Effects of an Electric Field of Sinusoidal Waves on the Amino Acid Biosynthesis by Azotobacter

1980 ◽  
Vol 35 (3-4) ◽  
pp. 258-261
Author(s):  
A. Martin Gonzalez ◽  
M. T. Izquierdo
Keyword(s):  
Amino Acids ◽  
Nitrogen Fixation ◽  
Amino Acid ◽  
Electric Field ◽  
Electric Fields ◽  
Culture Medium ◽  
Azotobacter Vinelandii ◽  
Amino Acid Biosynthesis ◽  
Medium Composition ◽  
Acid Biosynthesis

Abstract Electric Field Electric fields of sinusoidal waves have been applied in cultures of Azotobacter vinelandii, with potentials between 0 V and 10 V, intensities from 0 mA to 16 mA and frequencies between 5 Hz and 200 KHz. The influence of the electric field of sinusoidal waves on the nitrogen fixation on the post­ culture medium composition has a maximum at 5 V, 8 mA and 20 Hz. The rate of synthesis of specific amino acids by Azotobacter depends on the frequency and potential of the electric field applied. The concentration of each amino acid present in the post-culture medium is increased according to the electric field employed and the amino acid biosynthesis in culture medium is activated during the first days of incubation.

The Gcn2–eIF2α pathway connects iron and amino acid homeostasis in Saccharomyces cerevisiae

2018 ◽  
Vol 475 (8) ◽  
pp. 1523-1534 ◽  
Author(s):  
Marcos Caballero-Molada ◽  
María D. Planes ◽  
Helena Benlloch ◽  
Sergio Atares ◽  
Miguel A. Naranjo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Iron Content ◽  
Amino Acid Biosynthesis ◽  
Initiation Factor ◽  
Acid Biosynthesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Iron Sulfur ◽  
Amino Acid Homeostasis

In eukaryotic cells, amino acid biosynthesis is feedback-inhibited by amino acids through inhibition of the conserved protein kinase Gcn2. This decreases phosphorylation of initiation factor eIF2α, resulting in general activation of translation but inhibition of translation of mRNA for transcription factor (TF) Gcn4 in yeast or ATF4 in mammals. These TFs are positive regulators of amino acid biosynthetic genes. As several enzymes of amino acid biosynthesis contain iron–sulfur clusters (ISCs) and iron excess is toxic, iron and amino acid homeostasis should be co-ordinated. Working with the yeast Saccharomyces cerevisiae, we found that amino acid supplementation down-regulates expression of genes for iron uptake and decreases intracellular iron content. This cross-regulation requires Aft1, the major TF activated by iron scarcity, as well as Gcn2 and phosphorylatable eIF2α but not Gcn4. A mutant with constitutive activity of Gcn2 (GCN2c) shows less repression of iron transport genes by amino acids and increased nuclear localization of Aft1 in an iron-poor medium, and increases iron content in this medium. As Aft1 is activated by depletion of mitochondrial ISCs, it is plausible that the Gcn2–eIF2α pathway inhibits the formation of these complexes. Accordingly, the GCN2c mutant has strongly reduced activity of succinate dehydrogenase, an iron–sulfur mitochondrial enzyme, and is unable to grow in media with very low iron or with galactose instead of glucose, conditions where formation of ISCs is specially needed. This mechanism adjusts the uptake of iron to the needs of amino acid biosynthesis and expands the list of Gcn4-independent activities of the Gcn2–eIF2α regulatory system.

scholarly journals The genomic basis of electrotaxis in Dictyostelium discoideum: Electric field sensitive amino acids are dynamically encoded en masse for the streaming-stage proteome

2015 ◽  
Author(s):  
Albert J Erives
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Electric Field ◽  
Electric Fields ◽  
Large Scale ◽  
Genetic Model ◽  
Critical Role ◽  
Axon Growth ◽  
Amino Acid Residues ◽  
Signaling Systems

Electrotaxis plays a critical role in developmental cell migration, axon growth cone guidance, epithelial wound healing, tissue regeneration, and the degree of invasiveness characterizing different cancer cell lines. During electrotaxis in a direct current electric field (EF), a cell migrates preferentially either towards the anode or cathode depending on the cell-type. However, the types and ranges of mechanisms coupling trans-cellular electric fields to cellular EF-sensitive signaling systems are largely unknown. To address this cell biological phenomenon, I use transcriptomic data from a developmental genetic model in which multicellular social aggregation is induced by starvation of amoeboid cells. I find that the developmental proteome expressed during the streaming aggregation stage is measurably and substantially enriched in charged and highly polar amino acids relative to the proteomes of either the unicellular amoeboid or the multicellular fruiting body. This large-scale coding augmentation of EF-sensitive amino acid residues in thousands of streaming-specific proteins is accompanied by a proportional coding decrease in the number of small, uncharged amino acid residues. I also confirm an expected coding increase of biosynthetically costly amino acids in the proteome of the satiated feeding-stage amoeboid. These findings suggest that electrotactic capability is encoded broadly in the genetically regulated deployment of a developmental proteome with augmented EF-sensitivity. These results signify that extreme, nonuniform, evolutionary constraints can be exerted on the amino acid composition of an organism’s proteome.

scholarly journals GapMind: Automated Annotation of Amino Acid Biosynthesis

mSystems ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Morgan N. Price ◽  
Adam M. Deutschbauer ◽  
Adam P. Arkin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Building Blocks ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Alternative Pathways ◽  
Automated Annotation ◽  
A Genome ◽  
Minimal Media

ABSTRACT GapMind is a Web-based tool for annotating amino acid biosynthesis in bacteria and archaea (http://papers.genomics.lbl.gov/gaps). GapMind incorporates many variant pathways and 130 different reactions, and it analyzes a genome in just 15 s. To avoid error-prone transitive annotations, GapMind relies primarily on a database of experimentally characterized proteins. GapMind correctly handles fusion proteins and split proteins, which often cause errors for best-hit approaches. To improve GapMind’s coverage, we examined genetic data from 35 bacteria that grow in defined media without amino acids, and we filled many gaps in amino acid biosynthesis pathways. For example, we identified additional genes for arginine synthesis with succinylated intermediates in Bacteroides thetaiotaomicron, and we propose that Dyella japonica synthesizes tyrosine from phenylalanine. Nevertheless, for many bacteria and archaea that grow in minimal media, genes for some steps still cannot be identified. To help interpret potential gaps, GapMind checks if they match known gaps in related microbes that can grow in minimal media. GapMind should aid the identification of microbial growth requirements. IMPORTANCE Many microbes can make all of the amino acids (the building blocks of proteins). In principle, we should be able to predict which amino acids a microbe can make, and which it requires as nutrients, by checking its genome sequence for all of the necessary genes. However, in practice, it is difficult to check for all of the alternative pathways. Furthermore, new pathways and enzymes are still being discovered. We built an automated tool, GapMind, to annotate amino acid biosynthesis in bacterial and archaeal genomes. We used GapMind to list gaps: cases where a microbe makes an amino acid but a complete pathway cannot be identified in its genome. We used these gaps, together with data from mutants, to identify new pathways and enzymes. However, for most bacteria and archaea, we still do not know how they can make all of the amino acids.

scholarly journals Filling Gaps in Bacterial Amino Acid Biosynthesis Pathways with High-throughput Genetics

2017 ◽  
Author(s):  
Morgan N. Price ◽  
Grant M. Zane ◽  
Jennifer V. Kuehl ◽  
Ryan A. Melnyk ◽  
Judy D. Wall ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
High Throughput ◽  
Heterotrophic Bacteria ◽  
Current Knowledge ◽  
Acid Synthesis ◽  
Amino Acid Biosynthesis ◽  
Desulfovibrio Vulgaris ◽  
Acid Biosynthesis ◽  
Amino Acid Synthesis

AbstractFor many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacteriumDesulfovibrio vulgarissynthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

scholarly journals The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Yasuyuki Yamamoto ◽  
Tamotsu Kanai ◽  
Tsuyoshi Kaneseki ◽  
Haruyuki Atomi
Keyword(s):  
Amino Acids ◽  
Transcriptional Regulation ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Aromatic Amino Acid Biosynthesis

ABSTRACT TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar (Thermococcales aromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensis. IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.

scholarly journals A Newly Determined Member of the meso-Diaminopimelate Dehydrogenase Family with a Broad Substrate Spectrum

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Xiuzhen Gao ◽  
Zheng Zhang ◽  
Ya'nan Zhang ◽  
Ying Li ◽  
Heng Zhu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Quaternary Structure ◽  
Amino Acid Biosynthesis ◽  
Type I ◽  
Type Ii ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Keto Acids ◽  
Substrate Spectrum

ABSTRACT meso-Diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH) is the first member of the meso-DAPDH family known to catalyze the asymmetric reductive amination of 2-keto acids to produce d-amino acids. It is important to understand the catalytic mechanisms of StDAPDH and other enzymes in this family. In this study, based on an evolutionary analysis and examination of catalytic activity, the meso-DAPDH enzymes can be divided into two types. Type I showed highly preferable activity toward meso-diaminopimelate (meso-DAP), and type II exhibited obviously reversible amination activity with a broad substrate spectrum. StDAPDH belongs to type II. A quaternary structure analysis revealed that insertions/deletions (indels) and a loss of quaternary structure resulted in divergence among members of the meso-DAPDH family. A structure alignment of StDAPDH with a representative of type I, the meso-DAPDH from Corynebacterium glutamicum (CgDAPDH), indicated that they had the same folding. Based on sequence and conservation analyses, two amino acid residues of StDAPDH, R35 and R71, were found to be highly conserved within type II while also distinct from each other between the subtypes. Site mutagenesis studies identified R71 as a substrate preference-related residue of StDAPDH, which may serve as an indicator of the amination preference of type II. These results deepen the present understanding of the meso-DAPDH family and provide a solid foundation for the discovery and engineering of meso-DAPDH for d-amino acid biosynthesis. IMPORTANCE The l-form of amino acids is typically more abundant than the d-form. However, the d-form has many important pharmaceutical applications. meso-Diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH) was the first member of meso-DAPDH known to catalyze the amination of 2-keto acids to produce d-amino acids. Accordingly, we analyzed the evolution of meso-DAPDH proteins and found that they form two groups, i.e., type I proteins, which show high preference toward meso-diaminopimelate (meso-DAP), and type II proteins, which show a broad substrate spectrum. We examined the differences in sequence, ternary structure, and quaternary structure to determine the mechanisms underlying the functional differences between the type I and type II lineages. These results will facilitate the identification of additional meso-DAPDHs and may provide guidance to protein engineering studies for d-amino acid biosynthesis.

scholarly journals DksA-Dependent Resistance of Salmonella enterica Serovar Typhimurium against the Antimicrobial Activity of Inducible Nitric Oxide Synthase

2012 ◽  
Vol 80 (4) ◽  
pp. 1373-1380 ◽  
Author(s):  
Calvin A. Henard ◽  
Andrés Vázquez-Torres
Keyword(s):  
Nitric Oxide ◽  
Amino Acids ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Amino Acid Biosynthesis ◽  
Biosynthetic Pathways ◽  
Innate Response ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTIn coordination with the ppGpp alarmone, the RNA polymerase regulatory protein DksA controls the stringent response of eubacteria, negatively regulating transcription of translational machinery and directly activating amino acid promoters andde novoamino acid biosynthesis. Given the effects of nitric oxide (NO) on amino acid biosynthetic pathways and the intimate relationship of DksA with amino acid synthesis and transport, we tested whether DksA contributes to the resistance ofSalmonellato reactive nitrogen species (RNS). Our studies show that the zinc finger predicted to position DksA in the secondary channel of the RNA polymerase is essential for the resistance ofSalmonella entericaserovar Typhimurium to RNS in a murine model of systemic salmonellosis. Despite exhibiting auxotrophies for various amino acids, ΔdksAmutantSalmonellastrains regain virulence in mice lacking inducible NO synthase (iNOS). DksA is also important for growth of this intracellular pathogen in the presence of NO congeners generated by iNOS during the innate response of murine macrophages. Accordingly,dksAmutantSalmonellastrains are hypersusceptible to chemically generated NO, a phenotype that can be prevented by adding amino acids. The DksA-dependent antinitrosative defenses do not rely on the Hmp flavohemoprotein that detoxifies NO to NO3−and appear to operate independently of the ppGpp alarmone. Our investigations are consistent with a model by which NO produced in the innate response toSalmonellaexerts considerable pressure on amino acid biosynthesis. The cytotoxicity of NO againstSalmonellaamino acid biosynthetic pathways is antagonized in great part by the DksA-dependent regulation of amino acid biosynthesis and transport.

scholarly journals Auxotrophy Accounts for Nodulation Defect of Most Sinorhizobium meliloti Mutants in the Branched-Chain Amino Acid Biosynthesis Pathway

2008 ◽  
Vol 21 (9) ◽  
pp. 1232-1241 ◽  
Author(s):  
Maria de las Nieves Peltzer ◽  
Nicolas Roques ◽  
Véréna Poinsot ◽  
O. Mario Aguilar ◽  
Jacques Batut ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Amino Acid Biosynthesis ◽  
Nodule Formation ◽  
Biosynthesis Pathway ◽  
Acid Biosynthesis ◽  
Branched Chain Amino Acid ◽  
Symbiotic Phenotype ◽  
Branched Chain

Some Sinorhizobium meliloti mutants in genes involved in isoleucine, valine, and leucine biosynthesis were previously described as being unable to induce nodule formation on host plants. Here, we present a reappraisal of the interconnection between the branched-chain amino acid biosynthesis pathway and the nodulation process in S. meliloti. We characterized the symbiotic phenotype of seven mutants that are auxotrophic for isoleucine, valine, or leucine in two closely related S. meliloti strains, 1021 and 2011. We showed that all mutants were similarly impaired for nodulation and infection of the Medicago sativa host plant. In most cases, the nodulation phenotype was fully restored by the addition of the missing amino acids to the plant growth medium. This strongly suggests that auxotrophy is the cause of the nodulation defect of these mutants. However, we confirmed previous findings that ilvC and ilvD2 mutants in the S. meliloti 1021 genetic background could not be restored to nodulation by supplementation with exogenous amino acids even though their Nod factor production appeared to be normal.

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