N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

The genetic organization of arginine biosynthesis in Pseudomonas aeruginosa

Summary Six loci coding for arginine biosynthetic enzymes in Pseudomonas aeruginosa strain PAO were identified by enzyme assay: argA (N-acetylglutamate synthase), argB (N-acetylglutamate 5-phosphotransferase), argC (N-acetylglutamate 5-semialdehyde dehydrogenase), argF (anabolic ornithine carbamoyltransferase), argG (argininosuccinate synthetase), and argH (argininosuccinase). One-step mutants which had a requirement for arginine and uracil were defective in carbamoylphosphate synthase, specified by a locus designated car. To map these mutations we used the sex factor FP2 in an improved interrupted mating technique as well as the generalized transducing phages F116L and G101. We confirmed earlier studies, and found no clustering of arg and car loci. However, argA, argH, and argB were mapped on a short chromosome segment (approx. 3 min long), and argF and argG were cotransducible, but not contiguous. N-Acetylglutamate synthase, the enzyme which replenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.     

精氨酸代谢调控蛋白ArgR调控嗜热链球菌胞外多糖合成

[目的] 研究精氨酸代谢调控蛋白ArgR对嗜热链球菌胞外多糖（EPS）合成的调控作用。[方法] 利用大肠杆菌异源表达嗜热链球菌ArgR蛋白,通过尿素变性-复性和Ni²⁺亲和层析纯化。采用凝胶电泳迁移（EMSA）和生物膜层干涉（BLI）分析ArgR和eps基因簇中P_epsA启动子的相互作用和动力学信息。构建过表达和弱化argR基因菌株,利用苯酚-硫酸法测定其合成EPS差异。[结果] 大肠杆菌异源表达的ArgR为包涵体,使用尿素变性-复性纯化可获得2.95 mg/mL可溶性蛋白;EMSA和BLI结果显示ArgR和启动子P_epsA有特异性结合,且结合因解离水平低而稳定;过表达argR基因可显著降低嗜热链球菌EPS合成,而弱化argR基因则提高EPS合成。[结论] 本研究表明ArgR能特异性结合嗜热链球菌eps基因簇启动子,并负调控EPS生物合成。     

Overproduction of pyoverdins by Fur mutants of Pseudomonas aeruginosa strains PAO1 and Fe10 in stirred bioreactors

Fur mutants FPA12 and FF13 of strains Pseudomonas aeruginosa PAO1 and Fe10, respectively, were prepared and their production of pyoverdin evaluated. The strains were cultivated in stirred bioreactor in iron-deficient and iron-supplemented medium containing Casamino acids (CA) or succinate as a source of carbon and energy. When the pyoverdin production rate reached its maximum, the demand of iron-depleted cultures for O₂ was decreased. Mutant FF13 overproduced pyoverdin in both iron-depleted (862 mg l^–1) and iron-supplemented (428 mg l^–1) CA medium and could also be used to produce pyoverdin when grown in a conventional stirred tank fermenter.     

The nifU, nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii

Summary Strains with mutations in 23 of the 30 genes and open reading frames in the major nif gene cluster of A. vinelandii were tested for ability to grow on N-free medium with molybdenum (Nif phenotype), with vanadium (Vnf phenotype), or with neither metal present (Anf phenotype). As reported previously, nifE, nifty, nifU, nifS and nifV mutants were Nif^– (failed to grow on molybdenum) while nifM mutants were Nif^–, Vnf^– and Anf^–. nifV, nifS, and nifU mutants were found to be unable to grow on medium with or without vanadium, i.e. were Vnf^– Anf–. Therefore neither vnf nor anf analogoues of nifU, nifS, nifV or nifM are expected to be present in A. vinelandii.     

R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa

Summary The R factor R68 readily promotes chromosome transfer in Pseudomonas aeruginosa strain PAT, but shows little such sex factor activity in strain PAO. A variant of this plasmid, R68.45, has been isolated which produces recombinants in PAO plate matings at frequencies of 10^-3–10^-5 per donor cell for markers in the 0–60 min region of the chromosome. Little or no chromosome transfer was shown in liquid media. The kinetics of chromosome transfer were studied by interrupting matings on solid media with nalidixic acid. Five chromosomal markers, mapping in widely spaced regions of the chromosome all entered 3–5 min after initiation of mating. These results, combined with linkage studies, indicate that R68.45, unlike the Pseudomonas sex factors FP2 and FP39, promotes chromosome transfer from a range of origin sites and can thus be used for mapping the region of the P. aeruginosa chromosome later than 40 min.R68.45 and other similar variants were isolated from rare chromosomal recombinants appearing in crosses between PAO(R68) donors and PAO recipients in which selection for argB ⁺ was made. Selection for other chromosomal markers did not result in such variants suggesting that plasmids of the R68.45 type arise by recombination of genetic material between the R68 plasmid and certain regions of the bacterial chromosome.     

Low- and intermediate-copy-number cloning vectors based on thePseudomonas plasmid pVS1

The cloning vector pME290 (6.8 kb), which is derived from thePseudomonas plasmid pVS1 and has about 7 copies, was mutagenized in vitro to provide derivatives with altered copy numbers. Thus, pME292 (about 1–3 copies) and pME294 (about 15–20 copies) were isolated. These vectors were used in the characterization of theP. aeruginosa argF gene encoding ornithine carbamoyltransferase.     

Amino acid utilization by the ruminal bacterium Synergistes jonesii strain 78-1

The ruminal bacterium Synergistes jonesii strain 78-1, which is able to degrade the pyridinediol toxin in the plant Leucaena leucephala, was studied for its ability to utilise amino acids. The organism used arginine, histidine and glycine from a complex mixture of amino acids, and both arginine and histidine supported growth in a semi-defined medium. The products of (U-¹⁴C)-arginine metabolism were CO₂ acetate, butyrate, citrulline and ornithine. The labelling pattern of end products from (U-¹⁴C)-histidine metabolism differed in that carbon also flowed into formate and propionate. Arginine was catabolised by the arginine deiminase pathway which was characterised by the presence of arginine deiminase, ornithine transcarbamylase and carbamate kinase. This is the first report of a rumen bacterium that uses arginine and histidine as major energy yielding substrates.     

Mutational analysis of the arginine repressor of Escherichia coli

Arginine biosynthesis in Escherichia coli is negatively regulated by a hexameric repressor protein, encoded by the gene argR and the corepressor arginine. By hydroxylamine mutagenesis two types of argR mutants were isolated and mapped. The first type is transdominant. In heterodiploids, these mutant polypeptides reduce the activity of the wild-type repressor, presumably by forming heteropolymers. Four mutant repressor proteins were purified. Two of these map in the N-terminal half of the protein. Gel retardation experiments showed that they bind poorly to DNA, but they could be precipitated by l -arginine at the same concentration as the wild-type repressor. The other two mutant repressors map in the C-terminal half of the protein. They are poorly precipitated by L-arginine and they bind poorly to DNA. In addition, one of these mutants appears to exist as a dimer. The second type of argR mutant repressor consists of super-repressors. Such mutants behave as arginine auxotrophs as a result of hyper-repression of arginine biosynthetic enzymes. They map at many locations throughout the argR gene. Three arginine super-repressor proteins were purified, in comparison with the wild-type repressor, two of them were shown to have a higher DNA-binding affinity in the absence of bound arginine, while the third was shown to have a higher DNA-binding affinity when bound to arginine.     

In vitro propagation of taro,with spermine,arginine, and ornithine

In vitro growth and multiplication of taro [Colocasia esculenta var. antiquorum cv. Keladi Birah] was improved considerably, when primary shoot apices were cultured on two modifications of Linsmaier and Skoog [1965] medium, containing 5.5 mg 1^–1 naphthaleneacetic acid and 0.2 mg 1^–1 kinetin or 1.85 mg 1^–1 naphthaleneacetic acid and 2 mg 1^–1 kinetin and supplemented with 10^–4 or 10^–3 mol·1^–1 of polyamine spermine or either of the precursors of polyamine putrescine—arginine and ornithine. Plantlets were regenerated directly from primary shoot apices, axillary buds and protocorm-like bodies [PLB]. Frequency of plantlet regeneration, rate of development and growth in height of main plantlets were enhanced by the addition of arginine and ornithine to the media. Secondary plantlet formation from axillary buds and PLB were promoted by spermine and arginine respectively.     

Molecular Characterization and Regulation of an Operon Encoding a System for Transport of Arginine and Ornithine and the ArgR Regulatory Protein in Pseudomonas aeruginosa

The complete nucleotide sequence for the aot operon of Pseudomonas aeruginosa PAO1 was determined. This operon contains six open reading frames. The derived sequences for four of these, aotJ, aotQ, aotM, and aotP, show high similarity to those of components of the periplasmic binding protein-dependent ABC (ATP binding cassette) transporters of enteric bacteria. Transport studies with deletion derivatives established that these four genes function in arginine-inducible uptake of arginine and ornithine but not lysine. The aotO gene, which encodes a polypeptide with no significant similarity to any known proteins, is not essential for arginine and ornithine uptake. The sixth and terminal gene in the operon encodes ArgR, which has been recently shown to function in regulation of arginine metabolism. Studies with an aotJ::lacZ translational fusion showed that expression of the aot operon is strongly induced by arginine and that this effect is mediated by ArgR. S1 nuclease and primer extension experiments showed the presence of two promoters, P1 and P2. The downstream promoter, P2, is induced by arginine and appears to be subject to carbon catabolite repression. The upstream promoter, P1, is induced by glutamate. Footprinting experiments established the presence of a 44-bp ArgR binding site that overlaps the −35 region for P2, as was shown to be the case for the arginine-inducible aru promoter, and the −10 region for P1, as was shown to be the case for arginine-repressible operons in P. aeruginosa. Sequence alignment confirms the architecture and the consensus sequence of the ArgR binding sites, as was previously reported.     

Acetylhistidine as substrate for acetylornithinase: a new system for the selection of arginine regulation mutants in Escherichia coli

Summary The utilisation of acetylhistidine by histidine auxotrophs of E. coli K-12 was found to require a functioning acetylornithinase. The growth, on acetylhistidine-containing media, of his mutants possessing this enzyme was inhibited by arginine or its precursors acetylornithine, ornithine and citrulline. Mutants able to overcome this inhibition belonged to two classes: those (selected on acetylhistidine+acetylornithine or arginine) in which the arginine biosynthetic enzymes were repressible, as in the parent strains; and those (selected on acetylhistidine+acetylornithine, ornithine, citrulline or arginine) in which these enzymes were formed at high, non-repressible levels. The altered properties of the first class were shown genetically not to result from mutation in the argR or argECBH regions; the data are consistent with the second class carrying mutations at the argR locus.It is supposed that arginine, ornithine or citrulline, by repressing the formation of acetylornithinase, diminish the rate at which acetylhistidine can be utilised (although an acetylhistidine uptake system under arginine control would equally explain the results); non-repressible mutants would escape this effect. The kinetic properties, in crude extracts, of acetylornithinase from the parent strains and from members of each mutant class, with acetylornithine and acetylhistidine as substrates, were investigated. It was tentatively concluded that, in accord with the genetic results, the first class do not possess an acetylornithinase altered to make it function better with acetylhistidine as substrate. It is suggested that arginine may affect acetylhistidine utilisation by affecting its uptake in a manner not shared with ornithine or citrulline, as well as by repressing proteins of the arginine system, and that this arginine-specific effect is inoperative in the first class of mutants. The nature of the changes leading to ability to grow on acetylhistidine+acetylornithine remains unknown. Possible applications of these findings to the selection of hitherto undiscovered but potentially informative mutant types are discussed.     

Genetic Factors Affecting Recovery of Nonpoint Mutations in the Region of a Gene Coding for Ornithine Transcarbamylase: Involvement of Both the F Factor in Its Chromosomal State and the recA Gene

Mutants of E. coli K12 that overproduce ornithine transcarbamylase can be identified in Car^- strains because they permit utilization of citrulline as a carbamyl phosphate source, due to reversal of the normal OTCase reaction; they are called Cut mutants (citrulline utilizers). Hfr strains that carry the F factor adjacent to argF (one of two duplicate genes that code for ornithine transcarbamylase in E. coli K12) yield more Cut mutants than do F⁺ or F^- strains, or Hfr strains in which the F factor is not adjacent to argF. When Hfr strains in which the F factor is integrated adjacent to argF are made recA, they yield few Cut mutants. Many of the Cut mutants recovered from one of the Hfr strains used in the investigation (Hfr P4X) are unstable; the properties of these unstable mutations suggest that they carry aberrations in the region of the argF gene. Thus, the increased yields of Cut mutants probably result from aberrations that occur when the F factor is integrated adjacent to argF. The nature of these aberrations is not yet known. The unstable Cut mutants are to a large extent stabilized by recA; such stabilization is one of the properties of duplications. Other data indicate that the aberrations may be more complex than simple gene duplications; in particular properties of segregants and some recombinants derived from unstable Cut mutants are most easily interpreted by assuming that segregation from, and possibly formation of, the unstable mutants occurs in several stages.     

Competitive nodulation blocking of Afghanistan pea is determined by nodDABC and nodFE alleles in Rhizobium leguminosarum

Summary One well-defined competitive interaction amongst rhizobia is that between compatible and non-compatible strains of Rhizobium leguminosarum with respect to the nodulation of some primitive pea genotypes. The Middle Eastern pea cv Afghanistan is nodulated effectively can R. leguminosarum TOM, but its capacity to nodulate can be blocked if a mixed inoculation is made with R. leguminosarum PF₂. This PF₂ phenotype (Cnb) is encoded by its symbiotic plasmid and cosmid clones thereof. We found that Cnb is also encoded by the well-characterized Sym plasmid pRL1JI of R. leguminosarum strain 248. We have isolated and characterized a 6.9 kb HindIII fragment of pSymPF₂ which confers the Cnb⁺ phentoype on other (Cnb^–) rhizobia. A Tn5 site-directed Cnb^– mutant was constructed by homogenotization and was also found to be Nod^– on the European pea cv Rondo. DNA hybridization and complementation analysis indicated that the 6.9 kb Cnb⁺ fragment contained the nodD, nodABC and nodFE operons. Analysis of the Cnb phenotype of nod::Tn5 alleles of pRL1JI showed that mutations of nodC, nodD or nodE all abolished Cnb activity whereas mutants in nodI and nodJ reduced activity to 50% of the wild-type level.     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

Molecular cloning, characterization and purification of ornithine carbamoyltransferase from Mycobacterium bovis BCG.

Summary A genomic library of Mycobacterium bovis BCG has been constructed by cloning DNA partially digested with Sau3A into the Escherichia coli expression vector pAS1. The gene coding for ornithine carbamoyltransferase (EC.2.1.3.3 ; OTCase), hereafter referred to as argF, was isolated from the library by complementation of a double argF-argI mutant of E. coli and its sequence was determined. The translation initiation codon used, GTG, was identified by comparing the amino acid sequence deduced from the gene with the N-terminal sequence of the corresponding purified protein. On this basis, the M. bovis BCG OTCase monomer consists of 307 amino acid residues and displays about 44% identity with other OTCases, the most closely related homologue being the anabolic enzyme of Pseudomonas aeruginosa. The native enzyme has an estimated molecular mass of 110 kDa, suggesting a trimeric structure as is the case for most of the anabolic OTCases known from various organisms.