Homocitrate Synthase Genes Research Articles

Rapid and Accurate Screening of Lysine-Producing Edible Mushrooms via the Homocitrate Synthase Gene as a Universal Molecular Marker.

Edible mushrooms are important nutraceutical sources of foods and drugs, which can produce various nutritional ingredients including all essential amino acids. The method of rapid screening for the strains producing specific functional components is very indispensable. Homocitrate synthase is one of the key enzymes in the α-aminoadipate pathway for lysine biosynthesis and has preferable sequence conservation in Agaricales. Based on the blast of homocitrate synthase homologous genes of strains of Agaricales, we achieved combinations of degenerate primers as molecular markers to rapidly screen the lysine-producing edible mushrooms. The experimental results revealed that the consistency between PCR amplification and HPLC analysis attained 82 and 75% in strains of Agaricales and Polyporales, respectively. The finding showed that the molecular marker has higher universality for screening edible mushroom resources of Agaricales. This PCR-based approach shows excellent potential in evaluating and discriminating edible wild-grown mushrooms with high lysine content in Agaricales.

Homocitrate Synthase Genes of Two Wide-Host-Range Bradyrhizobium Strains are Differently Required for Symbiosis Depending on Host Plants.

The nifV gene encodes homocitrate synthase, the enzyme that catalyzes the formation of homocitrate, which is essential for arranging the FeMo-cofactor in the catalytic center of nitrogenase. Some host plants, such as Lotus japonicus, supply homocitrate to their symbionts, in this case, Mesorhizobium loti, which lacks nifV. In contrast, Bradyrhizobium ORS285, a symbiont of Aeschynomene cross-inoculation (CI) groups 2 and 3, requires nifV for symbiosis with Aeschynomene species that belong to CI group 3, and some species belonging to CI group 2. However, it currently remains unclear whether rhizobial nifV is required for symbiosis with Aeschynomene species belonging to CI group 1 or with other legumes. We generated nifV-disruption (ΔnifV) mutants of two wide-host-range rhizobia, Bradyrhizobium SUTN9-2 and DOA9, to investigate whether they require nifV for symbiosis. Both ΔnifV mutant strains showed significantly less nitrogenase activity in a free-living state than the respective wild-type strains. The symbiotic phenotypes of SUTN9-2, DOA9, and their ΔnifV mutants were examined with four legumes, Aeschynomene americana, Stylosanthes hamata, Indigofera tinctoria, and Desmodium tortuosum. nifV was required for the efficient symbiosis of SUTN9-2 with A. americana (CI group 1), but not for that of DOA9. SUTN9-2 established symbiosis with all three other legumes; nifV was required for symbiosis with I. tinctoria and D. tortuosum. These results suggest that, in addition to Aeschynomene CI groups 2 and 3, CI group 1 and several other legumes require the rhizobial nifV for symbiosis.

Gene expression patterns of Glaciozyma antarctica PI12 in response to cold, and freeze stress

Psychrophilic yeast, Glaciozyma antarctica PI12 was isolated from the sea ice in the Southern Ocean. Several recent studies have revealed some of the strategies employed by G. antarctica PI12 to grow and survive at low temperatures, but those information are still limited. Hence this project was carried out to sequence the whole transcriptome to generate additional information on the cold-adaptation strategies of G. antarctica PI12. The yeast grown optimally in Saccharomyces cerevisiae minimal medium at 12 °C was subsequently exposed to cold-shock at 0 °C and freeze-shock at −12 °C for 6 h and 24 h. RNA from those cells were extracted, sequenced, and analyzed. Interestingly, the results showed that G. antarctica PI12 remained metabolically active at −12 °C. Two hundred and five genes were differentially expressed in the cells. Among them, 107 genes were upregulated while 98 genes were downregulated. In the first 6 h after the cells were exposed to cold- and freeze-shocks, CCR4-NOT (carbon catabolite repressed 4 - negative on TATA-less) core subunit cdc36, DNA repair protein Rad8, Elongation factor 1-gamma, 26s proteosome subunit 45, and Homocitrate synthase genes were commonly upregulated to perform several immediate and important tasks to ensure the cells survived. Apart from that, there were also genes upregulated and downregulated uniquely at 6 h and 24 h to facilitate the cells to adjust to the new temperatures. After overcoming the stress of the cold- and freeze-shocks for 24 h, the cells acclimatized to those temperatures and became metabolically active again, and the cell cycles related genes were up-regulated.

A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for Candida species identification

Candida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species-specific differentiation of Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of the terminal restriction fragment length polymorphism (t-RFLP) method in the molecular identification of six species of Candida, which are the most common causes of fungal infections. Specific for fungi homocitrate synthase gene was chosen as a molecular target for amplification. The use of three restriction enzymes, DraI, RsaI, and BglII, for amplicon digestion can generate species-specific fluorescence labeled DNA fragment profiles, which can be used to determine the diagnostic algorithm. The designed method can be a cost-efficient high-throughput molecular technique for the identification of six clinically important Candida species.

Homocitrate synthase expression and lysine content in fruiting body of different developmental stages in Flammulina velutipes.

Homocitrate synthase (EC 2.3.3.14) regulates the first step of fungal lysine biosynthesis. The gene encoding homocitrate synthase was identified in whole genomic sequencing of Flammulina velutipes and contains seven introns. The homocitrate synthase gene of F. velutipes strain W23 (Fvhcs) is 1780 bp in length and encodes a 464 amino acid protein with a predicted molecular weight 50.7 kDa. Phylogenetic analysis of Fvhcs and other homocitrate synthase proteins from diverse fungi produced a topology congruent with the current best estimate of organismal phylogeny. Analysis of protein domains by InterProScan and a motif search found that Fvhcs gene encodes homocitrate synthase protein conserved across Agaricomycotina. In addition, we sequenced the transcriptome of different developmental stages and structures of the fruiting body to analyze the expression levels of the Fvhcs gene. The data showed a correlation between Fvhcs gene expression and lysine values in different developmental stages and structures of F. velutipes.

Evolutionary Mechanisms of Microbial Genomes 2012

What is the driving force in the course of microbial genome evolution? What is the mechanism for distinguishing self-genome from others? These fundamental questions remain elusive although rigorous studies are underway by using comparative genomics. The special issue “Evolutionary mechanisms of microbial genomes” has been launched in 2011 and presented 11 original papers. Here, this new version in 2012 presents 10 papers (one review and nine research articles). Two papers are presented in phylogenomics. K. Oshima et al. revealed a close relationship of Aquificales to Thermotogales based on the whole-genome comparison in “Phylogenetic position of Aquificales based on the whole genome sequences of six Aquificales species.” An extensive and elaborate review of fish pathogenic bacteria has been presented by P. S. Sudheesh et al. in “Comparative pathogenomics of bacteria causing infectious diseases in fish.” Two papers are presented on subjects related to evolution of base composition in genomes. H. Nishida et al. in “Genome signature difference between Deinococcus radiodurans and Thermus thermophilus” observed distinct tetranucleotide frequencies between the genomes of D. radiodurans and T. thermophilus, potentially reflecting different evolutionary backgrounds of the two species after divergence from common ancestor. H. Nishida in “Comparative analyses of base compositions, DNA sizes, and dinucleotide frequency profiles in archaeal and bacterial chromosomes and plasmids” reported lower GC content (by up to ~10%) of plasmids compared to their host chromosomes and higher correlation of GC content and chromosome size in bacteria than in archaea. Two papers are presented about horizontal gene transfer in genome evolution. M. Jalasvuori in “Vehicles, replicators, and intercellular movement of genetic information: Evolutionary dissection of a bacterial cell” discussed a hypothesis that any given biosphere comprising prokaryotic cell vehicles and genetic replicators may naturally evolve toward possessing horizontally moving replicators of various types. V. S. Pylro et al. described horizontal gene transfer events of the gene dszC involved in the cleavage of carbon-sulfur bonds in “Detection of horizontal gene transfers from phylogenetic comparisons.” An article about DNA mutation is presented by Y. Shiwa et al. in “Whole-genome profiling of a novel mutagenesis technique using proofreading-deficient DNA polymerase δ.” They compared mutations created by the chemical mutagen ethyl methanesulfonate (EMS) and the proofreading-deficient DNA polymerase δ and found that the mutations created by the proofreading-deficient DNA polymerase δ generated more diverse amino acid substitution patterns than those by EMS. Three papers are presented on subjects related to metabolic pathway. H. Nishida in “Comparative analyses of homocitrate synthase genes of ascomycetous yeasts” described gene duplications of the homocitrate synthase which have occurred multiple times during evolution of the ascomycetous yeasts. H. Nishida and M. Nishiyama in “Evolution of lysine biosynthesis in the phylum Deinococcus-Thermus” reported that bacterial lysine biosynthesis genes of the common ancestor of the Deinococcus-Thermus phylum used the α-aminoadipate pathway instead of the diaminopimelate pathway. K. Ueda et al. in “Dispensabilities of carbonic anhydrase in Proteobacteria” analyzed the distribution of carbonic anhydrase (CA) in proteobacteria, compared CA-retaining and CA-deficient genomes, and found absence of coding sequence in some strains and frame shifts in others. In closing this introduction to the special issue, we would like to express our full appreciation to all the authors and reviewers for their enormous efforts that have made the timely completion of our assignment successful. We sincerely hope that this special issue will stimulate further the investigation of evolutionary mechanisms of microbial genomes. Hiromi Nishida Shinji Kondo Hideaki Nojiri Ken-ichi Noma Kenro Oshima

Metabolic Pathways for Photobiological Hydrogen Production by Nitrogenase- and Hydrogenase-containing Unicellular Cyanobacteria Cyanothece

Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.

Effects of Disruption of Homocitrate Synthase Genes on Nostoc sp. Strain PCC 7120 Photobiological Hydrogen Production and Nitrogenase

In the case of nitrogenase-based photobiological hydrogen production systems of cyanobacteria, the inactivation of uptake hydrogenase (Hup) leads to significant increases in hydrogen production activity. However, the high-level-activity stage of the Hup mutants lasts only a few tens of hours under air, a circumstance which seems to be caused by sufficient amounts of combined nitrogen supplied by active nitrogenase. The catalytic FeMo cofactor of nitrogenase binds homocitrate, which is required for efficient nitrogen fixation. It was reported previously that the nitrogenase from the homocitrate synthase gene (nifV) disruption mutant of Klebsiella pneumoniae shows decreased nitrogen fixation activity and increased hydrogen production activity under N2. The cyanobacterium Nostoc sp. strain PCC 7120 has two homocitrate synthase genes, nifV1 and nifV2, and with the delta hupL variant of Nostoc sp. strain PCC 7120 as the parental strain, we have constructed two single mutants, the delta hupL delta nifV1 strain (with the hupL and nifV1 genes disrupted) and the delta hupL delta nifV2 strain, and a double mutant, the delta hupL delta nifV1 delta nifV2 strain. Diazotrophic growth rates of the two nifV single mutants and the double mutant were decreased moderately and severely, respectively, compared with the rates of the parent delta hupL strain. The hydrogen production activity of the delta hupL delta nifV1 mutant was sustained at higher levels than the activity of the parent delta hupL strain after about 2 days of combined-nitrogen step down, and the activity in the culture of the former became higher than that in the culture of the latter. The presence of N2 gas inhibited hydrogen production in the delta hupL delta nifV1 delta nifV2 mutant less strongly than in the parent delta hupL strain and the delta hupL delta nifV1 and delta hupL delta nifV2 mutants. The alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria.

ホモクエン酸合成酵素遺伝子破壊によるニトロゲナーゼ活性中心クラスターの改変がAnabaena PCC 7120の水素生産性に与える効果

ホモクエン酸合成酵素遺伝子破壊によるニトロゲナーゼ活性中心クラスターの改変がAnabaena PCC 7120の水素生産性に与える効果

IV. Yeast sequencing reports. New open reading frames, one of which is similar to the nifV gene of Azotobacter vinelandii, Found on a 12·5 kbp fragment of chromosome IV of Saccharomyces cerevisiae

AbstractThe nucleotide sequence of a 12·5 kbp segment of the left arm of chromosome IV is described. Five open reading frames (ORFs) longer than 100 amino acids were detected, all of which are completely confined to the 12·5 kbp region. Two ORFs (D1271 and D1286) correspond to previously sequenced genes (PPH22 and VMA1 or TFP1, respectively). ORF D1298 shows the characteristics of α‐isopropylmalate and homocitrate synthase genes and is similar to the nifV gene of Azotobacter vinelandii. Two more ORFs have no apparent homologue in the data libraries. Conversely, two smaller ORFs of 25 and 85 amino acids encoding the ribosomal protein YL41A and an ATPase inhibitor, respectively, were detected. Although a substantial part of the 12·5 kbp fragment apparently lacks protein‐coding characteristics, no other elements, such as tRNA genes or transposons, were found.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the Accession Number X83276.