205

RESUMO

Exposure time, metal bio-accumulation, and upregulation of ascorbate-glutathione (AsA-GSH) cycle are the key factor that provide tolerance against heavy metal stress. Thus, the current study is an endeavor to prove our hypothesis that regulation of arsenate (AsV: 50, 100, and 150 mM) and arsenite (AsIII: 50, 100, and 150 µM) toxicity is time dependent (48-96 h) due to modulation in bio-accumulation pattern, AsA-GSH cycle, and non-enzymatic antioxidants in two paddy field cyanobacteria Nostoc muscorum ATCC27893 and Anabaena sp. PCC7120. After 48 h, reduction in growth associated with increased sensitivity index, As bio-accumulation, and oxidative stress was observed which further intensified after 96 h but the degree of damage was lesser than 48 h. It denotes a significant recovery in growth after 96 h which is correlated with decreased As bio-accumulation and oxidative stress due to increased efficiency of AsA-GSH cycle and non-enzymatic antioxidants. Both the species of As caused significant rise in oxidative biomarkers as evident by in -vitro analysis of O2·-, H2O2, and MDA equivalent contents despite appreciable rise in the activity antioxidative enzymes APX, DHAR, and GR. The study concludes that among both forms of arsenic, AsIII induced more toxic effect on growth by over-accumulating the ROS as evident by weak induction of AsA-GSH cycle to overcome the stress as compared to AsV. Further, with increasing the time exposure, apparent recovery was noticed with the lower doses of AsV, i.e., 50 and 100 mM and AsIII, i.e., 50 and 100 µM; however, the toxicity further aggravated with higher dose of both AsV and AsIII. Study proposes the deleterious impact of AsV and AsIII on cyanobacteria N. muscorum and Anabaena sp. but the toxicity was overcome by time-dependent recovery.


Assuntos
Anabaena , Arsenitos , Cianobactérias , Nostoc muscorum , Anabaena/metabolismo , Antioxidantes , Arseniatos/toxicidade , Arsenitos/toxicidade , Cianobactérias/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio , Nostoc muscorum/metabolismo , Estresse Oxidativo
5.
Curr Microbiol ; 78(6): 2429-2439, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33983480

RESUMO

Agricultural productivity is threatened by increasing incidence of drought and the drought tolerant cyanobacteria offer a better solution in the restoration of soil fertility and productivity. The present study describes the comparative physiological response of the cyanobacterium Anabaena sp. acclimated and un-acclimated to desiccation stress induced by polyethylene glycol (10% PEG). While, the acclimated cyanobacterial cells grew luxuriantly with optimal chlorophyll content, photosynthetic activities and nitrogen fixation, the un-acclimated cells exhibited reduced growth rate, chlorophyll content, photosynthetic activities and nitrogen fixation. Distinct differences in the accumulation of lipid peroxidation products, proline and activity of superoxide dismutase were observed under identical growth conditions in the acclimated and un-acclimated cells. Desiccation-acclimated and un-acclimated cyanobacteria showed significant alterations in the abundance of important proteins in the proteome. Two-dimensional gel electrophoresis followed by MALDI-TOF-MS/MS analysis identified twelve proteins. The acclimated cells showed the up regulation of proteins such as Rubisco, fructose-bis-phosphate aldolase, fructose 1-6 bisphosphatase, phosphoglycerate dehydrogenase and elongation factors Tu and Ts as compared to un-acclimated cells. Therefore, the ability to maintain photosynthesis, antioxidants and increased accumulation of proteins related to energy metabolism helped the acclimated cyanobacterium Anabaena sp. to grow optimally under desiccation stress conditions.


Assuntos
Anabaena , Proteômica , Anabaena/genética , Anabaena/metabolismo , Dessecação , Fotossíntese , Proteoma/metabolismo , Estresse Fisiológico , Espectrometria de Massas em Tandem
6.
Aquat Toxicol ; 236: 105839, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34015754

RESUMO

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.


Assuntos
Venenos de Cnidários/toxicidade , Anabaena/metabolismo , Cianobactérias/metabolismo , Ecossistema , Transporte de Elétrons , Oxirredução , Fotossíntese/efeitos dos fármacos , Complexo de Proteína do Fotossistema I/metabolismo , Ficobilinas , Ficocianina , Espécies Reativas de Oxigênio/metabolismo , Poluentes Químicos da Água/toxicidade
7.
J Microbiol Biotechnol ; 31(5): 645-658, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-33879642

RESUMO

Porins are essential for the viability of Gram-negative bacteria. They ensure the uptake of nutrients, can be involved in the maintenance of outer membrane integrity and define the antibiotic or drug resistance of organisms. The function and structure of porins in proteobacteria is well described, while their function in photoautotrophic cyanobacteria has not been systematically explored. We compared the domain architecture of nine putative porins in the filamentous cyanobacterium Anabaena sp. PCC 7120 and analyzed the seven candidates with predicted OprB-domain. Single recombinant mutants of the seven genes were created and their growth capacity under different conditions was analyzed. Most of the putative porins seem to be involved in the transport of salt and copper, as respective mutants were resistant to elevated concentrations of these substances. In turn, only the mutant of alr2231 was less sensitive to elevated zinc concentrations, while mutants of alr0834, alr4741 and all4499 were resistant to high manganese concentrations. Notably the mutant of alr4550 shows a high sensitivity against harmful compounds, which is indicative for a function related to the maintenance of outer membrane integrity. Moreover, the mutant of all5191 exhibited a phenotype which suggests either a higher nitrate demand or an inefficient nitrogen fixation. The dependency of porin membrane insertion on Omp85 proteins was tested exemplarily for Alr4550, and an enhanced aggregation of Alr4550 was observed in two omp85 mutants. The comparative analysis of porin mutants suggests that the proteins in parts perform distinct functions related to envelope integrity and solute uptake.


Assuntos
Anabaena/metabolismo , Porinas/genética , Anabaena/genética , Anabaena/crescimento & desenvolvimento , Antibacterianos/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Metais/metabolismo , Mutação , Nitrogênio/metabolismo , Fenótipo , Porinas/metabolismo , Sais/metabolismo , Estresse Fisiológico/genética
8.
J Bacteriol ; 203(13): e0008121, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33846119

RESUMO

Multicellular heterocyst-forming cyanobacteria, such as Anabaena, grow as chains of cells forming filaments that, under diazotrophic conditions, contain two cell types: vegetative cells that perform oxygenic photosynthesis and N2-fixing heterocysts. Along the filament, the intercellular septa contain a thick peptidoglycan layer that forms septal disks. Proteinaceous septal junctions connect the cells in the filament traversing the septal disks through nanopores. The fraCDE operon encodes proteins needed to make long filaments in Anabaena. FraC and FraD, located at the intercellular septa, are involved in the formation of septal junctions. Using a superfolder-green fluorescent protein (GFP) fusion, we found in this study that FraE is mainly localized to the poles of the heterocysts, consistent with the requirement of FraE for constriction of the heterocyst poles to form the "heterocyst neck." A fraE insertional mutant was impaired by 22% to 38% in transfer of fluorescent calcein from vegetative cells to heterocysts. Septal disks were inspected in murein sacculi from heterocyst-enriched preparations. Unexpectedly, the diameter of the nanopores in heterocyst septa was about 1.5- to 2-fold larger than in vegetative cell septa. The number of these nanopores was 76% and 6% of the wild-type number in fraE and fraC fraD mutants, respectively. Our results show that FraE is mainly involved in heterocyst maturation, whereas FraC and FraD are needed for the formation of the large nanopores of heterocyst septa, as they are for vegetative cell nanopores. Additionally, arrays of small pores conceivably involved in polysaccharide export were observed close to the septal disks in the heterocyst murein sacculus preparations. IMPORTANCE Intercellular communication, an essential attribute of multicellularity, is required for diazotrophic growth in heterocyst-forming cyanobacteria such as Anabaena, in which the cells are connected by proteinaceous septal junctions that are structural analogs of metazoan connexons. The septal junctions allow molecular intercellular diffusion traversing the septal peptidoglycan through nanopores. In Anabaena the fraCDE operon encodes septal proteins involved in intercellular communication. FraC and FraD are components of the septal junctions along the filament, whereas here we show that FraE is mainly present at the heterocyst poles. We found that the intercellular septa in murein sacculi from heterocysts contain nanopores that are larger than those in vegetative cells, establishing a previously unknown difference between heterocyst and vegetative cell septa in Anabaena.


Assuntos
Anabaena/metabolismo , Cianobactérias/metabolismo , Nanoporos , Anabaena/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cianobactérias/genética , Citoesqueleto/metabolismo , Difusão , Regulação Bacteriana da Expressão Gênica , Mutagênese Insercional , Óperon , Peptidoglicano/metabolismo
9.
J Bacteriol ; 203(13): e0010821, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33846118

RESUMO

patU, one of the genes specifically found in filamentous cyanobacteria, is required for the pattern formation in heterocyst-forming species. In Anabaena sp. strain PCC 7120, patU is split into patU5 and patU3, and only patU3 is involved in heterocyst patterning. Here, we report that PatU3 is also involved in control of cell size. A patU3 deletion mutant showed remarkably smaller cell size and much higher heterocyst frequency than the wild type. Yeast two-hybrid and pulldown assays demonstrated a direct interaction between PatU3 and the cell division protein Ftn6. Without the N-terminal 16-amino-acid (aa) portion (MQERFQAVIKRRLQIH [the identified octapeptide is underlined]), PatU3 was no longer able to interact with Ftn6. This portion of PatU3 is also required for the interaction with PatN, a protein related to heterocyst differentiation/patterning. Addition of the 16-aa peptide or AVIKRRLQ-containing peptides restored the cell size and heterocyst frequency of a patU3 deletion mutant to normal or nearly wild-type levels. PatU3(1-16aa)-GFP, the N-terminal 16-aa sequence fused with green fluorescent protein (GFP), formed polar aggregates and peripheral patches in heterocysts of Anabaena sp. strain PCC 7120, whereas PatU3(1-198aa)-GFP showed a homogeneous distribution in the cytoplasm of all cells. The N-terminal AVIKRRLQ-containing sequence may function in intact PatU3, as a separate peptide, or both. IMPORTANCE PatU (or split into PatU5 and PatU3) is distributed in almost all filamentous cyanobacteria, including those that do not form heterocysts (except Pseudanabaena); however, its functions other than heterocyst differentiation/patterning have not been reported before. In this study, we found that PatU3 in Anabaena sp. strain PCC 7120 is involved in cell size determination. The N-terminal 16-aa sequence of PatU3 is required for the control of cell size and interaction with the cell division protein Ftn6, and an octapeptide (aa 7 to aa 14) within the 16-aa sequence can restore the cell size (and heterocyst frequency) of a patU3 deletion mutant to normal. Such a peptide, if generated from PatU or PatU3 in vivo, may promote intercellular coordination in filamentous cyanobacteria.


Assuntos
Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Tamanho Celular , Peptídeos/metabolismo , Anabaena/genética , Proteínas de Bactérias/genética , Divisão Celular , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Proteínas de Fluorescência Verde , Mutação , Peptídeos/genética
10.
J Hazard Mater ; 411: 124822, 2021 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-33858073

RESUMO

Cyanobacteria, the first photoautotrophs have remarkable adaptive capabilities against most abiotic stresses, including Cd. A model cyanobacterium, Anabaena sp. PCC 7120 has been commonly used to understand cyanobacterial plasticity under different environmental stresses. However, very few studies have focused on the acute Cd toxicity. In this context, Anabaena was subjected to 100 µM Cd for 48 h (acute Cd stress, ACdS) and then transferred into the fresh medium for post-stress recovery (PSR). We further investigated the dynamics of morpho-ultrastructure, physiology, cytosolic proteome, thylakoidal complexes, chelators, and transporters after ACdS, as well as during early (ER), mid (MR), and late (LR) phases of PSR. The findings revealed that ACdS induced intracellular Cd accumulation and ROS production, altered morpho-ultrastructure, reduced photosynthetic pigments, and affected the structural organization of PSII, which subsequently hindered photosynthetic efficiency. Anabaena responded to ACdS and recovered during PSR by reprogramming the expression pattern of proteins/genes involved in cellular defense and repair; CO2 access, Calvin-Benson cycle, glycolysis, and pentose phosphate pathway; protein biosynthesis, folding, and degradation; regulatory functions; PSI-based cyclic electron flow; Cd chelation; and efflux. These modulations occurred in an integrated and coordinated manner that facilitated Anabaena to detoxify Cd and repair ACdS-induced cellular damage.


Assuntos
Anabaena , Cianobactérias , Anabaena/genética , Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Cádmio/toxicidade , Cianobactérias/metabolismo , Fotossíntese , Proteoma
11.
PLoS One ; 16(3): e0248155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33662009

RESUMO

Nitrogen assimilation is strictly regulated in cyanobacteria. In an inorganic nitrogen-deficient environment, some vegetative cells of the cyanobacterium Anabaena differentiate into heterocysts. We assessed the photosynthesis and nitrogen-fixing capacities of heterocysts and vegetative cells, respectively, at the transcriptome level. RNA extracted from nitrogen-replete vegetative cells (NVs), nitrogen-deprived vegetative cells (NDVs), and nitrogen-deprived heterocysts (NDHs) in Anabaena sp. strain PCC 7120 was evaluated by transcriptome sequencing. Paired comparisons of NVs vs. NDHs, NVs vs. NDVs, and NDVs vs. NDHs revealed 2,044 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs showed that carbon fixation in photosynthetic organisms and several nitrogen metabolism-related pathways were significantly enriched. Synthesis of Gvp (Gas vesicle synthesis protein gene) in NVs was blocked by nitrogen deprivation, which may cause Anabaena cells to sink and promote nitrogen fixation under anaerobic conditions; in contrast, heterocysts may perform photosynthesis under nitrogen deprivation conditions, whereas the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Immunofluorescence analysis of nitrogenase iron protein suggested that the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Our findings provide insight into the molecular mechanisms underlying nitrogen fixation and photosynthesis in vegetative cells and heterocysts at the transcriptome level. This study provides a foundation for further functional verification of heterocyst growth, differentiation, and water bloom control.


Assuntos
Anabaena/citologia , Anabaena/genética , Regulação Bacteriana da Expressão Gênica , Fixação de Nitrogênio , Anabaena/metabolismo , Anabaena/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Nitrogênio/metabolismo , Transcriptoma
12.
J Biochem ; 169(6): 709-719, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-33537746

RESUMO

To understand the physiological role of NADPH-thioredoxin reductase C (NTRC) in cyanobacteria, we investigated an NTRC-deficient mutant strain of Anabaena sp., PCC 7120, cultivated under different regimes of nitrogen supplementation and light exposure. The deletion of ntrC did not induce a change in the cell structure and metabolic pathways. However, time-dependent changes in the abundance of specific proteins and metabolites were observed. A decrease in chlorophyll a was correlated with a decrease in chlorophyll a biosynthesis enzymes and photosystem I subunits. The deletion of ntrC led to a deregulation of nitrogen metabolism, including the NtcA accumulation and heterocyst-specific proteins while nitrate ions were available in the culture medium. Interestingly, this deletion resulted in a redox imbalance, indicated by higher peroxide levels, higher catalase activity and the induction of chaperones such as MsrA. Surprisingly, the antioxidant protein 2-CysPrx was downregulated. The deficiency in ntrC also resulted in the accumulation of metabolites such as 6-phosphogluconate, ADP and ATP. Higher levels of NADP+ and NADPH partly correlated with higher G6PDH activity. Rather than impacting protein expression levels, NTRC appears to be involved in the direct regulation of enzymes, especially during the dark-to-light transition period.


Assuntos
Anabaena/genética , Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , NADP/metabolismo , Nitrogênio/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Anabaena/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Clorofila A/metabolismo , Luz , Tiorredoxina Dissulfeto Redutase/genética
13.
Plant Cell Environ ; 44(6): 1885-1907, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33608943

RESUMO

Nitrogen sources are all converted into ammonium/ia as a first step of assimilation. It is reasonable to expect that molecular components involved in the transport of ammonium/ia across biological membranes connect with the regulation of both nitrogen and central metabolism. We applied both genetic (i.e., Δamt mutation) and environmental treatments to a target biological system, the cyanobacterium Anabaena sp PCC 7120. The aim was to both perturb nitrogen metabolism and induce multiple inner nitrogen states, respectively, followed by targeted quantification of key proteins, metabolites and enzyme activities. The absence of AMT transporters triggered a substantial whole-system response, affecting enzyme activities and quantity of proteins and metabolites, spanning nitrogen and carbon metabolisms. Moreover, the Δamt strain displayed a molecular fingerprint indicating nitrogen deficiency even under nitrogen replete conditions. Contrasting with such dynamic adaptations was the striking near-complete lack of an externally measurable altered phenotype. We conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. This analysis provides evidence for a potential role of AMT transporters in the regulatory/signalling network of nitrogen metabolism and the existence of a novel fourth regulatory mechanism controlling glutamine synthetase activity.


Assuntos
Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Nitrogênio/metabolismo , Anabaena/genética , Anabaena/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Deleção de Genes , Mutação , Transdução de Sinais
14.
Sci Rep ; 11(1): 2893, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536576

RESUMO

In the present study, defensive strategies of H2O2 mediated NO signaling were analyzed in Cd stressed Nostoc muscorum and Anabaena sp. Exogenously supplied SNP (10 µM) and H2O2 (1 µM) lessen the toxicity of Cd (6 µM) but without NO; H2O2 was unable to release the stress from cyanobacterial cells potentially. The reduced contents of exopolysaccharide, protein content, endogenous NO and enzymatic antioxidants (SOD, POD, CAT, and GST) due to Cd toxicity, were found increased significantly after exogenous application of H2O2 and SNP thereafter, cyanobacterial calls flourished much better after releasing toxic level of Cd. Moreover, increased level of ROS due to Cd stress also normalized under exogenous application of H2O2 and SNP. However, chelation of NO hindered the signaling mechanism of H2O2 that diminished its potential against Cd stress while signaling of NO has not been hindered by chelation of H2O2 and NO potentially released the Cd stress from cyanobacterial cells. In conclusion, current findings demonstrated the synergistic signaling between H2O2 and NO towards the improvement of cyanobacterial tolerance to Cd stress, thereby enhancing the growth and antioxidant defense system of test cyanobacteria that improved fertility and productivity of soil even under the situation of metal contamination.


Assuntos
Anabaena/efeitos dos fármacos , Cádmio/toxicidade , Nostoc muscorum/efeitos dos fármacos , Oryza/crescimento & desenvolvimento , Poluentes do Solo/toxicidade , Anabaena/metabolismo , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Nostoc muscorum/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Microbiologia do Solo
15.
Nat Commun ; 12(1): 851, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558474

RESUMO

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy's action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.


Assuntos
Trifosfato de Adenosina/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Periplásmicas/metabolismo , Anabaena/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Azotobacter/metabolismo , Escherichia coli/metabolismo , Flavodoxina/química , Flavodoxina/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Conformação Molecular , Proteínas Mutantes/metabolismo , Proteínas Periplásmicas/química , Ligação Proteica , Dobramento de Proteína , Especificidade por Substrato
16.
Appl Biochem Biotechnol ; 193(5): 1447-1468, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33484449

RESUMO

The wide applications, uniqueness, and high quality of cyanobacterial exopolysaccharides (EPSs) have attracted many biotechnologists. Despite it, the inducers and molecular determinants of EPS biosynthesis in cyanobacteria are lesser known. Although, studies revealed that environmental cues especially C/N ratio as the prime modulator, the factors like light, temperature, moisture, and nutrient availability, etc. have been overlooked. Due to this, the possibilities to modify cyanobacterial system for achieving higher quantity of EPS either by modifying growth medium or metabolic engineering are restricted to few optimisations. Therefore, the present work describes the impact of sulfate limitations on the EPS production and compositions in the cyanobacterium Anabaena sp. PCC 7120. Increased EPS production with enhanced expression of alr2882 was observed in lower sulfate supplementations; however, FTIR analysis depicted an altered composition of supramolecule. Furthermore, in silico analysis of Alr2882 depicted the presence of ExoD domain and three transmembrane regions, thereby indicating its membrane localisation and role in the EPS production. Additionally, the phylogeny and multiple sequence alignment showed vertical inheritance of exoD and conservation among cyanobacteria. The meta-threading template-based modelling and ab initio full atomic relaxation by LOMET and ModRefiner servers, respectively, also exhibited helical topology of Alr2882, with nine α-helices arranged antiparallel to the preceding one. Moreover, post-translational modifications predicted in Alr2882 indicated high order of molecular regulation underlining EPS production in Anabaena sp. PCC 7120. This study provides a foundation for understanding the EPS biosynthesis mechanism under sulfur limitation and the possible role of ExoD in cyanobacteria.


Assuntos
Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Anabaena/genética , Proteínas de Bactérias/genética , Cianobactérias/genética , Cianobactérias/metabolismo , Processamento de Proteína Pós-Traducional , Análise de Sequência de DNA , Espectroscopia de Infravermelho com Transformada de Fourier
17.
mSphere ; 6(1)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441411

RESUMO

In filamentous heterocyst-forming (N2-fixing) cyanobacteria, septal junctions join adjacent cells, mediating intercellular communication, and are thought to traverse the septal peptidoglycan through nanopores. Fluorescence recovery after photobleaching (FRAP) analysis with the fluorescent marker calcein showed that cultures of Anabaena sp. strain PCC 7120 grown in the presence of combined nitrogen contained a substantial fraction of noncommunicating cells (58% and 80% of the tested vegetative cells in nitrate- and ammonium-grown cultures, respectively), whereas cultures induced for nitrogen fixation contained far fewer noncommunicating cells (16%). A single filament could have communicating and noncommunicating cells. These observations indicate that all (or most of) the septal junctions in a cell can be coordinately regulated and are coherent with the need for intercellular communication, especially under diazotrophic conditions. Consistently, intercellular exchange was observed to increase in response to N deprivation and to decrease rapidly in response to the presence of ammonium in the medium or to nitrate assimilation. Proteins involved in the formation of septal junctions have been identified in Anabaena and include SepJ, FraC, and FraD. Here, we reevaluated rates of intercellular transfer of calcein and the number of nanopores in mutants lacking these proteins and found a strong positive correlation between the two parameters only in cultures induced for nitrogen fixation. Thus, whereas the presence of a substantial number of noncommunicating cells appears to impair the correlation, data obtained in diazotrophic cultures support the idea that the nanopores are the structures that hold the septal junctions.IMPORTANCE Multicellularity is found in bacteria as well as in eukaryotes, and the filamentous heterocyst-forming (N2-fixing) cyanobacteria represent a simple and ancient paradigm of multicellular organisms. Multicellularity generally involves cell-cell adhesion and communication. The cells in the cyanobacterial filaments are joined by proteinaceous septal junctions that mediate molecular diffusion. The septal junctions traverse the septal peptidoglycan, which bears holes termed nanopores. Our results show that the septal junctions can be coordinately regulated in a cell and emphasize the relationship between septal junctions and nanopores to build intercellular communication structures, which are essential for the multicellular behavior of heterocyst-forming cyanobacteria.


Assuntos
Anabaena/citologia , Anabaena/metabolismo , Citoesqueleto/metabolismo , Fixação de Nitrogênio , Anabaena/genética , Proteínas de Bactérias/genética , Citoesqueleto/ultraestrutura , Fluoresceínas/metabolismo , Regulação Bacteriana da Expressão Gênica , Microscopia Eletrônica de Transmissão , Nanoporos
18.
J Bacteriol ; 203(4)2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33257527

RESUMO

The outer membrane of Gram-negative bacteria acts as an initial diffusion barrier that shields the cell from the environment. It contains many membrane-embedded proteins required for functionality of this system. These proteins serve as solute and lipid transporters or as machines for membrane insertion or secretion of proteins. The genome of Anabaena sp. strain PCC 7120 codes for two outer membrane transporters termed TpsB1 and TpsB2. They belong to the family of the two-partner secretion system proteins which are characteristic of pathogenic bacteria. Because pathogenicity of Anabaena sp. strain PCC 7120 has not been reported, the function of these two cyanobacterial TpsB proteins was analyzed. TpsB1 is encoded by alr1659, while TpsB2 is encoded by all5116 The latter is part of a genomic region containing 11 genes encoding TpsA-like proteins. However, tpsB2 is transcribed independently of a tpsA gene cluster. Bioinformatics analysis revealed the presence of at least 22 genes in Anabaena sp. strain PCC 7120 putatively coding for substrates of the TpsB system, suggesting a rather global function of the two TpsB proteins. Insertion of a plasmid into each of the two genes resulted in altered outer membrane integrity and antibiotic resistance. In addition, the expression of genes coding for the Clp and Deg proteases is dysregulated in these mutants. Moreover, for two of the putative substrates, a dependence of the secretion on functional TpsB proteins could be confirmed. We confirm the existence of a two-partner secretion system in Anabaena sp. strain PCC 7120 and predict a large pool of putative substrates.IMPORTANCE Cyanobacteria are important organisms for the ecosystem, considering their contribution to carbon fixation and oxygen production, while at the same time some species produce compounds that are toxic to their environment. As a consequence, cyanobacterial overpopulation might negatively impact the diversity of natural communities. Thus, a detailed understanding of cyanobacterial interaction with the environment, including other organisms, is required to define their impact on ecosystems. While two-partner secretion systems in pathogenic bacteria are well known, we provide a first description of the cyanobacterial two-partner secretion system.


Assuntos
Anabaena/genética , Anabaena/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bactérias Gram-Negativas/metabolismo , Anabaena/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/metabolismo , Transporte Biológico , Cianobactérias , Resistência Microbiana a Medicamentos , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Glucosiltransferases , Proteínas de Membrana Transportadoras/genética , Sistemas de Secreção Tipo V/metabolismo
19.
FEBS J ; 288(10): 3197-3216, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33205554

RESUMO

Polymerizing and filament-forming proteins are instrumental for numerous cellular processes such as cell division and growth. Their function in stabilization and localization of protein complexes and replicons is achieved by a filamentous structure. Known filamentous proteins assemble into homopolymers consisting of single subunits - for example, MreB and FtsZ in bacteria - or heteropolymers that are composed of two subunits, for example, keratin and α/ß tubulin in eukaryotes. Here, we describe two novel coiled-coil-rich proteins (CCRPs) in the filament-forming cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena) that assemble into a heteropolymer and function in the maintenance of the Anabaena multicellular shape (termed trichome). The two CCRPs - Alr4504 and Alr4505 (named ZicK and ZacK) - are strictly interdependent for the assembly of protein filaments in vivo and polymerize nucleotide independently in vitro, similar to known intermediate filament (IF) proteins. A ΔzicKΔzacK double mutant is characterized by a zigzagged cell arrangement and hence a loss of the typical linear Anabaena trichome shape. ZicK and ZacK interact with themselves, with each other, with the elongasome protein MreB, the septal junction protein SepJ and the divisome associate septal protein SepI. Our results suggest that ZicK and ZacK function in cooperation with SepJ and MreB to stabilize the Anabaena trichome and are likely essential for the manifestation of the multicellular shape in Anabaena. Our study reveals the presence of filament-forming IF-like proteins whose function is achieved through the formation of heteropolymers in cyanobacteria.


Assuntos
Anabaena/genética , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Citoesqueleto/genética , Regulação Bacteriana da Expressão Gênica , Tricomas/genética , Anabaena/metabolismo , Anabaena/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Divisão Celular , Clonagem Molecular , Sequência Conservada , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/genética , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Polimerização , Multimerização Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tricomas/metabolismo , Tricomas/ultraestrutura
20.
ACS Synth Biol ; 9(12): 3364-3376, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33180461

RESUMO

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.


Assuntos
Anabaena/metabolismo , Edição de Genes/métodos , Oligopeptídeos/biossíntese , Produtos Biológicos/metabolismo , Cromatografia Líquida de Alta Pressão , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Família Multigênica , Oligopeptídeos/análise , Peptídeo Sintases/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Policetídeo Sintases/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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