The phylogeny of uptake hydrogenases in Frankia.

Frankia alni ACN14a, Frankia sp. CcI3 and Frankia sp. EAN1pec, which have different host specificity and geographical distribution, have two uptake hydrogenase syntons in their genome: hydrogenase synton#1 and hydrogenase synton#2. The organization of hydrogenase genes on these syntons also varies. Phylogenetic analysis of the structural genes of these syntons showed that they were significantly divergent and that hydrogenase synton#1 subunits of these Frankia strains were probably ancestral among the actinobacteria. Hydrogenase gene duplication might have occurred long before emergence of the three Frankia lineages. The structural subunits of hydrogenase HupS2 and HupL2 (synton#2) of F. alni ACN14a and Frankia sp. CcI3, which belong to phylogenetic Frankia cluster 1, were grouped closely together but away from Frankia sp. EAN1pec, which belongs to Frankia cluster 3. Phylogenetic analysis showed the occurrence of lateral transfer of hupL2 in Frankia sp. EAN1pec to or from Geobacter sulfurreducens. The transcript levels of hupS1 and hupL1 relative to hupS2 and hupL2 were higher in F. alni ACN14a grown under free‐living conditions. Under symbiotic conditions, transcript levels of hupS2 and hupL2 were higher than those of hupS1 and hupL1. Hydrogenase subunits of synton#1 are more expressed under free‐living conditions, whereas those of synton#2 are mainly involved in symbiotic interactions.

Hydrogenase in <i>Frankia</i> KB5: Expression of and relation to nitrogenase

У статті представлено результати впливу хімічних і біологічних протруйників на кількісний склад мікробіому ризосфери цукрової кукурудзи протягом її вегетації. Показано, що за використання хімічних препаратів у ризосфері рослин на порядок знижується кількість амоніфікаторів та мікроскопічних грибів у першій половині вегетаційного періоду культури. Вплив ксенобіотиків на вміст актиноміцетів, олігонітрофілів та педоторофів є менш токсичним. У порівнянні з контрольним варіантом вміст даних мікроорганізмів знижувався на 50-75 %, при цьому змін зазнає не лише кількісний, а й якісний склад ризосферної мікрофлори. У той же час досліджувані пестициди не впливали на вміст актиноміцетів і оліготрофів. По мірі розвитку рослин негативний вплив хімічних протруйників на мікрофлору ґрунту поступово нівелювався. Відбувалося відновлення складу ризосферної мікробіоти у кожній умовній функціональній групі мікроорганізмів і до завершення вегетації кукурудзи мікробний ценоз повертався до свого первинного кількісного і якісного складу. Застосування мікробіологічних засобів захисту не призводило до змін чисельності чи дисбалансу у складі ґрунтової мікробіоти. Її кількісний та якісний склад відповідав контрольному варіанту.

Previous studies have shown that the valanimycin producer Streptomyces viridifaciens contains two genes encoding proteins that are similar to seryl-tRNA synthetases (SerRSs). One of these proteins (SvsR) is presumed to function in protein biosynthesis, because it exhibits a high degree of similarity to the single SerRS of Streptomyces coelicolor. The second protein (VlmL), which exhibits a low similarity to the S. coelicolor SerRS, is hypothesized to play a role in valanimycin biosynthesis, because the vlmL gene resides within the valanimycin biosynthetic gene cluster. To investigate the role of VlmL in valanimycin biosynthesis, VlmL and SvsR have been overproduced in soluble form in Escherichia coli, and the biochemical properties of both proteins have been analyzed and compared. Both proteins were found to catalyze a serine-dependent exchange of 32P-labeled pyrophosphate into ATP and to aminoacylate total E. coli tRNA with L-serine. Kinetic parameters for the two enzymes show that SvsR is catalytically more efficient than VlmL. The results of these experiments suggest that the role of VlmL in valanimycin biosynthesis is to produce seryl-tRNA, which is then utilized for a subsequent step in the biosynthetic pathway. Orthologs of VlmL were identified in two other actinomycetes species that also contain orthologs of the S. coelicolor SerRS. The significance of these findings is herein discussed.

The enzymes directly involved in hydrogen metabolism in cyanobacteria are hydrogenases and nitrogenase. Functionally, two types of hydrogenase are known: uptake hydrogenase (Hup) and bidirectional hydrogenase (Hox). Distribution of hydrogenases among 15 filamentous cyanobacteria was studied by heterologous Southern hybridizations with hupL and hoxH probes from Anabaena PCC7120 and by direct assay of in vitro hydrogenase activities. All tested 15 strains showed hybridization with the hupL probe. With hoxH probe, hybridization bands are detected in 12 of them, but not in 3 of them. Moreover, the latter have no detectable in vitro Hox activity. The hupL and/or hoxH gene from Anabaena PCC7120 were inactivated. The extracts from the hoxH- strains were completely unable to perform Na2S2O4- and methyl viologen-dependent H2 evolution. H2 uptake with PMS by extracts from N2 fixing cells was undetectable in hupL-. The hupL- and hupL-hoxH- strains produced 4-7 times higher amounts of H2 than the wild-type strains. The nitrogenase activities of these hupL- strains were not reduced compared to the wild-type. Unexpectedly, the hoxH- strains were lower in H2 production activities by 20-50% compared to the wild-type.

Although nickel is a toxic metal for living organisms in its soluble form, its importance in many biological processes recently emerged. In this view, the investigation of the nickel-dependent enzymes urease and [NiFe]-hydrogenase, especially the mechanism of nickel insertion into their active sites, represent two intriguing case studies to understand other analogous systems and therefore to lead to a comprehension of the nickel trafficking inside the cell. Moreover, these two enzymes have been demonstrated to ensure survival and colonization of the human pathogen H. pylori, the only known microorganism able to proliferate in the gastric niche. The right nickel delivering into the urease active site requires the presence of at least four accessory proteins, UreD, UreE, UreF and UreG. Similarly, analogous process is principally mediated by HypA and HypB proteins in the [NiFe]-hydrogenase system. Indeed, HpHypA and HpHypB also have been proposed to act in the activation of the urease enzyme from H. pylori, probably mobilizing nickel ions from HpHypA to the HpUreE-HpUreG complex. A complete comprehension of the interaction mechanism between the accessory proteins and the crosstalk between urease and hydrogenase accessory systems requires the determination of the role of each protein chaperone that strictly depends on their structural and biochemical properties. The availability of HpUreE, HpUreG and HpHypA proteins in a pure form is a pre-requisite to perform all the subsequent protein characterizations, thus their purification was the first aim of this work. Subsequently, the structural and biochemical properties of HpUreE were investigated using multi-angle and quasi-elastic light scattering, as well as NMR and circular dichroism spectroscopy. The thermodynamic parameters of Ni2+ and Zn2+ binding to HpUreE were principally established using isothermal titration calorimetry and the importance of key histidine residues in the process of binding metal ions was studied using site-directed mutagenesis. The molecular details of the HpUreE-HpUreG and HpUreE-HpHypA protein-protein assemblies were also elucidated. The interaction between HpUreE and HpUreG was investigated using ITC and NMR spectroscopy, and the influence of Ni2+ and Zn2+ metal ions on the stabilization of this association was established using native gel electrophoresis, light scattering and thermal denaturation scanning followed by CD spectroscopy. Preliminary HpUreE-HpHypA interaction studies were conducted using ITC. Finally, the possible structural architectures of the two protein-protein assemblies were rationalized using homology modeling and docking computational approaches. All the obtained data were interpreted in order to achieve a more exhaustive picture of the urease activation process, and the correlation with the accessory system of the hydrogenase enzyme, considering the specific role and activity of the involved protein players. A possible function for Zn2+ in the chaperone network involved in Ni2+ trafficking and urease activation is also envisaged.

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this 'waste' product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under freeliving conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).

Studies of an effective strain of Frankia from Allocasuarina lehmanniana of the Casuarinaceae.- In vitro production of specialized reproductive torulose hyphae by Frankia strain ORS 021001 isolated from Casuarina junghuhniana root nodules.- Characterization and infectivity of a spontaneous variant isolated from Frankia sp. WEY 0131391.- Restriction pattern analysis of genomic DNA of Frankia isolates.- Restriction enzyme digestion patterns of Frankia plasmids.- Host range of Frankia endophytes.- Preinfection events in the establishment of Alnus-Frankia symbiosis: Development of a spot inoculation technique.- Effect of juglone on growth in vitro of Frankia isolates and nodulation of Alnus glutinosa in soil.- Nitrogen fixation and respiration by root nodules of Alnus rubra Bong.: Effects of temperature and oxygen concentration.- Effect of spring flooding on endophyte differentiation, nitrogenase activity, root growth and shoot growth in Myrica gale.- Performance of in vitro propagated Alnus glutinosa (L.) Gaertn. clones inoculated with Frankiae.- Variation in response among three Alnus spp. clones to progressive water stress.- In vitro propagation and nodulation by Frankia of actinorhizal Russian Olive (Elaeagnus angustifolia L.).- Inoculation and production of container-grown red alder seedlings.- Seed germination, seedling inoculation and establishment of Alnus spp. in containers in greenhouse trials.- Large scale inoculation of actinorhizal plants with Frankia.- Biomass production by alders on four abandoned agricultural soils in Quebec.- Nitrogen cycling in dense plantings of hybrid poplar and black alder.

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong ermE* promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.

Frankia [NiFe] uptake hydrogenases and genome reduction: different lineages of loss.

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The [FeFe]-hydrogenases, although share common features when compared to other metal containing hydrogenases, clearly have independent evolutionary origins. Examples of [FeFe]-hydrogenases have been characterized in detail by biochemical and spectroscopic approaches and the high resolution structures of two examples have been determined. The active site H-cluster is a complex bridged metal assembly in which a [4Fe-4S] cubane is bridged to a 2Fe subcluster with unique non-protein ligands including carbon monoxide, cyanide, and a five carbon dithiolate. Carbon monoxide and cyanide ligands as a component of a native active metal center is a property unique to the metal containing hydrogenases and there has been considerable attention to the characterization of the H-cluster at the level of electronic structure and mechanism as well as to defining the biological means to synthesize such a unique metal cluster. The chapter describes the structural architecture of [FeFe]-hydrogenases and key spectroscopic observations that have afforded the field with a fundamental basis for understanding the relationship between structure and reactivity of the H-cluster. In addition, the results and ideas concerning the topic of H-cluster biosynthesis as an emerging and fascinating area of research, effectively reinforcing the potential linkage between iron-sulfur biochemistry to the role of iron-sulfur minerals in prebiotic chemistry and the origin of life.

[FeFe]-hydrogenases (HYDA) link the production of molecular H(2) to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H(2)-photoproduction activity observed is as O(2) sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.

Mannitol biosynthesis in algae: more widespread and diverse than previously thought.

[NiFe(Se)]-hydrogenases are hetero-dimeric enzymes present in many microorganisms where they catalyze the oxidation of molecular hydrogen or the reduction of protons. Like the other two types of hydrogen-metabolizing enzymes, the [FeFe]- and [Fe]-hydrogenases, [NiFe]-hydrogenases have a Fe(CO)(x) unit in their active sites that is most likely involved in hydride binding. Because of their complexity, hydrogenases require a maturation machinery that involves several gene products. They include nickel and iron transport, synthesis of CN(-) (and maybe CO), formation and insertion of a FeCO(CN(-))(2) unit in the apo form, insertion of nickel and proteolytic cleavage of a C-terminal stretch, a step that ends the maturation process. Because the active site is buried in the structure, electron and proton transfer are required between this site and the molecular surface. The former is mediated by either three or one Fe/S cluster(s) depending on the enzyme. When exposed to oxidizing conditions, such as the presence of O(2), [NiFe]-hydrogenases are inactivated. Depending on the redox state of the enzyme, exposure to oxygen results in either a partially reduced oxo species probably a (hydro)peroxo ligand between nickel and iron or a more reduced OH(-) ligand instead. Under some conditions the thiolates that coordinate the NiFe center can be modified to sulfenates. Understanding this process is of biotechnological interest for H(2) production by photosynthetic organisms.