Artículos (Instituto de Bioquímica Vegetal y Fotosíntesis IBVF – CIC Cartuja)

URI permanente para esta colecciónhttps://hdl.handle.net/11441/10949

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Thioredoxins m are major players in the multifaceted light-adaptive response in Arabidopsis thaliana
(Wiley, 2021-07-20) Serrato, Antonio J.; Rojas González, José A.; Torres Romero, Diego; Vargas, Paola; Mérida, Ángel; Sahrawy, Mariam; Bioquímica Vegetal y Biología Molecular; Ministerio de Economía y Competitividad (MINECO). España; Ministerio de Ciencia e Innovación (MICIN). España; European Union (UE)
Thioredoxins (TRXs) are well-known redox signalling players, which carry out post-translational modiﬁca-tions in target proteins. Chloroplast TRXs are divided into different types and have central roles in lightenergy uptake and the regulation of primary metabolism. The isoforms TRX m1, m2, and m4 from Arabidop-sis thaliana are considered functionally related. Knowing their key position in the hub of plant metabolism,we hypothesized that the impairment of the TRX m signalling would not only have harmful consequenceson chloroplast metabolism but also at different levels of plant development. To uncover the physiologicaland developmental processes that depend on TRX m signalling, we carried out a comprehensive study ofArabidopsis single, double, and triple mutants defective in the TRX m1, m2, and m4 proteins. As light andredox signalling are closely linked, we investigated the response to high light (HL) of the plants that aregradually compromised in TRX m signalling. We provide experimental evidence relating the lack of TRX mand the appearance of novel phenotypic features concerning mesophyll structure, stomata biogenesis, andstomatal conductance. We also report new data indicating that the isoforms of TRX m ﬁne-tune theresponse to HL, including the accumulation of the protective pigment anthocyanin. These results revealnovel signalling functions for the TRX m and underline their importance for plant growth and fulﬁlment ofthe acclimation/response to HL conditions.
The Heterocyst-Specific Small RNA NsiR1 Regulates the Commitment to Differentiation in Nostoc
(American Society for Microbiology, 2022-03-01) Brenes-Álvarez, Manuel; Vioque Peña, Agustín; Muro Pastor, Alicia M.; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España; European Union (UE)
Heterocysts are specialized cells that ﬁlamentous cyanobacteria differentiatefor the ﬁxation of atmospheric nitrogen when other nitrogen sources are not available.Heterocyst differentiation at semiregular intervals along the ﬁlaments requires complexstructural and metabolic changes that are under the control of the master transcriptionalregulator HetR. NsiR1 (nitrogen stress-induced RNA 1) is a HetR-dependent noncoding RNAthat is expressed from multiple chromosomal copies, some identical, some slightly divergentin sequence, speciﬁcally in heterocysts from very early stages of differentiation. We havepreviously shown that NsiR1 inhibits translation of the overlapping hetF mRNA by anantisense mechanism. Here, we identify alr3234, a hetP-like gene involved in the regula-tion of commitment (point of no return) to heterocyst differentiation, as a target of NsiR1.A strain overexpressing one of the identical copies of NsiR1 commits to heterocyst devel-opment earlier than the wild type. The posttranscriptional regulation exerted by NsiR1 onthe expression of two genes involved in heterocyst differentiation and commitment, hetFand alr3234, adds a new level of complexity to the network of transcriptional regulationand protein-protein interactions that participate in heterocyst differentiation. IMPORTANCE Heterocysts are nitrogen-ﬁxing specialized cells that appear at semiregularintervals along cyanobacterial ﬁlaments upon nitrogen starvation. The differentiation andpatterning of heterocysts is a model for the study of cell differentiation in multicellularprokaryotes. The regulation of differentiation, which is only partially understood, includestranscriptional changes, factor diffusion between cells, and protein-protein interactions. Thiswork describes the identiﬁcation of a novel target for NsiR1, a small RNA (sRNA) encodedin multiple slightly divergent copies, and shows how different copies of “sibling” sRNAs reg-ulate the expression of different targets involved in one of the few examples of a differen-tiation process in prokaryotes.
Photosynthetic assimilation of CO2 regulates TOR activity
(National Academy of Sciences, 2022-01-07) Mallén Ponce, Manuel J.; Pérez Pérez, María Esther; Crespo, José L.; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia y Tecnología (MCYT). España
The target of rapamycin (TOR) kinase is a master regulator that integrates nutrient signals to promote cell growth in all eukaryotes. It is well established that amino acids and glucose are major regulators of TOR signaling in yeast and metazoan, but whether and how TOR responds to carbon availability in photosynthetic organisms is less understood. In this study, we showed that photosynthetic assimilation of CO2 by the Calvin–Benson–Bassham (CBB) cycle regulates TOR activity in the model single-celled microalga Chlamydomonas reinhardtii. Stimulation of CO2 fixation boosted TOR activity, whereas inhibition of the CBB cycle and photosynthesis down-regulated TOR. We uncovered a tight link between TOR activity and the endogenous level of a set of amino acids including Ala, Glu, Gln, Leu, and Val through the modulation of CO2 fixation and the use of amino acid synthesis inhibitors. Moreover, the finding that the Chlamydomonas starch-deficient mutant sta6 displayed disproportionate TOR activity and high levels of most amino acids, particularly Gln, further connected carbon assimilation and amino acids to TOR signaling. Thus, our results showed that CO2 fixation regulates TOR signaling, likely through the synthesis of key amino acids.
Soil Bacteriome Shifts along a Cultivation Gradient in Southwestern Spanish Wetlands
(Springer, 2025-11-29) González Pimentel, José Luis; Cuecas Morano, María de Piedras Alba; Álvarez Núñez, Consolación; Bioquímica Vegetal y Biología Molecular
Understanding how long-term agricultural practices affect soil bacteriome is essential for sustainable land management. In the Guadalquivir Marshes of southwestern Spain, which encompass both Doñana National Park and one of Europe’s most productive rice cultivation areas, decades of rice farming have transformed natural wetlands into artificial agroecosystems. Although bacterial degradation in cultivated soils has been previously suggested, comparative analyses between rice paddies and adjacent natural wetlands remain scarce. Here, we characterized the soil bacteriome across a cultivation gradient by comparing undisturbed natural marshes, within Doñana National Park, with rice fields cultivated for 25 years (Cantarita) and 80 years (Mínima 2). Using full 16S rRNA gene via long-read metabarcoding and standardized soil physicochemical assays, we analysed taxonomic composition, environmental associations, and predicted functional profiles. Our results reveal a progressive restructuring of bacterial communities with increased cultivation time, notably a significant enrichment of Chloroflexota (especially Anaerolineae) and a decline in Actinomycetota and Planctomycetota in paddy soils. Functional predictions indicated a higher potential for denitrification in cultivated soils—likely involving Chloroflexota taxa—compared to more diverse nitrogen pathways in natural sites. These shifts were strongly associated with changes in pH, electrical conductivity, calcium carbonate, and nitrate levels. Remarkably, most bacterial differences were already evident within the first 25 years of cultivation, underscoring the rapid ecological impact of intensive rice cultivation. Notably, we identified specific bacterial groups (Anaerolineae and Nocardioides in paddy soils; Euzebya, Rubrobacter, and Planctomycetota in natural wetlands), whose enrichment was associated with soil type. This approach highlights the value of integrating bacterial-based assessments into sustainable wetland management strategies.
Hydrogen sulfide improves performance under suppressed photorespiration in Arabidopsis thaliana and orchestrates molecular reprogramming to alleviate stress
(Elsevier, 2026-01-08) Luque, C.; García Calderón, Margarita; Gotor, C.; Márquez Cabeza, Antonio José; Aroca Aguilar, Ángeles; Bioquímica Vegetal y Biología Molecular; European Union (UE); Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
High levels of atmospheric carbon dioxide result in suppression of plant photorespiration. The non-photorespiratory conditions (NPC) result in unbalancing the C/N metabolism, overproducing reactive oxygen species (ROS), and reducing stomatal activity. In plant stress responses, hydrogen sulfide (H2S) has been identified as an important signaling molecule through persulfidation of specific proteins. Previous works demonstrated that H₂S protects Arabidopsis thaliana against NPC-induced stress, and this work investigates the molecular basis of such protection. H₂S modulates a metabolic reprogramming influencing elemental homeostasis of C/N ratio, amino acids profile, central carbon metabolites and accumulation of polyunsaturated fatty acids (PUFAs). Persulfidation level under NPC was also restored after H₂S treatment. At the transcriptomic level, several well-known hypoxia marker genes, such as plant CYSTEINE OXIDASE 1 and 2, ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR ERF71 AND ETHYLENE RECEPTOR 2, are induced under NPC, and sulfide treatment decreases their expression levels to the ones in active photorespiration conditions (APC). H₂S also negatively regulates ABA signaling by targeting genes controlling ion transport and stomatal development which are involved in stomatal function. These integrated responses across metabolism, redox regulation and developmental programming emphasize the key contribution of H₂S to orchestrating plant adaptation to high CO₂ environments, positioning it as a master regulator that ensures plant resilience in the face of climate change.
Dihydroxyacetone phosphate generated in the chloroplast mediates the activation of TOR by CO2 and light
(American Association for the Advancement of Science, 2025-04-18) Mallén Ponce, Manuel J.; Quintero Moreno, Andrea M.; Gámez Arcas, Samuel; Grossman, Arthur R.; Pérez Pérez, María Esther; Crespo, José L.; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España
Light and CO2 assimilation activate the target of rapamycin (TOR) kinase in photosynthetic cells, but how thesesignals are transmitted to TOR is unknown. Using the green alga Chlamydomonas reinhardtii as a model system,we identified dihydroxyacetone phosphate (DHAP) as the key metabolite regulating TOR in response to carbonand light cues. Metabolomic analyses of synchronized cells revealed that DHAP levels change more than any oth-er metabolite between dark- and light-grown cells and that the addition of the DHAP precursor, dihydroxyacetone(DHA), was sufficient to activate TOR in the dark. We also demonstrated that TOR was insensitive to light or inor-ganic carbon but not to exogenous DHA in a Chlamydomonas mutant defective in the export of DHAP from thechloroplast. Our results provide a metabolic basis for the mode of TOR control by light and inorganic carbon andindicate that cytoplasmic DHAP is an important metabolic regulator of TOR.
Redox partner interactions in the ATG8 lipidation system in microalgae
(Elsevier, 2023-04-06) Mallén Ponce, Manuel J.; Gámez Arcas, Samuel; Pérez Pérez, María Esther; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España
Autophagy is a catabolic pathway that functions as a degradative and recycling process to maintain cellular homeostasis in most eukaryotic cells, including photosynthetic organisms such as microalgae. This process involves the formation of double-membrane vesicles called autophagosomes, which engulf the material to be degraded and recycled in lytic compartments. Autophagy is mediated by a set of highly conserved autophagyrelated (ATG) proteins that play a fundamental role in the formation of the autophagosome. The ATG8 ubiquitin-like system catalyzes the conjugation of ATG8 to the lipid phosphatidylethanolamine, an essential reaction in the autophagy process. Several studies identified the ATG8 system and other core ATG proteins in photosynthetic eukaryotes. However, how ATG8 lipidation is driven and regulated in these organisms is not fully understood yet. A detailed analysis of representative genomes from the entire microalgal lineage revealed a high conservation of ATG proteins in these organisms with the remarkable exception of red algae, which likely lost ATG genes before diversification. Here, we examine in silico the mechanisms and dynamic interactions between different components of the ATG8 lipidation system in plants and algae. Moreover, we also discuss the role of redox post-translational modifications in the regulation of ATG proteins and the activation of autophagy in these organisms by reactive oxygen species.
Redox-mediated activation of ATG3 promotes ATG8 lipidation and autophagy progression in Chlamydomonas reinhardtii
(Oxford University Press, 2023-09-29) Mallén Ponce, Manuel J.; Pérez Pérez, María Esther; Bioquímica Vegetal y Biología Molecular; Ministerio de Economía y Competitividad (MINECO). España; Ministerio de Ciencia e Innovación (MICIN). España
Autophagy is one of the main degradative pathways used by eukaryotic organisms to eliminate useless or damaged intracellular material to maintain cellular homeostasis under stress conditions. Mounting evidence indicates a strong interplay between the generation of reactive oxygen species and the activation of autophagy. Although a tight redox regulation of autophagy has been shown in several organisms, including microalgae, the molecular mechanisms underlying this control remain poorly understood. In this study, we have performed an in-depth in vitro and in vivo redox characterization of ATG3, an E2-activating enzyme involved in ATG8 lipidation and autophagosome formation, from 2 evolutionary distant unicellular model organisms: the green microalga Chlamydomonas (Chlamydomonas reinhardtii) and the budding yeast Saccharomyces cerevisiae. Our results indicated that ATG3 activity from both organisms is subjected to redox regulation since these proteins require reducing equivalents to transfer ATG8 to the phospholipid phosphatidylethanolamine. We established the catalytic Cys of ATG3 as a redox target in algal and yeast proteins and showed that the oxidoreductase thioredoxin efficiently reduces ATG3. Moreover, in vivo studies revealed that the redox state of ATG3 from Chlamydomonas undergoes profound changes under autophagyactivating stress conditions, such as the absence of photoprotective carotenoids, the inhibition of fatty acid synthesis, or high light irradiance. Thus, our results indicate that the redox-mediated activation of ATG3 regulates ATG8 lipidation under oxidative stress conditions in this model microalga.
Lipid turnover through lipophagy in the newly identiﬁedextremophilic green microalga Chlamydomonas urium
(Wiley, 2024-07-05) Pérez Pérez, María Esther; Mallén Ponce, Manuel J.; Odriozola Gil, Yosu; Rubio, Alejandro; Salas, Joaquín J.; Martínez Force, Enrique; Pérez Pulido, Antonio J.; Crespo, José Luis; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Junta de Andalucía; Consejo Superior de Investigaciones Científicas (CSIC)
Autophagy is a central degradative pathway highly conserved among eukaryotes, includingmicroalgae, which remains unexplored in extremophilic organisms. In this study, we describedand characterized autophagy in the newly identiﬁed extremophilic green microalga Chlamy-domonas urium, which was isolated from an acidic environment. The nuclear genome of C. urium was sequenced, assembled and annotated in order toidentify autophagy-related genes. Transmission electron microscopy, immunoblotting, meta-bolomic and photosynthetic analyses were performed to investigate autophagy in this extre-mophilic microalga. The analysis of the C. urium genome revealed the conservation of core autophagy-relatedgenes. We investigated the role of autophagy in C. urium by blocking autophagic ﬂux withthe vacuolar ATPase inhibitor concanamycin A. Our results indicated that inhibition of autop-hagic ﬂux in this microalga resulted in a pronounced accumulation of triacylglycerols and lipiddroplets (LDs). Metabolomic and photosynthetic analyses indicated that C. urium cells withimpaired vacuolar function maintained an active metabolism. Such effects were not observedin the neutrophilic microalga Chlamydomonas reinhardtii. Inhibition of autophagic ﬂux in C. urium uncovered an active recycling of LDs through lipo-phagy, a selective autophagy pathway for lipid turnover. This study provided the metabolicbasis by which extremophilic algae are able to catabolize lipids in the vacuole.
Retention of a SulP-family bicarbonate transporter in a periplasmic N2-fixing cyanobacterial endosymbiont of an open ocean diatom
(Oxford University Press, 2025-09-04) Nieves Morión, Mercedes; Romero García, Rubén; Bardi, Sepehr; López Maury, Luis; Hagemann, Martin; Flores, Enrique; Foster, Rachel A.; Bioquímica Vegetal y Biología Molecular; Swedish Research Council; Junta de Andalucía; Knut and Alice Wallenberg Foundation
Symbioses between diatoms and the N2-fixing, heterocyst-forming cyanobacteria Richelia spp. are widespread and contribute to primary production. Unique to these symbioses is a variation in the symbiont location: one lives in the host cytoplasm (endobiont) vs. residing between the host frustule and plasmalemma (periplasmic endobiont). Both partners are photosynthetic, yet how the partners acquire, share, or compete for bicarbonate necessary for their photosynthesis is unknown. The genomes of both endobionts (ReuHH01 and RintRC01, respectively) contain genes encoding SulP-family proteins, which are oxyanion transporters. To study the possible involvement of these transporters in bicarbonate uptake, we used complementation in a Synechocystis sp. PCC 6803 mutant that is unable to grow at air levels of CO2 because all five of its inorganic carbon uptake systems have been inactivated. Of the five genes tested, only one (RintRC_3892) from the periplasmic endobiont complemented the mutant to grow with air levels of CO2 or at low bicarbonate concentrations. The complemented strain showed strong sodium-dependent and low-affinity bicarbonate uptake that was consistent with bicarbonate concentrations expected in the diatom periplasm. Additionally, all the amino acids involved in the bicarbonate binding site of BicA from Synechocystis sp. PCC 6803 are conserved in RintRC_3892. Finally, the importance of the RintRC_3892 protein was confirmed by the consistent detection of its transcripts in wild Richelia populations from three different oceans. Combined our results showed no evidence for a bicarbonate transporter in the cytoplasmic endobiont, whereas the periplasmic endobiont has retained a SulP-type bicarbonate transporter for its own photosynthesis.
Two opposing redox signals mediated by 2-cys peroxiredoxin shape the redox proteome during photosynthetic induction
(Elsevier, 2025-08-05) Doron, Shani; Lampl, Nardy; Savidor, Alon; Pri-Or, Amir; Katina, Corine; Cejudo Fernández, Francisco Javier; Levin, Yishai; Rosenwasser, Shilo; Bioquímica Vegetal y Biología Molecular; European Research Council (ERC); Israel Science Foundation
Photosynthetic induction, characterized by the lag in CO2 assimilation rates during transition from darkness to light, has traditionally been attributed to Rubisco activase activity and stomatal opening. Yet, the faster induction of photosynthesis in the 2-Cys peroxiredoxins (Prxs) mutant (2cpab) suggested a role for oxidative signals in regulating photosynthetic rates, although the underlying molecular mechanism remains unclear. SPEAR, a redox proteomics approach, was used to systematically map redox changes occurring during photosynthesis induction and to unravel the role of 2-Cys Prxs in shaping these redox alterations. No significant difference was observed in protein expression levels between WT and 2cpab plants, suggesting that protein abundance does not account for the 2cpab phenotype. During the transition from dark to low light, 82 and 54 cysteine-containing peptides were reduced or oxidized, respectively, in WT plants. Most redox-regulated cysteines in photosynthetic proteins were found oxidized in the dark and became reduced in response to light. A reverse pattern was observed among redox-regulated cysteines in proteins involved in starch degradation and chloroplast glycolysis, which shifted from a reduced to an oxidized state in response to light. These findings demonstrate the initiation of two opposing redox responses, affecting distinct sets of metabolic proteins during the induction phase. Remarkably, a significantly lower number of cysteines were reduced or oxidized in 2cpab plants, highlighting the crucial role 2-Cys Prxs play in shaping both signals. Taken together, rotational shifts between metabolic pathways during the photosynthesis induction phase are regulated by two opposing redox signals mediated by 2-Cys Prx activity.
Redox regulation of membrane-associated processes mediated by chloroplastic thioredoxins
(Elsevier, 2026-02) Vargas, Paola; Torres Romero, Diego; Mérida, Ángel; Sahrawy, Mariam; Serrato, Antonio J.; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Ministerio de Ciencia e Innovación (MICIN). España
Plant chloroplasts are complex organelles that house a plethora of redox-controlled metabolic processes. However, our understanding of membrane-level redox signalling remains limited. In order to expand our knowledge of redox regulation in these photosynthetic subcellular compartments, we carried out in vitro and in vivo experimental approaches focused on the analysis of processes taking place at the membrane level. In addition to the classic stromal localization, these approaches have revealed that chloroplastic thioredoxins (TRXs) from Arabidopsis thaliana are also membrane-associated proteins, having a network of non-stromal interactors. We have identified 185 putative chloroplastic targets, with 80 % predicted to be located at the envelope or thylakoid membranes, and classified into 18 functional categories, with the most prevalent one being related to photosynthesis and, notably, metabolite/ion transport, a novel finding in redox regulation. Direct in vivo interactions between TRXs m and three integral proteins involved in protein import and metabolite transport, as well as one thylakoid membrane-bound protein that regulates proteolytic processes, were confirmed. Moreover, the role of TRXs m appears to extend beyond the regulation of the primary process of photosynthesis, such as during the establishment of greening cotyledons and the protection against high-light intensities. These findings provide a novel perspective on the function of TRXs as multifaceted regulators. The present study aims to address a current knowledge gap by exploring redox signalling in the membranes of plant chloroplasts.
The Inorganic Nutrient Regime and the mre Genes Regulate Cell and Filament Size and Morphology in the Phototrophic Multicellular Bacterium Anabaena
(American Society for Microbiology, 2020) Velázquez Suárez, Cristina; Luque, I.; Herrero, A.; Genética; Agencia Estatal de Investigación. España; European Union (UE); Gobierno de España
ABSTRACT The model cyanobacterium Anabaena sp. PCC 7120 exhibits a phototrophic metabolism relying on oxygenic photosynthesis and a complex morphology. The organismic unit is a filament of communicated cells that may include cells specialized in different nutritional tasks, thus representing a paradigm of multicellular bacteria. In Anabaena, the inorganic carbon and nitrogen regime influenced not only growth, but also cell size, cell shape, and filament length, which also varied through the growth cycle. When using combined nitrogen, especially with abundant carbon, cells enlarged and elongated during active growth. When fixing N2, which imposed lower growth rates, shorter and smaller cells were maintained. In Anabaena, gene homologs to mreB, mreC, and mreD form an operon that was expressed at higher levels during the phase of fastest growth. In an ntcA mutant, mre transcript levels were higher than in the wild type and, consistently, cells were longer. Negative regulation by NtcA can explain that Anabaena cells were longer in the presence of combined nitrogen than in diazotrophic cultures, in which the levels of NtcA are higher. mreB, mreC, and mreD mutants could grow with combined nitrogen, but only the latter mutant could grow diazotrophically. Cells were always larger and shorter than wild-type cells, and their orientation in the filament was inverted. Consistent with increased peptidoglycan width and incorporation in the intercellular septa, filaments were longer in the mutants, suggesting a role for MreB, MreC, and MreD in the construction of septal peptidoglycan that could affect intercellular communication required for diazotrophic growth. IMPORTANCE Most studies on the determination of bacterial cell morphology have been conducted in heterotrophic organisms. Here, we present a study of how the availability of inorganic nitrogen and carbon sources influence cell size and morphology in the context of a phototrophic metabolism, as found in the multicellular cyanobacterium Anabaena. In Anabaena, the expression of the MreB, MreC, and MreD proteins, which influence cell size and length, are regulated by NtcA, a transcription factor that globally coordinates cellular responses to the C-to-N balance of the cells. Moreover, MreB, MreC, and MreD also influence septal peptidoglycan construction, thus affecting filament length and, possibly, intercellular molecular exchange that is required for diazotrophic growth. Thus, here we identified new roles for Mre proteins in relation to the phototrophic and multicellular character of a cyanobacterium, Anabaena.
Antimicrobial Activity of the Circular Bacteriocin AS-48 against Clinical Multidrug-Resistant Staphylococcus aureus
(MDPI, 2021) Velázquez Suárez, Cristina; Cebrián, R.; Gasca Capote, C.; Sorlózano Puerto, A.; Gutiérrez Fernández, J.; Martínez Bueno, M.; Maqueda, M.; Valdivia, E.; Genética; Ministerio de Economia, Industria y Competitividad (MINECO). España; Gobierno de España
The treatment and hospital-spread-control of methicillin-resistant Staphylococcus aureus (MRSA) is an important challenge since these bacteria are involved in a considerable number of nosocomial infections that are difficult to treat and produce prolonged hospitalization, thus also increasing the risk of death. In fact, MRSA strains are frequently resistant to all β-lactam antibiotics, and co-resistances with other drugs such as macrolides, aminoglycosides, and lincosamides are usually reported, limiting the therapeutical options. To this must be added that the ability of these bacteria to form biofilms on hospital surfaces and devices confer high antibiotic resistance and favors horizontal gene transfer of genetic-resistant mobile elements, the spreading of infections, and relapses. Here, we genotypically and phenotypically characterized 100 clinically isolated S. aureus for their resistance to 18 antibiotics (33% of them were OXA resistant MRSA) and ability to form biofilms. From them, we selected 48 strains on the basis on genotype group, antimicrobial-resistance profile, and existing OXA resistance to be assayed against bacteriocin AS-48. The results showed that AS-48 was active against all strains, regardless of their clinical source, genotype, antimicrobial resistance profile, or biofilm formation capacity, and this activity was enhanced in the presence of the antimicrobial peptide lysozyme. Finally, we explored the effect of AS-48 on formed S. aureus biofilms, observing a reduction in S. aureus S-33 viability. Changes in the matrix structure of the biofilms as well as in the cell division process were observed with scanning electron microscopy in both S-33 and S-48 S. aureus strains.
The Role of MreB, MreC and MreD in the Morphology of the Diazotrophic Filament of Anabaena sp. PCC 7120
(MDPI, 2022) Velázquez Suárez, Cristina; Luque, I.; Herrero, A.; Genética; Junta de Andalucía; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Gobierno de España
The cyanobacterium Anabaena sp. PCC 7120 forms filaments of communicating cells. Under conditions of nitrogen scarcity, some cells differentiate into heterocysts, allowing the oxygen-sensitive N2-reduction system to be expressed and operated in oxic environments. The key to diazotrophic growth is the exchange of molecules with nutritional and signaling functions between the two types of cells of the filament. During heterocyst differentiation, the peptidoglycan sacculus grows to allow cell enlargement, and the intercellular septa are rebuilt to narrow the contact surface with neighboring cells and to hold specific transport systems, including the septal junction complexes for intercellular molecular transfer, which traverse the periplasm between heterocysts and neighboring vegetative cells through peptidoglycan nanopores. Here we have followed the spatiotemporal pattern of peptidoglycan incorporation during heterocyst differentiation by Van-FL labeling and the localization and role of proteins MreB, MreC and MreD. We observed strong transitory incorporation of peptidoglycan in the periphery and septa of proheterocysts and a maintained focal activity in the center of mature septa. During differentiation, MreB, MreC and MreD localized throughout the cell periphery and at the cell poles. In mreB, mreC or mreD mutants, instances of strongly increased peripheral and septal peptidoglycan incorporation were detected, as were also heterocysts with aberrant polar morphology, even producing filament breakage, frequently lacking the septal protein SepJ. These results suggest a role of Mre proteins in the regulation of peptidoglycan growth and the formation of the heterocyst neck during differentiation, as well as in the maintenance of polar structures for intercellular communication in the mature heterocyst. Finally, as previously observed in filaments growing with combined nitrogen, in the vegetative cells of diazotrophic filaments, the lack of MreB, MreC or MreD led to altered localization of septal peptidoglycan-growth bands reproducing an altered localization of FtsZ and ZipN rings during cell division.
The Role of Mre Factors and Cell Division in Peptidoglycan Growth in the Multicellular Cyanobacterium Anabaena
(American Society for Microbiology, 2022) Velázquez Suárez, Cristina; Valladares, A.; Luque, I.; Herrero, A.; Genética; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER)
Bacteria in general serve two main tasks: cell growth and division. Both processes include peptidoglycan extension to allow cell expansion and to form the poles of the daughter cells, respectively. The cyanobacterium Anabaena forms filaments of communicated cells in which the outer membrane and the peptidoglycan sacculus, which is engrossed in the intercellular regions between contiguous cells, are continuous along the filament. During the growth of Anabaena, peptidoglycan incorporation was weak at the cell periphery. During cell division, midcell peptidoglycan incorporation matched the localization of the divisome, and incorporation persisted in the intercellular septa, even after the division was completed. MreB, MreC, and MreD were located throughout the cell periphery and, in contrast to other bacteria, also to the divisome all along midcell peptidoglycan growth. In Anabaena mutants bearing inactivated mreB, mreC, or mreD genes, which showed conspicuous alterations in the filament morphology, consecutive septal bands of peptidoglycan growth were frequently not parallel to each other and were irregularly spaced along the filament, reproducing the disposition of the Z-ring. Both lateral and septal growth was impaired in strains down-expressing Z-ring components, and MreB and MreD appeared to directly interact with some divisome components. We propose that, in Anabaena, association with the divisome is a way for localization of MreB, MreC, and MreD at the cell poles, where they regulate lateral, midcell, and septal peptidoglycan growth with the latter being involved in localization and maintenance of the intercellular septal-junction protein structures that mediate cell-cell communication along the filament. IMPORTANCE Peptidoglycan surrounds the bacterial cell, being essential for the determination of the bacterium-specific morphology and survival. Peptidoglycan growth has been thoroughly investigated in some model rod-shaped bacteria, and more recently some representatives with disparate morphologies became into focus, revealing that patterns of peptidoglycan growth are much more diverse than previously anticipated. Anabaena forms filaments of communicated cells exhibiting features of multicellular organisms, such as the production of morphogens and coupled circadian oscillations. Here, we showed that Anabaena presented a distinct pattern of peptidoglycan growth characterized by continuous incorporation of material at the polar intercellular regions, contributing to assembling and maintaining the protein complexes that expand the septal peptidoglycan mediating intercellular molecular exchange in the filament.
SepT, A Novel Protein Specific to Multicellular Cyanobacteria, Influences Peptidoglycan Growth and Septal Nanopore Formation in Anabaena sp. PCC 7120
(American Society for Microbiology, 2002) Velázquez Suárez, Cristina; Springstein, B. L.; Nieves Morión, M.; Helbig, A. O.; Kieninger, A. K.; Maldener, I.; Nürnberg, D. J.; Stucken, K.; Luque, I.; Dagan, T.; Herrero, A.; Genética; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía; German Science Foundation (DFG)
Anabaena sp. PCC 7120 grows by forming filaments of communicating cells and is considered a paradigm of bacterial multicellularity. Molecular exchanges between contiguous cells in the filament take place through multiprotein channels that traverse the septal peptidoglycan through nanopores connecting their cytoplasms. Besides, the septal-junction complexes contribute to strengthen the filament. In search for proteins with coiled-coil domains that could provide for cytoskeletal functions in Anabaena, we identified SepT (All2460). SepT is characteristic of the phylogenetic clade of filamentous cyanobacteria with the ability to undergo cell differentiation. SepT-GFP fusions indicate that the protein is located at the cell periphery and, conspicuously, in the intercellular septa. During cell division, the protein is found at midcell resembling the position of the divisome. The bacterial adenylate cyclase two-hybrid analysis shows SepT interactions with itself and putative elongasome (MreB, RodA), divisome (FtsW, SepF, ZipN), and septal-junction (SepJ)-related proteins. Thus, SepT appears to rely on the divisome for localization at mature intercellular septa to form part of intercellular protein complexes. Two independently obtained mutants lacking SepT showed alterations in cell size and impaired septal and peripheral peptidoglycan incorporation during cell growth and division. Notably, both mutants showed conspicuous alterations in the array of nanopores present in the intercellular peptidoglycan disks, including aberrant nanopore morphology, number, and distribution. SepT appears, therefore, to be involved in the control of peptidoglycan growth and the formation of cell-cell communication structures that are at the basis of the multicellular character of this group of cyanobacteria. IMPORTANCE Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism.
An Ancient Bacterial Zinc Acquisition System Identified from a Cyanobacterial Exoproteome
(Public Library of Science, 2024) ; Ochoa de Alda, Jesús A. G.; Velázquez Suárez, Cristina; Rubio, M. A.; Gómez Baena, G.; Fillat, M. F.; Luque, I.; Genética; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Gobierno de Aragón; Agencia Estatal de Investigación. España; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER)
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.
Quantitative Assessment of Hormogonia Induction in Nostoc punctiforme by a Fluorescent Reporter Strain
(Oxford University Press, 2025-05-13) Neubauer, Anna; Iniesta Pallarés, Macarena; Álvarez Núñez, Consolación; Bailly, Aurélien; Szövényi, Péter; Mariscal, Vicente; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Universidad de Sevilla
While symbiotic plant–cyanobacteria interactions hold significant potential for revolutionizing agricultural practices by reducing the application of artificial nitrogen fertilizers, the genetic underpinnings of the symbiotic interaction between the plant host and the cyanobiont remain poorly understood. In particular, the molecular mechanisms through which host plants induce the formation of motile cyanobacterial filaments (hormogonia), essential for colonization and initiation of symbiosis, are not well characterized. In this study, we present a novel yet objective method for quantifying hormogonia induction, addressing limitations of traditional qualitative approaches. We have developed a reporter strain of Nostoc punctiforme PCC 73102 capable of quantifying hormogonia induction in response to diverse biotic and abiotic stimuli. This reporter strain, generated via triparental mating conjugation transformation, contains the promoter sequence of prepilin pilA fused to a green fluorescent protein (GFP) and enables quantitative and high-throughput monitoring of hormogonia induction using a microplate reader. Our innovative approach, employing a cyanobacterial hormogonia reporter strain, allows high-throughput screening of the hormogonia-inducing effect of a wide array of environmental and plant signals. This method is expected to greatly advance our understanding of the genetic determinants underpinning plant–cyanobacteria symbioses.
New Role for Thioredoxins in Plants: Implication of TRXo1 in Protein Depersulfidation
(Elsevier, 2025) De Brasi Velasco, Sabrina; Aroca Aguilar, Ángeles; Romero, Luis C.; Gotor, Cecilia; Sevilla, Francisca; Jiménez, Ana; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; European Union (UE); Comunidad Autónoma de Murcia; Junta de Andalucía
Persulfidation, a posttranslational modification of cysteines to persulfides, is the best characterized molecular mechanism of H2S signaling. This study is focused on new functions for thioredoxins (TRXs) in plants beyond those of thiol disulfide (S–S) exchange, including the regulation of protein persulfidation as it has been described in animal systems. To elucidate the impact of TRXo1 deficiency on the protein persulfidation pattern in plants of Arabidopsis thaliana L. wild type (WT) and two Attrxo1 T-DNA insertion mutants grown under non stress conditions, a quantitative proteomic approach was performed. The proteomic analysis revealed a higher number of proteins that were more persulfidated in the mutants compared to WT plants, suggesting a role for TRXo1 in protein depersulfidation. Interestingly, most of the differentially persulfidated proteins were located in the chloroplast, implying a coordination between chloroplast H2S-dependent persulfidation and mitochondrial TRXo1 depersulfidation. Among the differentially persulfidated proteins located in mitochondria, the antioxidant enzymes sAPX, DHAR1 and MDAR6 were selected for further studies. The effect of H2S-dependent persulfidation on their enzymatic activities and its reversibility by the NADPH/thioredoxin reductase (NTRB)/TRXo1 system was analyzed, as well as their persulfidation levels were quantified. Sulfide treatment brought about increases in the activity levels of the enzymes, that match with a raise on the persulfidation levels. Interestingly, both activations declining after treatment with the thioredoxin system, indicate the regulation of their persulfidation by TRXo1. These results point to a positive effect of persulfidation on the enzymatic activities and also to a new depersulfidase activity for TRXo1. All together these results give a new insight of the mechanism of elimination of –SSH groups in plants exerted by TRXo1, and the involvement of a redox regulation on the protein persulfidation.

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