Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes

The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440. A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine. Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement. When the wildtype strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed. Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil. It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines. In vitro regulation of aspartate transcarbamoylase activity in P. pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by P_i, PP_i, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP.     

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha ATCC 9890 was investigated and the pyrimidine biosynthetic pathway enzyme activities were affected by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. In pyrimidine-grown ATCC 9890 cells, the activities of four de novo enzymes could be depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the succinate-grown mutant strain cells, pyrimidine limitation of this strain caused dihydroorotase activity to increase about 3-fold while dihydroorotate dehydrogenase and orotidine 5'-monophosphate decarboxylase activities rose about 2-fold. Regulation of de novo pathway enzyme synthesis by pyrimidines appeared to be occurring. At the level of enzyme activity, aspartate transcarbamoylase activity in P. synxantha ATCC 9890 was strongly inhibited in vitro by pyrophosphate, UTP, ADP, ATP, CTP and GTP under saturating substrate concentrations.     

Regulation of pyrimidine synthesis in Pseudomonas resinovorans

AIMS: To investigate the regulation of de novo pyrimidine biosynthesis in the bacterium Pseudomonas resinovorans ATCC 14235. METHODS AND RESULTS: The pyrimidine biosynthetic pathway enzymes were measured in cell extracts from P. resinovorans ATCC 14235 and from an auxotroph lacking orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic pathway enzyme activities in ATCC 14235 were affected by the addition of pyrimidine bases to the culture medium. The de novo enzyme activities of the phosphoribosyltransferase mutant strain increased after pyrimidine starvation indicating possible repression of the pathway by a pyrimidine-related compound. Aspartate transcarbamoylase activity in ATCC 14235 was inhibited in vitro by ATP, UTP and pyrophosphate. CONCLUSIONS: Pyrimidine biosynthesis in P. resinovorans was regulated at the level of enzyme synthesis and at the level of activity for aspartate transcarbamoylase. Its regulation of enzyme synthesis seemed to be similar to what has been observed in the taxonomically related species Pseudomonas oleovorans. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found that pyrimidine biosynthesis is regulated in P. resinovorans. This could prove helpful to future studies investigating polyhydroxyalkanoate production by the bacterium.     

Regulation of the Pyrimidine Biosynthetic Pathway in <Emphasis Type="Italic">Pseudomonas mucidolens</Emphasis>

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.     

Regulation of pyrimidine nucleotide formation in Pseudomonas reptilivora

AIMS: To study the regulation of de novo pyrimidine biosynthesis in the pathogenic bacterium Pseudomonas reptilivora ATCC 14836. METHODS AND RESULTS: The pyrimidine biosynthetic pathway enzymes were assayed in extracts of Ps. reptilivora ATCC 14836 cells and of cells from an auxotroph lacking aspartate transcarbamoylase activity. Pyrimidine biosynthetic pathway enzyme activities in ATCC 14836 were influenced by the addition of pyrimidine bases to the culture medium with orotic acid addition inducing dihydroorotase activity. Pyrimidine starvation of the transcarbamoylase mutant strain increased its de novo enzyme activities suggesting that the de novo pathway was also subject to repression by a pyrimidine-related compound. Aspartate transcarbamoylase activity in ATCC 14836 was inhibited in vitro by pyrophosphate and ATP. CONCLUSIONS: Regulation of pyrimidine biosynthesis in Ps. reptilivora was observed at the level of enzyme synthesis and at the level of activity for aspartate transcarbamoylase. Its regulation of enzyme synthesis seemed to be more highly controlled than what was observed in the related species Ps. fluorescens. SIGNIFICANCE AND IMPACT OF THE STUDY: This investigation found that pyrimidine biosynthesis is controlled in Ps. reptilivora. This could prove helpful to future studies exploring its pathogenicity.     

Degradation of diphenylether by Pseudomonas cepacia

The microbial degradation of hard coal implies the cleavage of diaryl ether linkages in the coal macromolecule. We investigated the biodegradation of diphenylether as a model compound representing this substructure of coal. A bacterial strain isolated from soil and identified as Pseudomonas cepacia, was able to grow with diphenylether as sole source of carbon. During microbial growth, three metabolites were detected in the culture supernatant by high pressure liquid chromatography. As product of ring hydroxylation and subsequent rearomatization, 2,3-dihydroxydiphenylether was identified by UV, mass and nuclear magnetic resonance spectrometry and gas chromatography analyses. The cleavage of the ether linkage led to the formation of phenol and 2-pyrone-6-carboxylic acid, the latter being not further degraded by Pseudomonas cepacia. The possible cleavage mechanism of the ether linkage is discussed.Non-standard abbreviations DPE diphenylether - PCA 2-pyrone-6-carboxylic acid - GC gas chromatography - MS mass spectrometry - HPLC high pressure liquid chromatography     

Branched chain amino acid aminotransferase isoenzymes of Pseudomonas cepacia

Pseudomonas cepacia grew rapidly using a mixture of all three branched chain amino acids as carbon source, but failed to use individual branched chain amino acids as sole carbon source. Extracts of bacteria grown on branched chain amino acids had between 2- and 3-fold higher levels of -ketoglutarate-dependent branched chain amino acid aminotransferase activity than extracts of glucose-grown bacteria. The increase in enzyme activity was due to the presence of a second aminotransferase not detected in extracts of glucose-grown bacteria. The enzyme, which presumably plays a role in branched chain amino acid degradation, had an apparent molecular weight (mol. wt.) of 75,000. The other aminotransferase was formed constitutively and apparently functions in synthesis of branched chain amino acids. It was more stable than the 75,000 mol.wt. enzyme, and was purified to homogeneity and found to be a 180,000 mol.wt. oligomer containing 6 subunits of approximately 30,000 mol.wt. Antiserum prepared against the purified enzyme inhibited its activity but failed to influence the activity of the 75,000 mol.wt. aminotransferase, suggesting that the two isoenzymes are encoded by different genes.     

Molecular cloning and expression of perhydrolase genes from Pseudomonas aeruginosa and Burkholderia cepacia in Escherichia coli

Two bacterial perhydrolase genes, perPA and perBC, were cloned from Pseudomonas aeruginosa and Burkholderia cepacia, respectively, using PCR amplification with primers designed to be specific for conserved amino acid sequences of the already-known perhydrolases. The amino acid sequence of PerPA was identical to a putative perhydrolase of P. aeruginosa PAO1 genome sequences, whereas PerBC of B. cepacia was a novel bacterial perhydrolase showing similarity of less than 80% with all other existing perhydrolases. Most importantly, the perPA gene was expressed as a soluble intracellular form to an extent of more than 50% of the total protein content in Escherichia coli. Two perhydrolase enzymes were confirmed to exhibit the halogenation activity towards Phenol Red and monochlorodimedone. These results suggested that we successfully obtained the newly identified members of the bacterial perhydrolase family, expanding the pool of available perhydrolases.     

Copper biosorption by Pseudomonas cepacia and other strains

Biosorption of copper by Pseudomonas cepacia was found to be dependent on added copper concentration. Copper uptake by the cells was rapid over the range of copper concentrations tested and complete within the first 10 min of incubation time. The effect of pH on copper uptake by P. cepacia was determined using overlapping buffers over the pH range 3–8, and copper biosorption from a 10 mM copper solution was greatest at pH 7. Copper uptake (measured by analysis of cell digests) was unaffected by cyanide and azide (up to 30 mM) and by incubation of cells with a 10 mM copper solution at 4 °C. Evidence from these results suggested that copper uptake by P. cepacia cells involves surface binding and not intracellular accumulation by active transport. Biosorption of copper by various Pseudomonas isolates from metal-contaminated environments agreed well with copper biosorption by Pseudomonas strains from the National Collection of Type Cultures (NCTC).     

Replicon fusions promoted by insertion sequences on Pseudomonas cepacia plasmid pTGL6

Summary Plasmid pMR5 (pRP1ts) failed to replicate in Pseudomonas cepacia at 47° C. Selection at this temperature for maintenance of tetracycline resistance associated with this plasmid allowed isolation of cointegrate plasmids formed by fusion of pMR5 with pTGL6, a 170 kb plasmid harbored by P. cepacia 249. In the cointegrate plasmids pTGL100, pTGL101, and pTGL102, different regions of pTGL6 were involved in fusion with the same tra-2-containing region of pMR5. Formation of all three plasmids was promoted by insertion sequences on pTGL6, which were also represented in the chromosome.Two different copies of a 1.3 kb element, IS401, were involved in formation of pTGL100 and pTGL101. Another insertion sequence, IS402 (1 kb), promoted the fusion which formed pTGL102. Southern hybridization experiments indicated that each of the cointegrate plasmids contained an additional copy of the fusion mediating element. Plasmid pTGL100 was observed to resolve into two independent replicons: pTGL6 and pTGL105 (pMR5::IS401), a novel derivative of pMR5 containing a copy of IS401.The third cointegrate plasmid, pTGL102, evolved in two steps: fusion of pTGL6 and pMR5 mediated by IS402, and transposition of IS411 (1.9 kb) to a region of pMR5 distinct from that involved in the fusion. Plasmid pTGL6 contained one copy of IS402 and IS411 while pTGL102 contained two copies of each of these elements.     

Influence of environmental factors on 2,4-dichlorophenoxyacetic acid degradation by Pseudomonas cepacia isolated from peat

A Pseudomonas cepacia, designated strain BRI6001, was isolated from peat by enrichment culture using 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. BRI6001 grew at up to 13 mM 2,4-D, and degraded 1 mM 2,4-D at an average starting population density as low as 1.5 cells/ml. Degradation was optimal at acidic pH, but could also be inhibited at low pH, associated with chloride release from the substrate, and the limited buffering capacity of the growth medium. The only metabolite detected during growth on 2,4-D was 2,4-dichlorophenol (2,4-DCP), and degradation of the aromatic nucleus was by intradiol cleavage. Growth lag times prior to the on-set of degradation, and the total time required for degradation, were linearly related to the starting population density and the initial 2,4-D concentration. BRI6001, grown on 2,4-D, oxidized a variety of structurally similar chlorinated aromatic compounds accompanied by stoichiometric chloride release.     

Growth and survival of Pseudomonas cepacia DBO1(pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10⁷ CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days. In general, the strain survived best in sterile soil. When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil. In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days.The influence of 0,0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P. cepacia DBO1(pRO101) inoculated to a level of 6×10⁴, 6×10⁶ or 1×10⁸ CFU/g soil was studied in non-sterile soil. Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P. cepacia DBO1(pRO101). At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels. P. cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature. During this starvation, cells shrunk to about one third the volume of exponentially growing cells.Abbreviations AODC acridine orange direct count - CFU colony forming units - PTYG-Agar peptone, tryptone, yeast & glucose agar - TET tetracycline - LB Luria Bertani medium     

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The K _m-value of 1 M for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with ¹⁸O₂ and H₂ ¹⁸O indicated the incorporation of ¹⁸O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).Non-standard abbreviations DPE diphenylether - 2,3-dihydroxy-DPE 2,3-dihydroxydiphenylether - PCA 2-pyrone-6-carboxylic acid - 2,3-dihydroxy-BP dioxygenase 2,3-dihydroxybiphenyl dioxygenase - GC gas chromatography     

Modeling allosteric regulation of de novo pyrimidine biosynthesis in Escherichia coli

With the emergence of multifaceted bioinformatics-derived data, it is becoming possible to merge biochemical and physiological information to develop a new level of understanding of the metabolic complexity of the cell. The biosynthetic pathway of de novo pyrimidine nucleotide metabolism is an essential capability of all free-living cells, and it occupies a pivotal position relative to metabolic processes that are involved in the macromolecular synthesis of DNA, RNA and proteins, as well as energy production and cell division. This regulatory network in all enteric bacteria involves genetic, allosteric, and physiological control systems that need to be integrated into a coordinated set of metabolic checks and balances. Allosterically regulated pathways constitute an exciting and challenging biosynthetic system to be approached from a mathematical perspective. However, to date, a mathematical model quantifying the contribution of allostery in controlling the dynamics of metabolic pathways has not been proposed. In this study, a direct, rigorous mathematical model of the de novo biosynthesis of pyrimidine nucleotides is presented. We corroborate the simulations with experimental data available in the literature and validate it with derepression experiments done in our laboratory. The model is able to faithfully represent the dynamic changes in the intracellular nucleotide pools that occur during metabolic transitions of the de novo pyrimidine biosynthetic pathway and represents a step forward in understanding the role of allosteric regulation in metabolic control.     

Purification and characterization of dihydroorotase fromPseudomonas putida

Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK _m andV _max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min⁻¹ mg⁻¹, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min⁻¹ mg⁻¹, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn²⁺. However, excessive Zn²⁺ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK _i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK _i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.     

Coordinately elevated pyrimidine biosynthetic enzymes in fluorouracil resistant mutants ofNocardia lactamdurans

Summary Nocardia lactamdurans, which produces the antibiotics efrotomycin and cephamycin C, exhibits essentially unregulated pyrimidine synthesis. Neither the first nor the second step is subject to feedback regulation. In a series of strains, comprising the original soil isolate, a set of spontaneous fluorouracil-resistant (FUR) mutants and the first improved efrotomycin producer, three of the pyrimidine biosynthetic enzymes were found to be coordinately regulated over a tenfold range. This amplification of enzyme activity accounts for the pyrimidine-related properties but not, at present, for their improved efrotomycin capacity.     

防御假单胞菌双突变体H78ΔrsmA/E中hmgA基因对藤黄绿菌素生物合成的抑制

【背景】防御假单胞菌(Pseudomonas protegens) H78是分离于油菜根际的一株生防菌,其能合成藤黄绿菌素(pyoluteorin,Plt)等多种广谱抗生素,H78的rsmA/E双突变体中Plt合成被完全抑制。【目的】通过转座子诱变技术,筛选H78ΔrsmA/E双突变体中重新激活Plt合成的下游调控因子。【方法】通过同源重组的方法在pltL基因下游插入红色荧光蛋白(redfluorescentprotein,RFP)基因来指示Plt操纵子表达的激活情况;利用转座子随机插入突变、半随机PCR技术筛选并定位目标基因;通过基因回补等方法进一步验证基因功能。【结果】从约2万株H78ΔrsmA/E的转座子突变体中筛选到一株高产Plt和某种黑色素的菌株,并确定其插入位点为hmgA基因,hmgA基因回补能重新抑制H78ΔrsmA/E的Plt合成。【结论】假单胞菌双突变体H78ΔrsmA/E中hmgA基因对Plt的合成存在强烈抑制作用,是潜在的RsmA/E下游调控基因。本研究为进一步阐明Plt合成的调控机制与网络及通过基因工程提高Plt产量奠定了基础。