scholarly journals Engineering the Genotype of Acinetobacter sp. Strain ADP1 To Enhance Biosynthesis of Cyanophycin

2006 ◽  
Vol 72 (2) ◽  
pp. 1410-1419 ◽  
Author(s):  
Yasser Elbahloul ◽  
Alexander Steinbüchel
Keyword(s):  
Dry Matter ◽  
Regulatory Protein ◽  
Fold Increase ◽  
Sole Source ◽  
Luria Bertani ◽  
Mineral Medium ◽  
Phosphate Limitation ◽  
Carbamoylphosphate Synthetase ◽  
Genes Encoding ◽  
Phosphate Regulon

ABSTRACT To study the importance of arginine provision and phosphate limitation for synthesis and accumulation of cyanophycin (CGP) in Acinetobacter sp. strain ADP1, genes encoding the putative arginine regulatory protein (argR) and the arginine succinyltransferase (astA) were inactivated, and the effects of these mutations on CGP synthesis were analyzed. The inactivation of these genes resulted in a 3.5- or 7-fold increase in CGP content, respectively, when the cells were grown on glutamate. Knockout mutations in both genes led to a better understanding of the effect of the addition of other substrates to arginine on CGP synthesis during growth of the cells of Acinetobacter sp. strain ADP1. Overexpression of ArgF (ornithine carbamoyltransferase), CarA-CarB (small and large subunits of carbamoylphosphate synthetase), and PepC (phosphoenolpyruvate carboxylase) triggered synthesis of CGP if amino acids were used as a carbon source whereas it was not triggered by gluconate or other sugars. Cells of Acinetobacter sp. strain ADP1, which is largely lacking genes for carbohydrate metabolism, showed a significant increase in CGP contents when grown on mineral medium supplemented with glutamate, aspartate, or arginine. The Acinetobacter sp. ΔastA(pYargF) strain is unable to utilize arginine but synthesizes more arginine, resulting in CGP contents as high as 30% and 25% of cell dry matter when grown on protamylasse or Luria-Bertani medium, respectively. This recombinant strain overcame the bottleneck of the costly arginine provision where it produces about 75% of the CGP obtained from the parent cells grown on mineral medium containing pure arginine as the sole source of carbon. Phosphate starvation is the only known trigger for CGP synthesis in this bacterium, which possesses the PhoB/PhoR phosphate regulon system. Overexpression of phoB caused an 8.6-fold increase in CGP content in comparison to the parent strain at a nonlimiting phosphate concentration.

DIGESTION OF NUTRIENTS IN THE SMALL INTESTINE OF PIGS FED DIETS CONTAINING RAW AND AUTOCLAVED FABA BEANS

1976 ◽  
Vol 56 (3) ◽  
pp. 451-456 ◽  
Author(s):  
M. IVAN ◽  
J. P. BOWLAND
Keyword(s):  
Amino Acids ◽  
Small Intestine ◽  
Beneficial Effect ◽  
Dietary Protein ◽  
Dry Matter ◽  
Latin Square ◽  
Sole Source ◽  
Intestinal Uptake ◽  
Gross Energy ◽  
Faba Beans

Four castrated pigs, each fitted with a re-entrant cannula in the terminal ileum, were used to study digestion in the small intestine. A nitrogen-free diet was used for the estimation of metabolic nitrogen and amino acids. Faba beans, as the sole source of dietary protein, were used raw or after autoclaving for 30 or 60 min. The four diets were fed to the pigs in a 4 × 4 latin square experiment. The pigs were fed each diet for 6 days prior to a 24-hr collection of total ileal contents. Autoclaving of faba beans had no significant effect on digestibility of dry matter, gross energy, nitrogen and individual amino acids except arginine, which was significantly increased. The intestinal uptake of arginine was the highest and of cystine the lowest in all faba bean diets. It was concluded that autoclaving faba beans had no beneficial effect on the digestion of nutrients in the small intestine of the pig.

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

Blood ◽  
2001 ◽  
Vol 98 (8) ◽  
pp. 2555-2562 ◽  
Author(s):  
Mark Loyevsky ◽  
Timothy LaVaute ◽  
Charles R. Allerson ◽  
Robert Stearman ◽  
Olakunle O. Kassim ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Regulatory Protein ◽  
Protein S ◽  
Fold Increase ◽  
Binding Activity ◽  
Mobility Shift ◽  
Iron Responsive Element ◽  
Element Binding Protein ◽  
Responsive Element ◽  
Electrophoretic Mobility Shift Assays

Abstract This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

Analysis of wound-induced gene expression in Nicotiana species with contrasting alkaloid profiles

2004 ◽  
Vol 31 (7) ◽  
pp. 721 ◽  
Author(s):  
Steven J. Sinclair ◽  
Richard Johnson ◽  
John D. Hamill
Keyword(s):  
Fold Increase ◽  
Transcript Abundance ◽  
Nicotiana Sylvestris ◽  
Nicotiana Glauca ◽  
Transcriptional Responses ◽  
Transcript Levels ◽  
Solanaceae Species ◽  
Genes Encoding ◽  
Root Tissues ◽  
Alkaloid Profiles

We determined the capacity of three Nicotiana (Solanaceae) species with very different alkaloid profiles (Nicotiana sylvestris Speg & Comes, Nicotiana alata Link & Otto and Nicotiana glauca Grah.) to increase their alkaloid contents in both leaf and root tissues following foliage damage. We also investigated the transcriptional responses of genes encoding enzymes important for alkaloid biosynthesis, namely quinolinate phosphoribosyltransferase (QPT), putrescine N-methyltransferase (PMT), ornithine decarboxylase (ODC) and the putative alkaloid biosynthetic gene A622. In response to wounding of foliage in the well studied ‘model’ species N. sylvestris, a rise, approximately 2-fold, in leaf nicotine levels was observed several days after a 4–5-fold increase in the transcript levels of all genes in the roots. In contrast, leaf tissues of the ornamental tobacco N. alata showed very low levels of any pyridine alkaloid, even when analysed 1 week after wounding, correlating with a general lack of transcript abundance representing any of these genes in leaves or roots following foliage damage. However, addition of methyl jasmonate to cultured roots of N. alata did produce elevated levels of nicotine and anatabine raising the possibility that components of the leaf–root wound signalling system in N. alata are different from those in N. sylvestris. Wounding of the tree tobacco N. glauca, was followed by a 2-fold increase in anabasine levels several days later. This increase followed a large rise in transcript levels of ODC, QPT and A622, though not PMT, in wounded leaves, but not in non-wounded leaves or roots. These data support the hypothesis that N. glauca is able to produce increased anabasine levels following wounding in its foliage, setting it apart from N. sylvestris where induced alkaloid production takes place in roots. We discuss the possibility that increased transcript levels detected by ODC and A622 probes play important roles in anabasine synthesis in N. glauca.

scholarly journals INJECTABLE COPPER AND ZINC FOR GRAZING YEARLING STEERS

1987 ◽  
Vol 67 (4) ◽  
pp. 1033-1041
Author(s):  
F. A-R. SANKOH ◽  
R. J. BOILA
Keyword(s):  
Body Weight ◽  
Dry Matter ◽  
Phase 1 ◽  
Sole Source ◽  
The Body ◽  
Mineral Supplements ◽  
Copper Zinc ◽  
Group 2 ◽  
Cu And Zn ◽  
Group 1

Injectable Cu and Zn were evaluated as mineral supplements using 37 purebred Herefords (Group 1) and 23 Angus-Charolais-North Devon crosses (Group 2). The 60 steers were carried through a 113-d grazing phase. This was followed by a 79-d finishing phase for all steers of Group 1 and seven steers of Group 2. Treatments were combinations of the non-dosing (0Cu, 0Zn) and dosing (+Cu, +Zn) of steers with injectable Cu or Zn at the start of the grazing phase: (1) 0Cu, 0Zn; (2) +Cu, 0Zn; (3) 0Cu, +Zn; and (4) +Cu, +Zn. Injectables were the sole source of supplementary Cu and Zn during grazing and finishing phases. Means (SE) for Cu and Zn, respectively, in forages as milligrams per kilogram dry matter (DM) during the grazing phase were: grasses, 4.4 (0.17), 15.7 (0.46); alfalfa, 6.9 (0.18), 16.5 (1.49); and birdsfoot trefoil 5.0 (0.76), 15.0 (3.41). Injectable Cu did not influence (P > 0.05) body weight during the grazing phase. Body weight responses were lower (P < 0.05) for +Cu steers in the finishing phase, when dietary Cu was low, but steers could not be classified as Cu-deficient based on liver Cu. Liver Cu increased to means greater than 140 mg kg−1 DM in all steers, but was higher (P < 0.05) in +Cu steers during the grazing phase. Serum Cu varied widely during the grazing phase, but was higher (P < 0.05) for +Cu steers only on day 84 of the grazing phase. Neither liver Cu nor serum Cu was influenced (P > 0.05) by injectable Zn. Injectable Zn decreased growth rates in the latter part of the grazing phase but did not affect (P > 0.05) the body weight response during the finishing phase. The concentrations of Zn in liver and serum were not influenced (P > 0.05) by injectable Cu or Zn. Key words: Cattle, grazing, copper, zinc, injectable copper, injectable zinc

scholarly journals Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from Pseudomonas aeruginosa

1999 ◽  
Vol 181 (17) ◽  
pp. 5426-5432 ◽  
Author(s):  
Martina M. Ochs ◽  
Chung-Dar Lu ◽  
Robert E. W. Hancock ◽  
Ahmed T. Abdelal
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Regulatory Protein ◽  
Basic Amino Acid ◽  
Sole Source ◽  
Activation Mechanism ◽  
Beta Lactam ◽  
Mobility Shift ◽  
Carbon And Nitrogen

ABSTRACT Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction ofoprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of theoprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression ofoprD is linked to both carbon and nitrogen metabolism ofPseudomonas aeruginosa.

scholarly journals Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

2018 ◽  
Author(s):  
Krithika Rajagopalan ◽  
Jonathan Dworkin
Keyword(s):  
Biochemical Characterization ◽  
Regulatory Protein ◽  
Biochemical Properties ◽  
Bacterial Genomes ◽  
Phosphatase Inhibitors ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Genes Encoding ◽  
Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

scholarly journals Identification of Novel Type III Secretion Effectors in Xanthomonas oryzae pv. oryzae

2009 ◽  
Vol 22 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Ayako Furutani ◽  
Minako Takaoka ◽  
Harumi Sanada ◽  
Yukari Noguchi ◽  
Takashi Oku ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Effector Proteins ◽  
Xanthomonas Oryzae ◽  
Dependent Manner ◽  
Plant Pathogenic Bacteria ◽  
Type Iii ◽  
Genes Encoding

Many gram-negative bacteria secrete so-called effector proteins via a type III secretion (T3S) system. Through genome screening for genes encoding potential T3S effectors, 60 candidates were selected from rice pathogen Xanthomonas oryzae pv. oryzae MAFF311018 using these criteria: i) homologs of known T3S effectors in plant-pathogenic bacteria, ii) genes with expression regulated by hrp regulatory protein HrpX, or iii) proteins with N-terminal amino acid patterns associated with T3S substrates of Pseudomonas syringae. Of effector candidates tested with the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter for translocation into plant cells, 16 proteins were translocated in a T3S system-dependent manner. Of these 16 proteins, nine were homologs of known effectors in other plant-pathogenic bacteria and seven were not. Most of the effectors were widely conserved in Xanthomonas spp.; however, some were specific to X. oryzae. Interestingly, all these effectors were expressed in an HrpX-dependent manner, suggesting coregulation of effectors and the T3S system. In X. campestris pv. vesicatoria, HpaB and HpaC (HpaP in X. oryzae pv. oryzae) have a central role in recruiting T3S substrates to the secretion apparatus. Secretion of all but one effector was reduced in both HpaB– and HpaP– mutant strains, indicating that HpaB and HpaP are widely involved in efficient secretion of the effectors.

scholarly journals Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

2001 ◽  
Vol 67 (2) ◽  
pp. 688-695 ◽  
Author(s):  
Jan Bohuslavek ◽  
Jason W. Payne ◽  
Yong Liu ◽  
Harvey Bolton ◽  
Luying Xun
Keyword(s):  
Gene Cluster ◽  
Genomic Library ◽  
Regulatory Protein ◽  
Chelating Agent ◽  
Environmental Pollutant ◽  
Flavin Mononucleotide ◽  
Bacterial Strains ◽  
System A ◽  
Monooxygenase Gene ◽  
Genes Encoding

ABSTRACT EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed inEscherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. TheemoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.

fra-1: a serum-inducible, cellular immediate-early gene that encodes a fos-related antigen

1988 ◽  
Vol 8 (5) ◽  
pp. 2063-2069 ◽  
Author(s):  
D R Cohen ◽  
T Curran
Keyword(s):  
Regulatory Protein ◽  
Transcriptional Response ◽  
Early Gene ◽  
Immediate Early ◽  
Cdna Clones ◽  
Protein Synthesis Inhibitors ◽  
Amino Acid Homology ◽  
Serum Stimulation ◽  
Fos Protein ◽  
Genes Encoding

A set of proteins antigenically related to the c-fos protein (Fos) are induced by serum in fibroblasts. To isolate cDNA clones of genes encoding such proteins, a lambda gt11 expression cDNA library constructed from serum-stimulated rat fibroblasts was screened with antibodies raised against a hydrophilic region (amino acids 127 to 152) of Fos. One of the positive clones identified, termed fra-1 (Fos-related antigen) was characterized. It encoded a protein that shared several regions of extensive amino acid homology with Fos (including the region that showed similarity to both the yeast GCN4 regulatory protein and the protein encoded by the jun oncogene), although its nucleotide sequence was considerably diverged from that of the c-fos gene. Only a subset of the agents and conditions that activated c-fos also induced fra-1. Induction of fra-1 expression following serum stimulation was delayed compared with that of c-fos. However, like c-fos, fra-1 was induced rapidly by serum in the presence of protein synthesis inhibitors. Thus, a family of Fos-related, inducible genes are involved in the cellular immediate-early transcriptional response to extracellular stimuli.

scholarly journals Biochemical and Genetic Analysis of 4-Hydroxypyridine Catabolism in Arthrobacter sp. Strain IN13

2020 ◽  
Vol 8 (6) ◽  
pp. 888
Author(s):  
Justas Vaitekūnas ◽  
Renata Gasparavičiūtė ◽  
Jonita Stankevičiūtė ◽  
Gintaras Urbelis ◽  
Rolandas Meškys
Keyword(s):  
Recombinant Expression ◽  
Expression Profiles ◽  
Peptide Mass Fingerprinting ◽  
Sole Source ◽  
Open Reading Frames ◽  
Expression Of Genes ◽  
Peptide Mass ◽  
Genes Encoding ◽  
Protein Expression Profiles ◽  
Reading Frames

N-Heterocyclic compounds are widely spread in the biosphere, being constituents of alkaloids, cofactors, allelochemicals, and artificial substances. However, the fate of such compounds including a catabolism of hydroxylated pyridines is not yet fully understood. Arthrobacter sp. IN13 is capable of using 4-hydroxypyridine as a sole source of carbon and energy. Three substrate-inducible proteins were detected by comparing protein expression profiles, and peptide mass fingerprinting was performed using MS/MS. After partial sequencing of the genome, we were able to locate genes encoding 4-hydroxypyridine-inducible proteins and identify the kpi gene cluster consisting of 16 open reading frames. The recombinant expression of genes from this locus in Escherichia coli and Rhodococcus erytropolis SQ1 allowed an elucidation of the biochemical functions of the proteins. We report that in Arthrobacter sp. IN13, the initial hydroxylation of 4-hydroxypyridine is catalyzed by a flavin-dependent monooxygenase (KpiA). A product of the monooxygenase reaction is identified as 3,4-dihydroxypyridine, and a subsequent oxidative opening of the ring is performed by a hypothetical amidohydrolase (KpiC). The 3-(N-formyl)-formiminopyruvate formed in this reaction is further converted by KpiB hydrolase to 3-formylpyruvate. Thus, the degradation of 4-hydroxypyridine in Arthrobacter sp. IN13 was analyzed at genetic and biochemical levels, elucidating this catabolic pathway.

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