Adaptation de Rubrivivax gelatinosus à la présence de l'oxygène (étude des différents mécanismes via l'expression de l'antenne LH2)

Nous nous sommes intéressés aux mécanismes d adaptation que possèdent la bactérie pourpre Rubrivivax gelatinosus pour maintenir en présence d oxygène la synthèse des complexes photosynthétiques et plus particulièrement celle de l antenne LH2. Les gènes pucBA (codant les protéines de LH2) codent deux transcrits, formés à partir du même promoteur et de deux terminateurs. Leur production est pratiquement identique en photosynthèse et en semi aérobie. En amont des gènes pucBA, est présent un site de fixation pour le facteur PpsR, identifié chez Rhodobacter comme étant une répresseur transcriptionnel des gènes photosynthétiques et notamment des gènes puc, en présence d oxygène. Nous nous sommes alors intéressés à la régulation des gènes puc et du gène crtI par le facteur PpsR. Cette étude a permis d identifier en semi aérobie un rôle activateur de PpsR sur l expression des gènes puc et un rôle de répresseur sur crtI. Le maintien de la synthèse des protéines structurales en présence d oxygène n est pas suffisant pour expliquer la formation du complexe LH2, il est nécessaire que les bactériochlorophylles soient également produites. Nous avons également identifié une stratégie qu utilise la bactérie pour maintenir la voie de biosynthèse des bactériochlorophylles, quelques soient les conditions d oxygénation. Cette stratégie s effectue via un mécanisme à deux enzymes, BchE et AcsF. BchE est active en anaérobie et en faible oxygénation, AcsF est active en forte oxygénation. La capacité de maintenir la synthèse des bactériochlorophylles en présence d oxygène permet à la bactérie de produire rapidement les photosystèmes lors des transitions aérobes-anaérobes.We have studied the adaptation mechanisms developed by the purple bacterium Rubrivivax gelatinosus to maintain the synthesis of the photosynthetic complexes in presence of oxygen; in particular the synthesis of the LH2 antenna complex. During this work, we have analysed the pucBA (encoding for the LH2 polypeptides) gene expression. We have identified two pucBA transcripts made from the same promoter and ending at two different terminators. The amount of the pucBA transcripts is the same between the semi aerobic and photosynthetic conditions. The role of PpsR, a putative transcription factor that regulates the pucBA expression was studied. In Rhodobacter, this factor was identified as a repressor of the puc genes under aerobic condition. We have analysed the puc and crtI genes regulation by PpsR under semi aerobic conditions. This work has shown an activator role of PpsR on pucBA genes expression and a repressor role on crtI gene. The presence of the structural protein under aerobic condition is not sufficient to explain the presence of the LH2 complex; the bacteriochlorophyll should also be synthesized. It was shown that the bacterium maintains the bacteriochlorophyll synthesis under different aerobic conditions by using two different enzymes, BchE and AcsF. We have shown that BchE is active under anaerobic or low oxygenation, while AcsF is active only under high oxygenation. We have suggested that the availability of bacteriochlorophylls under aerobic condition is a significant advantage to rapidly build up the photosystem, during the transition from aerobic to anaerobic conditions.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Regulation of Photosynthesis Genes in Rubrivivax gelatinosus: Transcription Factor PpsR Is Involved in both Negative and Positive Control

Induction of biosynthesis of the photosystem in anoxygenic photosynthetic bacteria occurs when the oxygen concentration drops. Control of this induction takes place primarily at the transcriptional level, with photosynthesis genes expressed preferentially under anaerobic conditions. Here, we report analysis of the transcriptional control of two photosynthesis promoters, pucBA and crtI, by the PpsR factor in Rubrivivax gelatinosus. This was accomplished by analyzing the photosystem production in the wild type and in the PPSRK (ppsR::Km) mutant grown under anaerobic and semiaerobic conditions and by assessing the β-galactosidase activity of lacZ transcriptionally fused to promoters possessing the putative PpsR-binding consensus sequences. It was found that under semiaerobic conditions, inactivation of the ppsR gene resulted in overproduction of carotenoid and bacteriochlorophyll pigments, while the production of LH2 was drastically reduced. The β-galactosidase activity showed that, in contrast to what has been found previously for Rhodobacter species, PpsR acts in R. gelatinosus as an aerobic repressor of the crtI gene while it acts as an activator for the expression of pucBA. Inspection of the putative PpsR-binding consensus sequences revealed significant differences that may explain the different levels of expression of the two genes studied

Silver and Copper Acute Effects on Membrane Proteins and Impact on Photosynthetic and Respiratory Complexes in Bacteria

The use of metal ions represents a serious threat to the environment and to all living organisms because of the acute toxicity of these ions. Nowadays, silver nanoparticles are one of the most widely used nanoparticles in various industrial and health applications. The antimicrobial effect of nanoparticles is in part related to the released Ag+ ions and their ability to interact with bacterial membranes. Here, we identify, both in vitro and in vivo, specific targets of Ag+ ions within the membrane of bacteria. This include complexes involved in photosynthesis, but also complexes involved in respiration.Silver (Ag+) and copper (Cu+) ions have been used for centuries in industry, as well as antimicrobial agents in agriculture and health care. Nowadays, Ag+ is also widely used in the field of nanotechnology. Yet, the underlying mechanisms driving toxicity of Ag+ ions in vivo are poorly characterized. It is well known that exposure to excess metal impairs the photosynthetic apparatus of plants and algae. Here, we show that the light-harvesting complex II (LH2) is the primary target of Ag+ and Cu+ exposure in the purple bacterium Rubrivivax gelatinosus. Ag+ and Cu+ specifically inactivate the 800-nm absorbing bacteriochlorophyll a (B800), while Ni2+ or Cd2+ treatment had no effect. This was further supported by analyses of CuSO4- or AgNO3-treated membrane proteins. Indeed, this treatment induced changes in the LH2 absorption spectrum related to the disruption of the interaction of B800 molecules with the LH2 protein. This caused the release of B800 molecules and subsequently impacted the spectral properties of the carotenoids within the 850-nm absorbing LH2. Moreover, previous studies have suggested that Ag+ can affect the respiratory chain in mitochondria and bacteria. Our data demonstrated that exposure to Ag+, both in vivo and in vitro, caused a decrease of cytochrome c oxidase and succinate dehydrogenase activities. Ag+ inhibition of these respiratory complexes was also observed in Escherichia coli, but not in Bacillus subtilis

Responses of a Thermophilic Synechococcus Isolate from the Microbial Mat of Octopus Spring to Light▿

Thermophilic cyanobacteria of the genus Synechococcus are major contributors to photosynthetic carbon fixation in the photic zone of microbial mats in Octopus Spring, Yellowstone National Park. Synechococcus OS-B′ was characterized with regard to the ability to acclimate to a range of different light irradiances; it grows well at 25 to 200 μmol photons m−2 s−1 but dies when the irradiance is increased to 400 μmol photons m−2 s−1. At 200 μmol photons m−2 s−1 (high light [HL]), we noted several responses that had previously been associated with HL acclimation of cyanobacteria, including cell bleaching, reduced levels of phycobilisomes and chlorophyll, and elevated levels of a specific carotenoid. Synechococcus OS-B′ synthesizes the carotenoids zeaxanthin and β,β-carotene and a novel myxol-anhydrohexoside. Interestingly, 77-K fluorescence emission spectra suggest that Synechococcus OS-B′ accumulates very small amounts of photosystem II relative to that of photosystem I. This ratio further decreased at higher growth irradiances, which may reflect potential photodamage following exposure to HL. We also noted that HL caused reduced levels of transcripts encoding phycobilisome components, particularly that for CpcH, a 20.5-kDa rod linker polypeptide. There was enhanced transcript abundance of genes encoding terminal oxidases, superoxide dismutase, tocopherol cyclase, and phytoene desaturase. Genes encoding the photosystem II D1:1 and D1:2 isoforms (psbAI and psbAII/psbAIII, respectively) were also regulated according to the light regimen. The results are discussed in the context of how Synechococcus OS-B′ may cope with high light irradiances in the high-temperature environment of the microbial mat

Discriminating Susceptibility of Xanthine Oxidoreductase Family to Metals

ABSTRACT The xanthine oxidoreductase (XOR) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and flavin adenine dinucleotide (FAD) for their catalytic activity. This large molybdoenzyme family includes xanthine, aldehyde, and CO dehydrogenases. XORs are widely distributed from bacteria to humans due to their key roles in the catabolism of purines, aldehydes, drugs, and xenobiotics, as well as interconversions between CO and CO2. Assessing the effect of excess metals on the Rubrivivax gelatinosus bacterium, we found that exposure to copper (Cu) or cadmium (Cd) caused a dramatic decrease in the activity of a high-molecular-weight soluble complex exhibiting nitroblue tetrazolium reductase activity. Mass spectrometry and genetic analyses showed that the complex corresponds to a putative CO dehydrogenase (pCOD). Using mutants that accumulate either Cu+ or Cd2+ in the cytoplasm, we show that Cu+ or Cd2+ is a potent inhibitor of XORs (pCOD and the xanthine dehydrogenase [XDH]) in vivo. This is the first in vivo demonstration that Cu+ affects Moco-containing enzymes. The specific inhibitory effect of these compounds on the XOR activity is further supported in vitro by direct addition of competing metals to protein extracts. Moreover, emphasis is given on the inhibitory effect of Cu on bovine XOR, showing that the XOR family could be a common target of Cu. Given the conservation of XOR structure and function across the tree of life, we anticipate that our findings could be transferable to other XORs and organisms. IMPORTANCE The high toxicity of Cu, Cd, Pb, As, and other metals arises from their ability to cross membranes and target metalloenzymes in the cytoplasm. Identifying these targets provides insights into the toxicity mechanisms. The vulnerability of metalloenzymes arises from the accessibility of their cofactors to ions. Accordingly, many enzymes whose cofactors are solvent exposed are likely to be targets of competing metals. Here, we describe for the first time, with in vivo and in vitro experiments, a direct effect of excess Cu on the xanthine oxidoreductase family (XOR/XDH/pCOD). We show that toxic metal affects these Moco enzymes, and we suggest that access to the Moco center by Cu ions could explain the Cu inhibition of XORs in living organisms. Human XOR activity is associated with hyperuricemia, xanthinuria, gout arthritis, and other diseases. Our findings in vivo highlight XOR as a Cu target and thus support the potential use of Cu in metal-based therapeutics against these diseases

Increasing the copper sensitivity of microorganisms by restricting iron supply, a strategy for bio-management practices

International audiencePollution by copper (Cu$^{2+}$) extensively used as antimicrobial in agriculture and farming represents a threat to the environment and human health. Finding ways to make microorganisms sensitive to lower metal concentrations could help decreasing the use of Cu$^{2+}$ in agriculture. In this respect, we showed that limiting iron (Fe) uptake makes bacteria much more susceptible to Cu$^{2+}$ or Cd$^{2+}$ poisoning. Using efflux mutants of the purple bacterium $Rubrivivax\ gelatinosus$, we showed that Cu$ç+$ and Cd$^{2+}$ resistance relies on the expression of the Fur-regulated FbpABC and Ftr iron transporters. To support this conclusion, inactivation of these Fe-importers in the Cu$^+$ or Cd$^{2+}$ -ATPase efflux mutants gave rise to hypersensitivity towards these ions. Moreover, in metal overloaded cells the expression of FbpA, the periplasmic iron-binding component of the ferric ion transport FbpABC system was induced, suggesting that cells perceived an 'iron-starvation' situation and responded to it by inducing Fe-importers. In this context, the Fe-Sod activity increased in response to Fe homoeostasis dysregulation. Similar results were obtained for Vibrio cholerae and Escherichia coli, suggesting that perturbation of Fe-homoeostasis by metal excess appeared as an adaptive response commonly used by a variety of bacteria. The presented data support a model in which metal excess induces Fe-uptake to support [4Fe-4S] synthesis and thereby induce ROS detoxification system

Cadmium and Copper Cross-Tolerance. Cu+^+ Alleviates Cd2+^{2+} Toxicity, and Both Cations Target Heme and Chlorophyll Biosynthesis Pathway in Rubrivivax gelatinosusRubrivivax\ gelatinosus

International audienceCadmium, although not redox active is highly toxic. Yet, the underlying mechanisms driving toxicity are still to be characterized. In this study, we took advantage of the purple bacterium Rubrivivax gelatinosus strain with defective Cd2+-efflux system to identify targets of this metal. Exposure of the 1cadA strain to Cd2+ causes a decrease in the photosystem amount and in the activity of respiratory complexes. As in case of Cu+ toxicity, the data indicated that Cd2+ targets the porphyrin biosynthesis pathway at the level of HemN, a S-adenosylmethionine and CxxxCxxC coordinated [4Fe-4S] containing enzyme. Cd2+ exposure therefore results in a deficiency in heme and chlorophyll dependent proteins and metabolic pathways. Given the importance of porphyrin biosynthesis, HemN represents a key metal target to account for toxicity. In the environment, microorganisms are exposed to mixture of metals. Nevertheless, the biological effects of such mixtures, and the toxicity mechanisms remain poorly addressed. To highlight a potential cross-talk between Cd2+ and Cu+ -efflux systems, we show (i) that Cd2+ induces the expression of the Cd2+-efflux pump CadA and the Cu+ detoxification system CopA and CopI; and (ii) that Cu+ ions improve tolerance towards Cd2+, demonstrating thus that metal mixtures could also represent a selective advantage in the environment

A periplasmic cupredoxin with a green CuT1.5 center is involved in bacterial copper tolerance

International audienceAbstract Importance of copper resistance pathways in pathogenic bacteria is now well recognized since macrophages use copper to fight bacterial infections. Additionally, considering the increase of antibiotic resistance, growing attention is given to the antimicrobial properties of copper. It is of primary importance to understand how bacteria deal with copper. The Cu resistant cuproprotein CopI is present in many Human bacterial pathogens and environmental bacteria and crucial under microaerobiosis (conditions for most pathogens to thrive within their host). Hence, understanding its mechanism of function is essential. CopI proteins share conserved histidine, cysteine and methionine residues that could be ligands for different copper binding sites among which acupredoxin center that could be involved in the protein function. Here, we demonstrated that, Vibrio cholerae and Pseudomonas aeruginosa CopI restore Cu resistance phenotype in Rubrivivax (R.) gelatinosus ΔcopI mutant. We identified that Cys125 (ligand in the cupredoxin center) and conserved histidines and methionines are essential for R. gelatinosus CopI (RgCopI) function. We also performed spectroscopic analyses of the purified RgCopI protein and showed that it is a green cupredoxin able to bind a maximum of three Cu(II) ions: (i) a green Cu site (CuT1.5), (ii) a type 2 Cu binding site (T2) located in the N-terminal region, (iii) a third site with a yet unidentified location. CopI is therefore one member of the poorly described CuT1.5 center cupredoxin family. It is unique, since it is a single domain cupredoxin with more than one Cu site involved in Cu resistance

EmbRS a new two-component system that inhibits biofilm formation and saves Rubrivivax gelatinosus from sinking

Photosynthetic bacteria can switch from planktonic lifestyle to phototrophic biofilm in mats in response to environmental changes. The mechanisms of phototrophic biofilm formation are, however, not characterized. Herein, we report a two-component system EmbRS that controls the biofilm formation in a photosynthetic member of the Burkholderiales order, the purple bacterium Rubrivivax gelatinosus. EmbRS inactivation results in cells that form conspicuous bacterial veils and fast-sinking aggregates in liquid. Biofilm analyses indicated that EmbRS represses the production of an extracellular matrix and biofilm formation. Mapping of transposon mutants that partially or completely restore the wild-type (WT) phenotype allowed the identification of two gene clusters involved in polysaccharide synthesis, one fully conserved only in Thauera sp., a floc-forming wastewater bacterium. A second two-component system BmfRS and a putative diguanylate cyclase BdcA were also identified in this screen suggesting their involvement in biofilm formation in this bacterium. The role of polysaccharides in sinking of microorganisms and organic matter, as well as the importance and the evolution of such regulatory system in phototrophic microorganisms are discussed