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Previous genomic analyses of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 have identified three ferric uptake regulator (Fur) homologs with low sequence identities and probably different functions in the cell. FurA is a constitutive protein that shares the highest homology with Fur from heterotrophic bacteria and appears to be essential for invitro growth. In this study, we have analysed the effects of FurA overexpression on the Anabaena sp. phenotype and investigated which of the observed alterations were directly operated by FurA. Overexpression of the regulator led to changes in cellular morphology, resulting in shorter filaments with rounded cells of different sizes. The furA-overexpressing strain showed a slower photoautotrophic growth and a marked decrease in the oxygen evolution rate. Overexpression of the regulator also decreased both catalase and superoxide dismutase activities, but did not lead to an increase in the levels of intracellular reactive oxygen species. By combining phenotypic studies, reverse transcription-PCR analyses and electrophoretic mobility shift assays, we identified three novel direct targets of FurA, including genes encoding a siderophore outer membrane transporter (schT), bacterial actins (mreBCD) and the PSII reaction center protein D1 (psbA). The affinity of FurA for these novel targets was markedly affected by the absence of divalent metal ions, confirming previous evidence of a critical role for the metal co-repressor in the function of the regulator invivo. The results unravel new cellular processes modulated by FurA, supporting its role as a global transcriptional regulator in Anabaena sp. PCC 7120

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.

Aim: To determine whether expression of a cyanobacterial flavodoxin in soil bacteria of agronomic interest confers protection against the widely used herbicides paraquat and atrazine. Methods and Results: The model bacterium Escherichia coli, the symbiotic nitrogen-fixing bacterium Ensifer meliloti and the plant growth-promoting rhizobacterium Pseudomonas fluorescens Aur6 were transformed with expression vectors containing the flavodoxin gene of Anabaena variabilis. Expression of the cyanobacterial protein was confirmed by Western blot. Bacterial tolerance to oxidative stress was tested in solid medium supplemented with hydrogen peroxide, paraquat or atrazine. In all three bacterial strains, flavodoxin expression enhanced tolerance to the oxidative stress provoked by hydrogen peroxide and by the reactive oxygen species-inducing herbicides, witnessed by the enhanced survival of the transformed bacteria in the presence of these oxidizing agents. Conclusions: Flavodoxin overexpression in beneficial soil bacteria confers tolerance to oxidative stress and improves their survival in the presence of the herbicides paraquat and atrazine. Flavodoxin could be considered as a general antioxidant resource to face oxidative challenges in different micro-organisms. Significance and Impact of the study: The use of plant growth-promoting rhizobacteria or nitrogen-fixing bacteria with enhanced tolerance to oxidative stress in contaminated soils is of significant agronomic interest. The enhanced tolerance of flavodoxin-expressing bacteria to atrazine and paraquat points to potential applications in herbicide-treated soils.

The binding affinity of NtcA towards promoter regions of the microcystin gene cluster from Microcystis aeruginosa PCC 7806 has been analyzed by band-shift assay (EMSA). The key nitrogen transcriptional regulator exhibits affinity for two fragments of the bidirectional mcyDA promoter, as well as for promoter regions of mcyE and mcyH. The presence of 2-oxoglutarate increased by 2.5 fold the affinity of NtcA for the mcyA promoter region. The 2-oxoglutarate effect peaked at 0.8 mM, a physiological concentration for this compound under nitrogen-limiting conditions. The results suggest that the 2-oxoglutarate level, as a signal of the C to N balance of the cells, regulates the microcystin gene cluster. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Reaction of the aminophosphane ligands 2-(diphenylphosphanylamino)pyridine (Ph2PNHpy, 1) and 3-(diphenylphosphanylamino)-1,2,4-triazole [Ph2PNH(Htrz), 2] with the gold(I) compound [AuCl(tht)] (tht = tetrahydrothiophene) gave the complexes [AuCl(Ph2PNHpy)] (3) and [AuCl{Ph2PNH(Htrz)}] (4), respectively. A series of new gold(I) thiolate derivatives were synthesized from the reactions of complex 3 or 4 with 1 equiv. of a deprotonated thiol ligand. Complexes [Au(SR)(PPh2NHpy)] (5–8) were prepared by reaction of complex 3 with the corresponding thiol HSR [HSR = 2-mercaptopyridine (2-HSpy), 2-mercaptonicotinic acid (2-H2-mna), 2-thiouracil (2-HTU) or 2-thiocytosine (2-HTC)] in the presence of sodium carbonate or triethylamine. Complexes [Au(SR){PPh2NH(Htrz)}] (9–11) were prepared by the treatment of complex 4 with triethylamine and the respective thiol HSR [HSR = 2-mercaptopyridine (2-HSpy), 2-thiocytosine (2-HTC) or 6-thioguanine (6-HTG)]. The susceptibility of Enterococcus faecalis ATCC25923, Staphylococcus aureus ATCC29213 and Escherichia coli TG1 towards the synthesized gold(I) complexes was evaluated. Some of them exhibit powerful anti-bacterial activity, being more efficient against Gram-positive microorganisms. Complexes 7–9 and 11 present excellent activity against E. faecalis, whereas the highest sensitivity of S. aureus was against complexes 7 and 8. The structures of complexes 4, 5, 8 and 9 were determined by X-ray structural analysis. None of the four compounds shows aurophilic interactions between the gold(I) centres in the crystals. The four complexes possess intermolecular hydrogen bonds that lead to supramolecular arrangements.

Previous genomic analyses of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 have identified three ferric uptake regulator (Fur) homologs with low sequence identities and probably different functions in the cell. FurA is a constitutive protein that shares the highest homology with Fur from heterotrophic bacteria and appears to be essential for invitro growth. In this study, we have analysed the effects of FurA overexpression on the Anabaena sp. phenotype and investigated which of the observed alterations were directly operated by FurA. Overexpression of the regulator led to changes in cellular morphology, resulting in shorter filaments with rounded cells of different sizes. The furA-overexpressing strain showed a slower photoautotrophic growth and a marked decrease in the oxygen evolution rate. Overexpression of the regulator also decreased both catalase and superoxide dismutase activities, but did not lead to an increase in the levels of intracellular reactive oxygen species. By combining phenotypic studies, reverse transcription-PCR analyses and electrophoretic mobility shift assays, we identified three novel direct targets of FurA, including genes encoding a siderophore outer membrane transporter (schT), bacterial actins (mreBCD) and the PSII reaction center protein D1 (psbA). The affinity of FurA for these novel targets was markedly affected by the absence of divalent metal ions, confirming previous evidence of a critical role for the metal co-repressor in the function of the regulator invivo. The results unravel new cellular processes modulated by FurA, supporting its role as a global transcriptional regulator in Anabaena sp. PCC 7120