Thioredoxin Dependent Changes in the Redox States of FurA from Anabaena sp. PCC 7120.

FurA is a multifunctional regulator in cyanobacteria that contains five cysteines, four of them arranged into two CXXC motifs. Lack of a structural zinc ion enables FurA to develop disulfide reductase activity. In vivo, FurA displays several redox isoforms, and the oxidation state of its cysteines determines its activity as regulator and its ability to bind different metabolites. Because of the relationship between FurA and the control of genes involved in oxidative stress defense and photosynthetic metabolism, we sought to investigate the role of type m thioredoxin TrxA as a potential redox partner mediating dithiol-disulfide exchange reactions necessary to facilitate the interaction of FurA with its different ligands. Both in vitro cross-linking assays and in vivo two-hybrid studies confirmed the interaction between FurA and TrxA. Light to dark transitions resulted in reversible oxidation of a fraction of the regulator present in Anabaena sp. PCC7120. Reconstitution of an electron transport chain using E. coli NADPH-thioredoxin-reductase followed by alkylation of FurA reduced cysteines evidenced the ability of TrxA to reduce FurA. Furthermore, the use of site-directed mutants allowed us to propose a plausible mechanism for FurA reduction. These results point to TrxA as one of the redox partners that modulates FurA performance.

2-oxoglutarate modulates the affinity of FurA for the ntcA promoter in Anabaena sp. PCC 7120.

2-oxoglutarate (2-OG) is a central metabolite that acts as a signaling molecule informing about the status of the carbon/nitrogen balance of the cell. In recent years, some transcriptional regulators and even two-component systems have been described as 2-OG sensors. In the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, two master regulators, NtcA and FurA, are deeply involved in the regulation of nitrogen metabolism. Both of them show a complex intertwined regulatory circuit to achieve a suitable regulation of nitrogen fixation. In this work, 2-OG is found to bind FurA, modulating the specific binding of FurA to the ntcA promoter. This study provides evidence of a new additional control point in the complex network controlled by the NtcA and FurA proteins.

Redox-Based Transcriptional Regulation in Prokaryotes: Revisiting Model Mechanisms.

SIGNIFICANCE: The successful adaptation of microorganisms to ever-changing environments depends, to a great extent, on their ability to maintain redox homeostasis. To effectively maintain the redox balance, cells have developed a variety of strategies mainly coordinated by a battery of transcriptional regulators through diverse mechanisms. Recent Advances: This comprehensive review focuses on the main mechanisms used by major redox-responsive regulators in prokaryotes and their relationship with the different redox signals received by the cell. An overview of the corresponding regulons is also provided. CRITICAL ISSUES: Some regulators are difficult to classify since they may contain several sensing domains and respond to more than one signal. We propose a classification of redox-sensing regulators into three major groups. The first group contains one-component or direct regulators, whose sensing and regulatory domains are in the same protein. The second group comprises the classical two-component systems involving a sensor kinase that transduces the redox signal to its DNA-binding partner. The third group encompasses a heterogeneous group of flavin-based photosensors whose mechanisms are not always fully understood and are often involved in more complex regulatory networks. FUTURE DIRECTIONS: Redox-responsive transcriptional regulation is an intricate process as identical signals may be sensed and transduced by different transcription factors, which often interplay with other DNA-binding proteins with or without regulatory activity. Although there is much information about some key regulators, many others remain to be fully characterized due to the instability of their clusters under oxygen. Understanding the mechanisms and the regulatory networks operated by these regulators is essential for the development of future applications in biotechnology and medicine.

Microcystin-LR Binds Iron, and Iron Promotes Self-Assembly.

The microcystin-producing Microcystis aeruginosa PCC 7806 and its close strain, the nonproducing Microcystis aeruginosa PCC 7005, grow similarly in the presence of 17 µM iron. Under severe iron deficient conditions (0.05 µM), the toxigenic strain grows slightly less than in iron-replete conditions, while the nonproducing microcystin strain is not able to grow. Isothermal titration calorimetry performed at cyanobacterial cytosol or meaningful environmental pHs values shows a microcystin-LR dissociaton constant for Fe2+ and Fe3+ of 2.4 µM. Using atomic force microscopy, 40% of microcystin-LR dimers were observed, and the presence of iron promoted its oligomerization up to six units. Microcystin-LR binds also Mo6+, Cu2+, and Mn2+. Polymeric microcystin binding iron may be related with a toxic cell colony advantage, providing enhanced iron bioavailability and perhaps affecting the structure of the gelatinous sheath. Inside cells, with microcystin implicated in the fitness of the photosynthetic machinery under stress conditions, the toxin would be involved in avoiding metal-dependent Fenton reactions when photooxidation causes disassembly of the iron-rich photosystems. Additionally, it could be hypothesized that polymerization-depolymerization dynamics may be an additional signal that could trigger changes (for example, in the binding of microcystin to proteins).

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA from Anabaena sp. PCC 7120.

AIMS: The ferric uptake regulator (Fur) is the main transcriptional regulator of genes involved in iron homeostasis in most prokaryotes. FurA from Anabaena sp. PCC 7120 contains five cysteine residues, four of them arranged in two redox-active CXXC motifs. The protein needs not only metal but also reducing conditions to remain fully active in vitro. Through a mutational study of the cysteine residues present in FurA, we have investigated their involvement in metal and DNA binding. RESULTS: Residue C101 that belongs to a conserved CXXC motif plays an essential role in both metal and DNA binding activities in vitro. Substitution of C101 by serine impairs DNA and metal binding abilities of FurA. Isothermal titration calorimetry measurements show that the redox state of C101 is responsible for the protein ability to coordinate the metal corepressor. Moreover, the redox state of C101 varies with the presence or absence of C104 or C133, suggesting that the environments of these cysteines are mutually interdependent. INNOVATION: We propose that C101 is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal corepressor. CONCLUSION: This mechanism supports a novel feature of a Fur protein that emerges as a regulator, which connects the response to changes in the intracellular redox state and iron management in cyanobacteria. Antioxid. Redox Signal. 00, 000-000.

γ-Lindane Increases Microcystin Synthesis in Microcystis aeruginosa PCC7806.

HCH factories, and the waste dumpsites associated to its production, have become a global environmental concern, and their runoff could pollute ground and surface waters with high levels of the pollutant. In this study, the influence of lindane (γ-HCH) on microcystin production has been investigated in Microcystis aeruginosa PCC7806. This toxic cyanobacterium is highly tolerant to γ-lindane (20 mg/L), and produces more toxin (microcystin) in the presence of the pollutant. Microcystis degrades γ-lindane and presence of γ-lindane induces genes involved in its own degradation (nirA). RT-PCRsq has been used to monitor changes in levels of transcripts encoded by the mcy operon (mcyD, mcyH and mcyJ), responsible for the microcystin synthesis machinery, as well as other genes involved in its transcriptional regulation, such as ntcA and fur family members. The presence of lindane in the culture media induces mcyD expression, as well as ntcA gene transcription, while other genes, such as mcyH, (putative ABC transporter), are downregulated. The amount of microcystin found in the cells and the culture media is higher when M. aeruginosa is treated with γ-lindane than in control cells. The results suggest that in a lindane polluted environment, Microcystis toxic strains may enhance their microcystin synthesis.

Mesoscopic model and free energy landscape for protein-DNA binding sites: analysis of cyanobacterial promoters.

The identification of protein binding sites in promoter sequences is a key problem to understand and control regulation in biochemistry and biotechnological processes. We use a computational method to analyze promoters from a given genome. Our approach is based on a physical model at the mesoscopic level of protein-DNA interaction based on the influence of DNA local conformation on the dynamics of a general particle along the chain. Following the proposed model, the joined dynamics of the protein particle and the DNA portion of interest, only characterized by its base pair sequence, is simulated. The simulation output is analyzed by generating and analyzing the Free Energy Landscape of the system. In order to prove the capacity of prediction of our computational method we have analyzed nine promoters of Anabaena PCC 7120. We are able to identify the transcription starting site of each of the promoters as the most populated macrostate in the dynamics. The developed procedure allows also to characterize promoter macrostates in terms of thermo-statistical magnitudes (free energy and entropy), with valuable biological implications. Our results agree with independent previous experimental results. Thus, our methods appear as a powerful complementary tool for identifying protein binding sites in promoter sequences.

Identification of free-living amoebae and amoeba-associated bacteria from reservoirs and water treatment plants by molecular techniques.

The occurrence of free-living amoebae (FLA) was investigated in 83 water samples from reservoirs and water treatment plants, with culture positive in 64 of them (77.1%). Polymerase chain reaction (PCR) of partial 18S rRNA gene and ITS region was performed in order to identify amoeba isolates, and the presence of Legionella pneumophila , Mycobacterium spp., Pseudomonas spp., and Microcystis aeruginosa was investigated in 43 isolates of amoebae by multiplex PCR. Of the isolated amoebae, 31 were Acanthamoeba spp., 21 were Hartmannella vermiformis, 13 were Naegleria spp., and one was Vanella spp. T2, T4, and T5 genotypes of Acanthamoeba have been identified, and T4 isolates were grouped into five subgenotypes and graphically represented with a Weblog application. Inside amoebae, L. pneumophila was detected in 13.9% (6/43) of the isolates, and Pseudomonas spp. and Mycobacterium spp. were detected in 32.6% (14/43) and 41.9% (18/43), respectively. No statistical correlation was demonstrated between FLA isolation and seasonality, but the presence of intracellular bacteria was associated with warm water temperatures, and also the intracellular presence of Mycobacterium spp. and Pseudomonas spp. were associated. These results highlight the importance of amoebae in natural waters as reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

Identification of three novel antisense RNAs in the fur locus from unicellular cyanobacteria.

The interplay between Fur (ferric uptake regulator) proteins and small, non-coding RNAs has been described as a key regulatory loop in several bacteria. In the filamentous cyanobacterium Anabaena sp. PCC 7120, a large dicistronic transcript encoding the putative membrane protein Alr1690 and an α-furA RNA is involved in the modulation of the global regulator FurA. In this work we report the existence of three novel antisense RNAs in cyanobacteria and show that a cis α-furA RNA is conserved in very different genomic contexts, namely in the unicellular cyanobacteria Microcystis aeruginosa PCC 7806 and Synechocystis sp. PCC 6803. Syα-fur RNA covers only part of the coding sequence of the fur orthologue sll0567, whose flanking genes encode two hypothetical proteins. Transcriptional analysis of fur and its adjacent genes in Microcystis unravels a highly compact organization of this locus involving overlapping transcripts. Maα-fur RNA spans the whole Mafur CDS and part of the flanking dnaJ and sufE sequences. In addition, Mafur seems to be part of a dicistronic operon encoding this regulator and an α-sufE RNA. These results allow new insights into the transcriptomes of two unicellular cyanobacteria and suggest that in M. aeruginosa PCC 7806, the α-fur and α-sufE RNAs might participate in a regulatory connection between the genes of the dnaJ-fur-sufE locus.

Oligomerization properties of FurA from the cyanobacterium Anabaena sp. PCC 7120: direct visualization by in situ atomic force microscopy under different redox conditions.

Fur proteins are global prokaryotic transcriptional regulators. Functional studies of FurA from the cyanobacterium Anabaena sp. PCC 7120 evidenced the influence of the redox environment in the activity of the regulator and its ability to aggregate through disulphide bridges. Atomic force microscopy allows single-molecule imaging and monitorization of the status of FurA under different redox conditions mimicking a physiological environment. The estimated FurA average diameter was of 4 nm. In the absence of reducing agents, the purified FurA is mainly associated as trimers, being 40 degrees the prevalent angle alpha conformed by protein monomers. Reducing conditions induces trimer rearrangement to protein monomers and a major fraction of FurA dimers. Disruption of the dimeric assemblies and appearance of higher order aggregates, namely trimers and tetramers are induced by oxidation with diamide or hydrogen peroxide. The homogeneity of the angles exhibited by the trimeric particles, as well as the occurrence of dimers in the presence of DTT, suggests the participation of relatively specific hydrophobic interactions maintaining the dimer. Direct visualization of the regulator under liquid phase at molecular resolution unravels the importance of non-polar interactions in FurA dynamics and shows that in Anabaena disulphide bridges are not essential for the dimerization of FurA.

Respiratory active mitochondrial supercomplexes.

The structural organization of the mitochondrial respiratory complexes as four big independently moving entities connected by the mobile carriers CoQ and cytochrome c has been challenged recently. Blue native gel electrophoresis reveals the presence of high-molecular-weight bands containing several respiratory complexes and suggesting an in vivo assembly status of these structures (respirasomes). However, no functional evidence of the activity of supercomplexes as true respirasomes has been provided yet. We have observed that (1) supercomplexes are not formed when one of their component complexes is absent; (2) there is a temporal gap between the formation of the individual complexes and that of the supercomplexes; (3) some putative respirasomes contain CoQ and cytochrome c; (4) isolated respirasomes can transfer electrons from NADH to O(2), that is, they respire. Therefore, we have demonstrated the existence of a functional respirasome and propose a structural organization model that accommodates these findings.

Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa.

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to produce fertilization of the oocyte in vivo and in vitro. Although this process results from a poorly understood series of morphological and molecular events, protein tyrosine phosphorylation has been associated with sperm capacitation in several mammalian species, but it still remains to be demonstrated in ram spermatozoa. Studies of capacitation in ram spermatozoa are of great interest, since several reports have suggested that the reduced fertility of cryopreserved spermatozoa is due to their premature capacitation. In this work, we report for the first time, to our knowledge, that tyrosine phosphorylation of ram sperm membrane proteins is related to the capacitation state of these cells. Capacitation induced tyrosine phosphorylation of some plasma membrane proteins of ram spermatozoa freed from seminal plasma by a dextran/swim-up procedure. It has also been proved that cold-shock induces protein tyrosine phosphorylation as well as a decrease in plasma membrane integrity. Addition of seminal plasma proteins prior to cold-shock not only improved sperm survival but also promoted a decrease in protein tyrosine phosphorylation.