Maria Luisa Peleato

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.

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same Km values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.

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.

Interaction of FurA from Anabaena sp. PCC 7120 with DNA: A Reducing Environment and the Presence of Mn2+ are Positive Effectors in the Binding to isiB and furA Promoters

The Fur (ferric uptake regulator) protein is a global regulator in most prokaryotes that controls a large number of genes. Fur is a classical repressor that uses ferrous iron as co-repressor and binds to specific DNA sequences (iron boxes) as a dimer. Three different genes coding for Fur homologues have been identified in Anabaena sp. PCC 7120. FurA controls the transcription of flavodoxin, the product of the isiB gene, and is moderately autoregulated. In this work, the promoter of the furA gene was defined and the FurA protected regions in the furA and isiB promoters were identified, showing that the binding sites for Anabaena FurA contain A/T-rich sequences with a variable arrangement compared to the conventional 19-base pair Fur consensus. The influence of different factors on the interaction between FurA and the promoters was evaluated in vitro. The affinity of FurA for the DNA targets was significantly affected by the redox status of this regulator and the presence of Mn2+. The optimal binding conditions were observed in the presence of both Mn2+ and DTT. Those results suggest that, in addition to iron availability, FurA–DNA interaction is modulated by redox conditions.

Increases in phosphoenolpyruvate carboxylase activity in iron-deficient sugar beet roots: Analysis of spatial localization and post-translational modification

Root tips of Fe-deficient and Fe-sufficient sugar beet plants grown in hydroponics have been used to study the changes in the amount and activity of the cytosolic enzyme phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31). Phosphoenolpyruvate carboxylase activity in extracts of the yellow Fe-deficient root tips was, at pH 7.3, 30-fold higher (when expressed on a FW basis) and 7.1-fold higher (when expressed on a protein basis) than that found in the extracts of Fe-sufficient root tips. The amount of phosphoenolpyruvate carboxylase protein determined by immuno-blotting was, on a protein basis, 35-fold larger in the yellow zone of Fe-deficient root tips than in the Fe-sufficient root tips. The inhibition of the phosphoenolpyruvate carboxylase activity by 500 μm malate was 41 and 58% in the extracts Fe-deficient and Fe-sufficient roots. The possibility that post-translational regulation of phosphoenolpyruvate carboxylase may occur mediated through phosphorylation, was studied by immunological detection of phosphoserine residues in root tip extracts.

Differential activities of heterocyst ferredoxin, vegetative cell ferredoxin, and flavodoxin as electron carriers in nitrogen fixation and photosynthesis in Anabaena sp.

In cyanobacteria an increasing number of low potential electron carriers is found, but in most cases their contribution to metabolic pathways remains unclear. In this work, we compare recombinant plant-type ferredoxins from Anabaena sp. PCC 7120, encoded by the genes petF and fdxH, respectively, and flavodoxin from Anabaena sp. PCC 7119 as electron carriers in reconstituted in vitro assays with nitrogenase, Photosystem I, ferredoxin-NADP+ reductase and pyruvate-ferredoxin oxidoreductase. In every experimental system only the heterocyst ferredoxin catalyzed an efficient electron transfer to nitrogenase while vegetative cell ferredoxin and flavodoxin were much less active. This implies that flavodoxin is not able to functionally replace heterocyst ferredoxin. When PFO-activity in heterocyst extracts was reconstituted under anaerobic conditions, both ferredoxins were more efficient than flavodoxin, which suggested that this PFO was of the ferredoxin dependent type. Flavodoxin, synthesized under iron limiting conditions, replaces PetF very efficiently in the electron transport from Photosystem I to NADP+, using thylakoids from vegetative cells.

A new pentaplex-nested PCR to detect five pathogenic bacteria in free living amoebae

Changes in water use and anthropogenic activity have major impacts on the quality of natural aquatic ecosystems, water distribution and wastewater plants. One of the main problems is the presence of some pathogenic microorganisms that are resistant to disinfection procedures when they are hosted by free living amoeba and that in many cases are hardly detectable by culture-based procedures. In this work we report a sensitive, low-cost procedure consisting of a pentaplex-nested PCR that allows simultaneous detection of Legionella pneumophila, Mycobacterium spp., Pseudomonas spp., Vibrio cholerae and the microcystin-producing cyanobacteria Microcystis aeruginosa. The method has been used to detect the presence of these pathogenic bacteria in water and inside free living amoeba. Its validation in 72 samples obtained from different water sources from Aragon (Spain) evidences that Mycobacterium and Pseudomonas spp are prevailing as amoeba-resistant bacteria.

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.

Exploring the interaction of microcystin-LR with proteins and DNA.

The physiological role of microcystin-LR is still under discussion, and since binding of microcystin-LR to proteins different from their main cellular targets was described, we have performed experiments in order to explore this interaction. A non-specific interaction of microcystin-LR with a variety of soluble proteins in vitro is disrupted when using organic solvents such as methanol. The isoelectric point of proteins is not affected by their interaction with microcystin-LR, even though the presence of microcystin-LR alters the pool of peptides obtained by tryptic digestions. Under the conditions tested, microcystin-LR does not exhibit affinity for DNA. Although it is unlikely that the non-specific binding of microcystin-LR to proteins has a physiological meaning, one must be aware of the fact that determinations of the toxin extracted from any biological sample may be affected by the presence of proteins in the extracts. Consequently, we strongly recommend use organic solvents and to lyophilise the tissue samples to guarantee the accessibility of these organic solvents to microcystin-LR when performing experiments with tissue or cell extracts.