Oxygen protection of nitrogenase in Frankia sp. HFPArI3

O₂ protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O₂ uptake and acetylene reduction rates in response to substrate concentration. O₂ of NH ₄ ⁺ -grown cells showed an apparent K _m O₂ of approximately 1M O₂. In N₂-fixing cultures a second K _m O₂ of about 215 M O₂ was observed. Thus, respiration remained unsaturated by O₂ at air-saturation levels. In vivo, the apparent K _m for acetylene was more than 10-fold greater than reported in vitro values. These data were inter oreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.     

Frankia vesicles provide inducible and absolute oxygen protection for nitrogenase

When Frankia HFPCcI3 was grown in culture at oxygen O₂ levels ranging from 2 to 70 kilopascals O₂, under nitrogen fixing conditions, nitrogenase activity adapted to ambient pO₂ and showed a marked optimum close to growth pO₂. Vesicles were thin walled at low pO₂ and very thick walled at high pO₂. Freeze fracture transmission electron microscopy confirmed that Frankia produces vesicles with outer walls thickened by multiple lipid-like monolayers, in proportion to ambient pO₂.     

Hydrogenase in Frankia KB5: expression of and relation to nitrogenase

The localization and expression of the hydrogenase in free-living Frankia KB5 was investigated immunologically and by monitoring activity, focusing on its relationships with nitrogenase and H2. Immunological studies revealed that the large subunit of the hydrogenase in Frankia KB5 was modified post-translationally, and transferred into the membrane after processing. The large subunit was constitutively expressed and no correlation was found between hydrogenase activity and synthesis. Although H2 was not needed for induction of hydrogenase synthesis, exogenously added H2 triggered hydrogen uptake in medium containing nitrogen, i.e., in the hyphae. A correlation between nitrogenase activity and hydrogen uptake was found in cultures grown in media without nitrogen, but interestingly the two enzymes showed no co-regulation.     

Effects of oxygen and chloramphenicol on Frankia nitrogenase activity

The kinetics of asymbiotic nitrogenase activity in three strains of the actinomycete Frankia were studied. Decay rates for enzyme activity were determined by adding chloramphenicol to active acetylene-reducing cells and measuring the time required for all activity to cease. Synthesis rates were measured by bubbling oxygen through actively-reducing cells (which totally destroyed all activity) and then measuring the time required for activity to return to normal. Decay rates (t _1/2) for these three strains were approximately 30 to 40 min. Synthesis rates were slower and initial nitrogenase activities were recorded about 110 min (DDB 011610) or 210 min (DDB 020210 and WgCc1.17) after return to air-equilibrated cultures. Frankia strain WgCc1.17 showed a greater sensitivity to oxygen and nitrogenase activity was totally lost when cells were bubbled only with atmospheric concentrations of oxygen. The results presented here indicate that nitrogenase activity turnover time is relatively rapid, on the order of minutes rather than hours or days. However, regulation of nitrogenase activity will differ from one strain to another and asmmbiotic characterization will be useful for understanding nitrogenase regulation in the bacterial-plant symbiosis.Contribution no. 879 from the Battelle-Kettering Laboratory     

Detection of nitrogenase components and other nonheme iron proteins in polyacrylamide gels

The two Fe-containing component proteins of nitrogenase in crude cell-free extracts of N₂-fixing bacteria can be detected as pink bands upon treatment of polyacrylamide gel with α, α-dipyridyl and mercaptoacetic acid.This technique is useful in studies of mutant strains lacking N₂-fixing activity. It might be of value in the analysis of other nonheme iron proteins.     

Effect of carbon source on growth,nitrogenase and uptake hydrogenase activities of Frankia isolates from Casuarina sp.

The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied. In addition, regulation of nitrogenase and uptake hydrogenase activity by carbon sources was investigated. For each of the three isolates, JCT287, KB5 and HFPCcI3, growth was greatest on the carbon sources pyruvate and propionate. In general the carbon sources which gave the greatest growth gave the highest levels of nitrogenase activity, but repressed the activity of uptake hydrogenase. The regulation of growth, uptake hydrogenase activity and nitrogenase activity is discussed.     

Structural analyses of enzymes involved in the O-GlcNAc modification

In order to study the O-GlcNAc modification in vivo, it is evident that a range of specific small molecule inhibitors would be a valuable asset. One strategy for the design of such compounds would be to utilise 3-D structural information in tandem with knowledge of catalytic mechanism. The last few years has seen major breakthroughs in our understanding of the 3-D structure of the enzymes involved in the O-GlcNAc modification notably from the study of the tetratricopeptide repeat (TPR) domain of the human O-GlcNAc transferase, of the bacterial homologs of the O-GlcNAc hydrolase and more latterly bacterial homologs of the O-GlcNAc transferase itself. Of particular note are the bacterial O-GlcNAc hydrolase homologs that provide near identical active centres to the human enzyme. These have informed the design and/or subsequent analysis of inhibitors of this enzyme which have found great use in the chemical dissection of the O-GlcNAc in vivo, as described by Macauley and Vocadlo elsewhere in this issue.     

Structural biology of proteins involved in nitrogen cycling

Microbial metabolic processes drive the global nitrogen cycle through sophisticated and often unique metalloenzymes that facilitate difficult redox reactions at ambient temperature and pressure. Understanding the intricacies of these biological nitrogen transformations requires a detailed knowledge that arises from the combination of a multitude of powerful analytical techniques and functional assays. Recent developments in spectroscopy and structural biology have provided new, powerful tools for addressing existing and emerging questions, which have gained urgency due to the global environmental implications of these fundamental reactions. The present review focuses on the recent contributions of the wider area of structural biology to understanding nitrogen metabolism, opening new avenues for biotechnological applications to better manage and balance the challenges of the global nitrogen cycle.     

Combining gene expression and genetic analyses to identify candidate genes involved in cold responses in pea

Cold stress affects plant growth and development. In order to better understand the responses to cold (chilling or freezing tolerance), we used two contrasted pea lines. Following a chilling period, the Champagne line becomes tolerant to frost whereas the Terese line remains sensitive.     

Similarities among nitrogenase proteins and amongnif-HDK genes ofMethanosarcina barkeri and of other diazotrophic bacteria

Similarities between the nitrogen-fixing systems of the archaebacteriumMethanosarcina barkeri (strain Fusaro) and a number of eubacteria were investigated. Using antibodies againstRhizobium leguminosarum nitrogenase and a probe of clonednif-HDK genes of this species, homology withM. barkeri was demonstrated on the protein level and to a greater extent on the DNA level.     

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The -glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.     

A grass-based diet favours muscle n-3 long-chain PUFA deposition without modifying gene expression of proteins involved in their synthesis or uptake in Charolais steers

N-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) are subject of growing interest as they are of particular relevance for meat quality and human health. However, their content in the muscles of cattle is generally low probably as the complex result of their biosynthesis from dietary n-3 PUFA in the muscle and/or in other tissues/organs and of their subsequent uptake by the muscle. In view of this, this study aimed at understanding whether the changes in the muscle n-3 LCPUFA content, depending on the diet (maize silage v. grass) or the muscle type (Rectus abdominis, RA v. Semitendinosus, ST) in 12 Charolais steers, were related to variations in the gene expression of proteins involved in n-3 LCPUFA biosynthesis or cellular uptake. Tissue fatty acid composition was analysed by gas-liquid chromatography and mRNA abundance of proteins by quantitative real-time PCR. The grass-based diet resulted in a 2.3-fold (P < 0.0002) increase in both RA and ST n-3 LCPUFA content compared with the maize silage-based diet, whereas no difference in the expression of genes involved in n-3 LCPUFA biosynthesis and uptake was observed between diets. ST exhibited a 1.5-fold higher n-3 LCPUFA content than RA (P < 0.003), whereas the gene expression of proteins involved in n-3 LCPUFA biosynthesis and uptake was 1.3- to 18-fold higher in RA than in ST (P < 0.05). In conclusion, diet- or muscle type-dependent changes in the muscle n-3 LCPUFA content of Charolais steers did not seem to be mediated by the gene expression regulation of proteins involved in the biosynthesis or uptake of these fatty acids.     

IGF-1 modulates gene expression of proteins involved in inflammation,cytoskeleton, and liver architecture

Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1’s effects on liver by comparing wild-type controls, heterozygous igf1^+/?, and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.     

Analysis of a pleiotropic gene region involved in formation of catalytically active hydrogenases in Alcaligenes eutrophus H16

In Alcaligenes eutrophus H16 a pleiotropic DNA-region is involved in formation of catalytically active hydrogenases. This region lies within the hydrogenase gene cluster of megaplasmid pHG1. Nucleotide sequence determination revealed five open reading frames with significant amino acid homology to the products of the hyp operon of Escherichia coli and other hydrogenase-related gene products of diverse organisms. Mutants of A. eutrophus H16 carrying Tn5 insertions in two genes (hypB and hypD) lacked catalytic activity of both soluble (SH) and membrane-bound (MBH) hydrogenase. Immunological analysis showed that the mutants contained SH-and MBH-specific antigen. Growing the cells in the presence of ⁶³Ni²⁺ yielded significantly lower nickel accumulation rates of the mutant strains compared to the wild-type. Analysis of partially purified SH showed only traces of nickel in the mutant protein suggesting that the gene products of the pleiotropic region are involved in the supply and/or incorporation of nickel into the two hydrogenases of A. eutrophus.     

Genomic, evolutionary, and expression analyses of cee, an ancient gene involved in normal growth and development

The cee (conserved edge expressed protein) gene was recently identified in a genome-wide screen to discover genes associated with myotube formation in fast muscle of pufferfish. Comparative genomic analyses indicate that cee arose some 1.6-1.8 billion years ago and is found as a single-copy gene in most eukaryotic genomes examined. The complexity of its structure varies from an intronless gene in yeast and tunicates to nine exons and eight introns in vertebrates. cee is particularly conserved among vertebrates and is located in a syntenic region within tetrapods and between teleosts and invertebrates. Low dN/dS ratios in the cee coding region (0.02-0.09) indicate that the Cee protein is under strong purifying selection. In Atlantic salmon, cee is expressed in the superficial layers of developing organs and tissues. These data, together with functional screens in yeast and Caenorhabditis elegans, indicate that cee has a hitherto uncharacterized role in normal growth and development.     

Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.     

Regulation of nitrogenase gene expression in anaerobic cultures of Anabaena variabilis.

Derepression of nitrogenase gene expression was studied at the mRNA and enzyme activity levels in anaerobic cultures of Anabaena variabilis 29413. Cells, previously grown with ammonium chloride, were incubated in the absence of fixed nitrogen compounds under an Ar atmosphere with dichlorophenyldimethyl-urea present to inhibit oxygen evolution. The appearance of nitrogenase mRNA (measured by dot blot hybridization analysis) and nitrogenase activity (measured as acetylene-reducing activity) was followed, and the cells were also observed by phase-contrast microscopy. Nitrogenase mRNA could be detected after 1.5 to 2.0 h of nitrogen starvation; enzyme activity appeared about 1 h later. Although enzyme activity increased for many hours, mRNA levels reached a steady state rapidly. Neither heterocysts nor proheterocysts formed under these conditions; however, the cells were observed to shrink and become chlorotic. When anaerobic, derepressed cultures were exposed to oxygen, nitrogenase mRNA levels decreased very rapidly.