Arginine Hydroxamate Research Articles

Constitutive Stringent Response Restores Viability of Bacillus subtilis Lacking Structural Maintenance of Chromosome Protein.

Bacillus subtilis mutants lacking the SMC-ScpAB complex are severely impaired for chromosome condensation and partitioning, DNA repair, and cells are not viable under standard laboratory conditions. We isolated suppressor mutations that restored the capacity of a smc deletion mutant (Δsmc) to grow under standard conditions. These suppressor mutations reduced chromosome segregation defects and abrogated hypersensitivity to gyrase inhibitors of Δsmc. Three suppressor mutations were mapped in genes involved in tRNA aminoacylation and maturation pathways. A transcriptomic survey of isolated suppressor mutations pointed to a potential link between suppression of Δsmc and induction of the stringent response. This link was confirmed by (p)ppGpp quantification which indicated a constitutive induction of the stringent response in multiple suppressor strains. Furthermore, sublethal concentrations of arginine hydroxamate (RHX), a potent inducer of stringent response, restored growth of Δsmc under non permissive conditions. We showed that production of (p)ppGpp alone was sufficient to suppress the thermosensitivity exhibited by the Δsmc mutant. Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.

Structure-based design of peptide inhibitors of botulinum neurotoxin serotype A proteolytic activity

P1'arginine of the substrate SNAP-25 is an absolute requirement for catalysis, and the active site of botulinum neurotoxin serotype A (BoNT/A) is populated by acidic residues. We reasoned that arginine derivatives, and basic peptides might bind to the proteolytic domain, and act as inhibitors. A systematic investigation of amino acids, derivatives and basic peptides provided proofs of our concept. D-arginine, arginine hydroxamate and basic peptides effectively inhibited BoNT/A activity. Optimisation of the peptide inhibitors resulted in a RRGC tetrapeptide displaying 90% inhibition at 20 microM. Our studies provide an inhibitor scaffold for potential development as a BoNT/A drug candidate.

Compartmentalization of transcription and translation in Bacillus subtilis.

Using fusions of green fluorescent protein to subunits of RNA polymerase (RNAP) and ribosomes, we have investigated the subcellular localization of the transcriptional and translational machinery in the bacterium Bacillus subtilis. Unexpectedly, we found that RNAP resides principally within the nucleoid. Conversely, ribosomes localized almost exclusively outside the nucleoid, concentrating particularly towards sites of cell division. This zonal localization was not dependent on cell division and is probably due, at least in part, to exclusion from the nucleoid. Dual labelling of RNAP and ribosomes was used to confirm the spatial separation of the two processes. We conclude that, even in the absence of a nuclear membrane, transcription and translation occur predominantly in separate functional domains. At higher growth rates, concentrations of RNAP developed, probably representing the sites of rRNA synthesis. These may represent a further spatial specialization, possibly equivalent to the eukaryotic nucleolus.

Synthesis of aqueous magnetic liquids by surface complexation of maghemite nanoparticles

Abstract Adsorption of various ligands onto maghemite particles has been investigated in aqueous medium aiming at the production of magnetic liquids (ferrofluids) stable in a large range of pH. The formation of complexes with the structural iron of the surface oxide causes drastic changes in the stability of the colloid dispersion. The flocculation ranges have been established as a function of pH for maghemite particles in presence of ligands such as α-hydroxy or thiol polycarboxylic acids and arginine hydroxamate. The stability behavior of the systems has been correlated with adsorption isotherms.

Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa PAO1.

Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginine-inducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.

Nucleoside intermediates in blasticidin S biosynthesis identified by the in vivo use of enzyme inhibitors

Intermediates in the biosynthesis of blasticidin S and its nucleoside co-metabolites were detected by altering fermentation conditions. Inhibitors of specific types of biochemical reactions that were expected to be involved in blasticidin biosynthesis were fed to Streptomyces griseochromogenes, in some cases with the inclusion of large quantities of the primary precursors of blasticidin S. The types of reactions and inhibitors used were (1) transaminase (aminooxyacetic acid and 2-methylglutamate), (2) amidotransferase (azaserine and 6-diazo-5-oxo-L-norleucine), (3) arginine biosynthesis (arginine hydroxamate), and (4) methyltransferase (ethionine). These manipulations apparently distorted the pools of precursors and (or) intermediates, and led to substantial accumulations of three known, previously minor, metabolites of S. griseochromogenes, cytosylglucuronic acid, pentopyranine C, and demethylblasticidin S, and of two new ones, pentopyranone and isoblasticidin S. New cytosyl metabolites were detected by HPLC with photodiode array detection. Fermentations to which arginine hydroxamate and cytosine had been added also produced three aberrant metabolites that were derived from pentopyranone and arginine hydroxamate.

Statistical Mixture Design Applied to RP-HPTLC Chromatographic Separations of Amino Acid Hydroxamates

Abstract Mixture Design and a Chromatographic Response Function were used to study the effect of different organic solvents as modifiers on the Reversed Phase (C-18) High Performance Thin Layer Chromatographic separation of three amino acid hydroxamates (Arginine hydroxamate, Threonine hydroxamate, and Histidine hydroxamate). Both 3 solvent (methanol, acetonitrile and tetrahydrofuran) and 4 solvent (methanol, acetonitrite, tetrahydrofuran and acetone) systems were studied.

The stringent response blocks DNA replication outside the ori region in Bacillus subtilis and at the origin in Escherichia coli

When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after growth at 45 °C, DNA replication is synchronized. Under these conditions, we have shown previously that DNA replication is inhibited when the Stringent Response is induced by the amino acid analogue, arginine hydroxamate. We have now shown, using DNA-DNA hybridization analysis, that substantial replication of the oriC region nevertheless occurs during the Stringent Response, and that replication inhibition is therefore implemented downstream from the origin. On the left arm, replication continues for at least 190 × 10 3 base-pairs to the gnt gene and for a similar distance on the right arm to the gerD gene. When the Stringent Response is lifted, DNA replication resumed downstream from oriC on both arms, confirming that DNA replication is regulated at a post-initiation level during the Stringent Response in B. subtilis. Resumption of DNA synthesis following the lifting of the Stringent Response did not require protein or RNA synthesis or the initiation protein DnaB. We suggest, therefore, that a specific control region, involving Stringent Control sites, facilitate reversible inhibition of fork movement downstream from the origin via modifications of a replisome component during the Stringent Response. In contrast, in Escherichia coli, induction of the Stringent Response appears to block initiation of DNA replication at oriC itself. No DNA synthesis was detected in the oriC region and, upon lifting the Stringent Response, replication occurred from oriC. Post-initiation control in B. subtilis therefore results in duplication of many key genes involved in growth and sporulation. We discuss the possibility that such a control might be linked to differentiation in this organism.

L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells.

L-Arginine is required for expression of the activated macrophage cytotoxic effector mechanism that causes inhibition of mitochondrial respiration, aconitase activity, and DNA synthesis in tumor target cells. This effector mechanism is active in the presence of L-arginine even when the cocultivation medium lacks all other amino acids and serum. Cytotoxic activated macrophage-induced inhibition of mitochondrial respiration in target cells is proportional to the concentration of L-arginine in the medium. L-Arginine must be present during the cocultivation period. Pretreatment of cytotoxic activated macrophages with L-arginine or posttreatment of the target cells after cocultivation is not effective. D-Arginine does not substitute for L-arginine and at high concentrations is a competitive inhibitor of the L-arginine-dependent effector mechanism. Other analogues that could not replace L-arginine include agmatine, argininic acid, arginine hydroxamate, and tosyl-L-arginine methyl ester. L-homoarginine, however, can effectively substitute for L-arginine. NG-monomethyl-L-arginine is a potent competitive inhibitor of this effector mechanism. High concentrations of lipopolysaccharide do not reverse inhibition of the L-arginine-dependent effector mechanism by NG-monomethyl-L-arginine. However, inhibition of the effector mechanism by NG-monomethyl-L-arginine can be overridden by increasing the concentration of L-arginine in the culture medium. We compared NGNG-dimethyl-L-arginine and NGN1G-dimethyl-L-arginine with NG-monomethyl-L-arginine as inhibitors of the L-arginine-dependent effector mechanism. The results show that the inhibitory effect of these guanidino methylated derivatives of L-arginine is highly determined by structure. Guanidine is a weak competitive inhibitor of the L-arginine-dependent effector mechanism. The requirement for L-arginine does not appear to be for protein synthesis, creatine biosynthesis, polyamine biosynthesis, or ADP ribosylation reactions. Bacterial lipopolysaccharide is effective as a second signal only when the cocultivation medium contains L-arginine, and this strict L-arginine dependency is not overridden by increasing the concentration of lipopolysaccharide. Bovine liver arginase, by competing for L-arginine in the cocultivation medium, inhibits the L-arginine-dependent activated macrophage cytotoxic effector mechanism.

Cloning inEscherichia coli of aBacillus subtilis arginine repressor gene through its ability to confer structural stability on a fragment carrying genes of arginine biosynthesis

The structural stability of a previously isolated recombinant plasmid pUL720 was examined. pUL720 contains an insert in pBR322 of 23.8 kbp comprising 4 EcoR1 fragments of sizes 12 kbp and 6 kbp, both of which are homologous to the B. subtilis genome, and 5 kbp and 0.8 kbp (of unknown origin). The 12 kbp fragment, which encodes the arginine biosynthesis genes argA-F-cpa, cannot be cloned in isolation in a high copy vector in E. coli but can be inserted into a low copy vector pGV1106 to generate pUL800. Deletion analysis of pUL720 indicated that the 5 kbp and 0.8 kbp fragments were not necessary to maintain plasmid stability. The 6 kbp fragment, when cloned into the EcoR1 site in pACYC184 to generate pUL2030, permitted the cloning in trans in pBR322 of the 12 kbp fragment or subclones containing the instability region. The minimum inhibitory concentration of kanamycin determined in the B. subtilis argC-neo transcriptional fusion pUL730 and expression of the argF gene product, ornithine carbamoyltransferase (OCTase), in pUL800 were reduced by approximately 3 and 2 fold respectively under conditions of arginine excess and in the presence of pUL2030. B. subtilis partial diploids were constructed by transforming parental and arginine hydroxamate resistant (Ahr) mutants with pUL2100, a plasmid generated by inserting the 6 kbp fragment into the integration vector pJH101. The 6 kbp fragment complemented and restored parental type levels of OCTase in ahrC mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

The detergent form of the major aminopeptidase from the plasma membrane of midgut caeca cells of Rhynchosciara americana (Diptera) larva

1. 1. Plasma membrane-bound aminopeptidases from Rhynchosciara americana midgut caeca cells may be solubilized by Triton X-100 or may be released by papain treatment. 2. 2. The major detergent solubilized aminopeptidase was purified by electrophoresis and was shown to have M r 169,000 and pI 7.8. 3. 3. K m values for two substrates ( l-arginine -p- nitroanilide and l-leucine -p- nitroanilide ) and K i values for four linear inhibitors (arginine hydroxamate, leucine hydroxamate, hydroxyl amine and isoamyl alcohol) corresponding to the major detergent-solubilized aminopeptidase are identical to those of the major papain released aminopeptidase, which was previously purified and characterized. 4. 4. Both aminopeptidase forms display the same pH optimum (7.2 in Tris-HCl buffer) and follow the same thermal inactivation kinetics (half-lives of 23 min at 50°C). 5. 5. Treatment of the major detergent form of the aminopeptidase with papain results in the appearance of a M r 97,000 aminopeptidase. 6. 6. The data support the proposal that the papain-form of the aminopeptidase (M r 207,000) results from the dimerization of the products of the papain action upon the major native (M r 169,000) aminopeptidase.

Minor aminopeptidases purified from the plasma membrane of midgut caeca cells of an insect ( Rhynchosciara americana) larva

Rhynchosciara americana midgut caecal cells display in their plasma membranes, as judged by electrophoresis, a major (T 1) and three minor (T 2, T 3 and T 4) aminopeptidases which are solubilized by Triton X-100, and a major (P 1) and two minor (P 2 and P 3) aminopeptidases which are released by papain treatment. Previous work showed that T 1 corresponds to P 1. Aminopeptidases T 1 and T 3 have the same M r value as determined by density-gradient centrifugation and by electrophoresis, and display different pI values, as judged by isoelectric focusing and electrophoresis. Furthermore, T 1 and T 3 purified by electrophoresis display identical K m values for arginine p- nitroanilide and leucine p- nitroanilide and the same K i values for leucine hydroxamate, hydroxyl amine and isoamyl alcohol. The data suggest that T 1 and T 3 differ only in net charge, implying that T 2 and T 4 should be related to P 2 and P 3. P 2 and P 3 purified by electrophoresis display identical K i values for arginine hydroxamate, leucine hydroxamate, hydroxylamine and isoamyl alcohol and also identical substrate specificities. P 2 and P 3 show a broad specificity in relation to the N-terminal aminoacyl residue and hydrolyses tetrapeptides more efficiently than tripeptides and much more efficiently than dipeptides. The data suggest that T 2 and T 4 as well as P 2 and P 3 are respectively different aggregation states of a minor native membrane bound aminopeptidase solubilized by detergent and released by papain. The native aminopeptidase is probably a tetramer of identical (or similar) subunits, which, due to its substrate specificities, produces short oligopeptides (mainly tripeptides) which are the preferred substrates for the major membrane-bound aminopeptidase.

Bacillus subtilis 168 mutants resistant to arginine hydroxamate in the presence of ornithine or citrulline.

Mutations in Bacillus subtilis 168 have been isolated that confer resistance to arginine hydroxamate in the presence, but not absence, of ornithine. Seven such Ahor mutants have been studied in detail. In common with certain classes of Ahr mutant (resistant to arginine hydroxamate in the absence of arginine precursors) described previously, these Ahor mutants showed little or no inducibility of enzymes of arginine catabolism. Mutants that showed no inducibility were unable to utilize arginine or ornithine as sole nitrogen source. The only biosynthetic enzyme to show any consistent differences in activity from the parent was ornithine carbamoyltransferase, whose level was slightly elevated in cells grown in the presence of ornithine or citrulline. PBS1 transduction crosses showed that two of the ahor mutations map at the ahrA locus, while a third (unique in its resistance to arginine hydroxamate in the presence of citrulline) mapped at a hitherto undescribed locus closely linked to metC, designated ahrD.

Map locations of some mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168.

Mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168 have been located on the genetic map by PBS1-mediated transduction. The majority of these mutations, belonging to classes 1, 2 and 4 of Harwood and Baumberg (1977) and affecting only expression of arginine catabolic enzymes, map at a locus designated ahr A cotransducible with cysA, purA and sacA. The order of markers in this region appears to be sacA-ahrA-purA-cysA. Certain anomalies were observed in the properties of Pur+ transductants from crosses with an Ahr donor and a purA recipient. A single ahr mutation (class 3), also affecting only arginine catabolism, maps between ctrA and sacA at a locus designated ahrB. Two others (class 6), affecting simultaneously enzymes of both arginine biosynthesis and catabolism, map between lys and aroD at a locus designated ahrC. Preliminary attempts to define the nature of functional products specified by these ahr loci suggest that a protein is encoded at ahrA.

Arginine Hydroxamate-resistant Mutants of Bacillus subtilis with Altered Control of Arginine Metabolism

Arginine hydroxamate inhibits the growth of Bacillus subtilis. From a large number of mutants isolated as resistant to this arginine analogue, 29 were chosen for further investigation. Most of these shared diminished ability to utilize arginine, citrulline and/or ornithine as sole nitrogen source. All 29 had reduced levels of the catabolic enzymes arginase and ornithine aminotransferase under various conditions in which these enzymes are induced in the parent. In some circumstances, five of the mutants also showed elevated levels of the biosynthetic enzyme ornithine carbamoyltransferase. On the basis of these data, the 29 mutants were divided into six phenotypic classes; in four of these, control of ornithine carbamoyltransferase was the same as in the wild type, while in the other two it was altered. It is suggested that the isolates carry regulatory mutations, and that certain of these may affect simultaneously the formation of arginine catabolic and biosynthetic enzymes. The implication of the latter is that in B. subtilis, as in yeast, controls of the catabolic and biosynthetic pathways are connected. Single representatives of five of the phenotypic classes carry mutations conferring arginine hydroxamate resistance linked to cysA by transduction with phage PBSI; this did not appear to be true for a representative of the sixth class.

Physical and Kinetic Studies of Arginyl Transfer Ribonucleic Acid Ligase of Neurospora: A SEQUENTIAL ORDERED MECHANISM

Abstract The arginyl-tRNA ligase of Neurospora has been purified 1,800-fold. The purified preparation catalyzed the esterification of 140 µmoles of arginine to tRNA per hour per mg of protein of 35°. Polyacrylamide gel electrophoresis revealed a single major band from which the enzyme was estimated to be 95% homogeneous. The apparent molecular weight of the enzyme is about 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration. No evidence for subunits was obtained. The enzyme required tRNAArg for the ATP-pyrophosphate exchange reaction, and this requirement was not satisfied by periodate-oxidized tRNA. Transfer RNA was also required for the arginine-dependent cleavage of pyrophosphate from ATP. These two reactions (pyrophosphate exchange and pyrophosphate cleavage from ATP) proceed at a faster rate than the esterification reaction. Other differences with the Escherichia coli arginyl-tRNA ligase, including molecular size, amino acid composition, tRNA substrate specificity, and order of substrate addition, were noted. dATP could fully replace ATP in both the pyrophosphate exchange and the esterification reactions. Canavanine could replace arginine, whereas arginine hydroxamate, arginine methyl ester, argininosuccinate, homoarginine, and α-amino-γ-guanidobutyrate were nonreacting competitive inhibitors. The addition of substrates and the release of products were investigated primarily by bisubstrate kinetics, deadend, and product inhibition studies. The kinetic patterns obtained indicated a unique sequential ordered mechanism of substrate addition; tRNAArg is bound first, followed by arginine, and then by ATP. Pyrophosphate and AMP may be released in a random fashion, and arginyl-tRNA is the last product to dissociate from the enzyme. The ligase catalyzed a tRNA-dependent pyrophosphorolysis of exogenous arginyladenylate. The enzyme could also use arginyladenylate as an arginine donor for the esterification of tRNA. The rates of pyrophosphorolysis and esterification with arginyladenylate were very similar to the ATPpyrophosphate exchange and over-all esterification rates, respectively.

Fermentative Production of l-Arginine

Most of the bacteria, which were examined for the sensitivity to l-arginine analogs (l-canavanine, l-homoarginine, d-arginine and arginine hydroxamate), were insensitive to the analogs at a concentration of 8 mg/ml. Corynebacterium glutamicum DSS-8 isolated as d-serine-sensitive mutant from an isoleucine auxotroph KY 10150, was found to be sensitive to d-arginine and arginine hydroxamate. Furthermore, DSS-8 produced l-arginine in a cultural medium. l-Arginine analog-resistant mutants were derived from DSS-8 by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment. Most of them were found to produce a large amount of l-arginine. An isoleucine revertant from one of these mutants produced 19.6 mg/ml of l-arginine in the medium containing 15% (as sugar) of molasses.The mechanism of the sensitivity to l-arginine analogs and that of the production of l-arginine in the d-serine-sensitive mutant, DSS-8, were investigated. DSS-8 seems to be a mutant having increased permeability to d- and l-arginine.