Pyrimidine base and ribonucleoside utilization by thePseudomonas alcaligenes group

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of thePseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth ofPseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supportedPseudomonas alcaligenes ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by eitherP. pseudoalcaligenes orP. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts ofP. pseudoalcaligenes andP. alcaligenes. InP. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.     

Pyrimidine ribonucleoside catabolic enzyme activities ofPseudomonas pickettii

Pyrimidine ribonucleoside catabolic enzyme activities of the opportunistic pathogenPseudomonas pickettii were examined. Of the pyrimidine and related compounds tested, only dihydrouracil (nitrogen source) and ribose (carbon source) supported growth. Thin-layer chromatographic separation of the uridine and cytidine catabolities produced byP. pickettii extracts indicated that this pseudomonad contained nucleoside hydrolase activity. Its presence was confirmed by enzyme assay. Hydrolase activity was elevated in both glucose- and ribose-grown cells relative to succinate-grown cells. Nucleoside hydrolase activity was depressed when dihydrouracil served as a nitrogen source. Cytosine deaminase activity was present in extracts prepared from succinate-, glucose- or ribose-grown cells when (NH₄)₂SO₄ served as the nitrogen source although cells grown on glucose or ribose exhibited a higher enzyme activity. Cytosine deaminase activity was not detected in extracts prepared from cells grown on dihydrouracil as a nitrogen source. Both dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were measurable inP. pickettii. The dehydrogenase activity was higher with NADH than with NADPH as its nicotinamide cofactor when uracil served as its substrate. Carbon source did not affect dehydrogenase or dihydropyrimidinase activity greatly but both activities were diminished in cells grown on the nitrogen source dihydrouracil.     

Pyrimidine base catabolism in Pseudomonas putida biotype B

Reductive catabolism of the pyrimidine bases uracil and thymine was found to occur in Pseudomonas putida biotype B. The pyrimidine reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl--alanine amidohydrolase activities were detected in this pseudomonad. The initial reductive pathway enzyme dihydropyrimidine dehydrogenase utilized NADH or NADPH as its nicotinamide cofactor. The source of nitrogen in the culture medium influenced the reductive pathway enzyme activities and, in particular, dihydropyrimidinase activity was highly affected by nitrogen source. The reductive pathway enzyme activities in succinate-grown P. putida biotype B cells were induced when uracil served as the nitrogen source.     

Degradation of pyrimidine ribonucleosides by Pseudomonas aeruginosa

Pyrimidine ribonucleoside degradation in the human pathogen Pseudomonas aeruginosa ATCC 15692 was investigated. Either uracil, cytosine, 5-methylcytosine, thymine, uridine or cytidine supported P. aeruginosa growth as a nitrogen source when glucose served as the carbon source. Using thin-layer chromatographic analysis, the enzymes nucleoside hydrolase and cytosine deaninase were shown to be active in ATCC 15692. Compared to (NH₄)₂SO₄-grown cells, nucleoside hydrolase activity in ATCC 15692 approximately doubled after growth on 5-methylcytosine as a nitrogen source while its cytosine deaminase activity increased several-fold after growth on the pyrimidine bases and ribonucleosides examined as nitrogen sources. Regulation at the level of protein synthesis by 5-methylcytosine was indicated for nucleoside hydrolase and cytosine deaminase in P. aeruginosa.     

Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa

Summary A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl--D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - Sm^r resistance to streptomycin - Km^r resistance to kanamycin - Ap^r resistance to ampicillin - Tc^r resistance to tetracycline - Cm^r resistance to chloramphenicol - CMCase carboxymethylcellulase     

The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens subsp. cellulosa

Summary The complete nucleotide sequence of the gene coding for one of the carboxymethycellulases (CMCase), expressed by Pseudomonas fluorescens subsp. cellulosa, has been determined. The structural gene consists of an open reading frame, commencing with an ATG start codon, of 2886 base pairs followed by a TAA stop codon. The gene was shown to code for a signal peptide which closely resembles the signal peptides of other secreted proteins. Unlike most pseudomonas genes, the CMCase sequence does not have a high G+C (51%) content and there is no marked preference for codons ending in G or C. Upstream of the structural gene there are no sequences which bear a strong resemblance to consensus Escherichia coli promoters. A sequence is present, however, which exhibits homology to the consensus DNA sequence that binds the catabolic activator protein (CAP). Bal31 deletions of the structural gene revealed the extent by which the gene could be modified and still encode a functional CMCase. Subclones of the cellulase gene have been constructed in pUC18 and pUC19. One of the resultant plasmids, pJHS1 directs a 20-fold increase in CMCase synthesis, when compared to the original construct, pJHH2. Analysis of cells harbouring pJHS1 showed the cellulase polypeptide to have a molecular weight of 106000. This is in close agreement with the predicted size of the enzyme deduced from the nucleotide sequence data.Abbreviations CMCase carboxymethylcellulase - PAGE polyacrylamide gel electrophoresis - IPTG isopropyl--D-thiogalactoside - CAT chloramphenicol acetyl transferase     

A relationship between RP4 plasmid acquisition and phenotypic changes in Pseudomonas fluorescens R2fN

The physiological behaviour of Pseudomonas fluorescens strain R2fN was compared to that of transconjugants [R2fN(RP4)], and two aggregation phenotypes were identified (Agr^– and Agr⁺). Agr⁺ phenotype is characterized by the appearance of macroscopic aggregates when cells are growing in liquid media. Transconjugants exhibited Agr⁺ phenotype whereas wild type strain represented Agr^–. Evidence is presented to support correlation between Agr⁺ phenotype acquisition and the presence of the broad-host range plasmid RP4 in strain R2fN. In addition, according to bacterial adherence to hydrocarbon test the transconjugant cells appeared to be very hydrophilic whereas wild type R2fN cells were hydrophobic.     

Pyrimidine base and ribonucleoside catabolic enzyme activities of the Pseudomonas diminuta group

Pyrimidine base and ribonucleoside catabolic enzyme activities of the two type strains of the Pseudomonas diminuta group were investigated for taxonomic classification purposes. The presence of the pyrimidine salvage enzyme nucleoside hydrolase was indicated in both type strains following thin-layer chromatographic analysis. The presence of the hydrolase was also confirmed by enzyme assay. In addition, the activities of the pyrimidine salvage enzymes dihydropyrimidine dehydrogenase and dihydropyrimidinase were measurable in cell-free extracts of both P. diminuta and P. vesicularis. An absence of cytosine deaminase activity was found when assaying extracts of the two type strains. Nucleoside hydrolase and dihydropyrimidine dehydrogenase levels in P. vesicularis were influenced by carbon source while dihydropyrimidinase activity was observed to increase after P. diminuta growth on dihydrothymine as a nitrogen source.     

Cloning and Sequencing of the Gene of Tryptophan-7-Halogenase from Pseudomonas fluorescens Strain CHA0

The gene of tryptophan-7-halogenase from the Pseudomonas fluorescens strain CHA0, a producer of the halogenated antibiotic pyrrolnitrin, has been cloned and sequenced.     

Effect of copper (II) on survival of Pseudomonas fluorescens and transfer of plasmid RP4 in soil

Survival of Pseudomonas fluorescens (R2f, RP4), P. fluorescens (R2f) and plasmid RP4 transfer efficiency depended on the concentration of Cu (II) and the time of incubation. The indigenous heterotrophic bacteria were more tiolerant to Cu (II) than the introduced strains. Although transconjugants were present for the first 3 days, the frequencies of conjugation and absence of transconjugants at 14 days indicate that conjugation occurred early in the incubation and that transconjugants were unable to survive for very long.The author is with the Department of Microbiology, Sllesian University, Jagiellonska 28, 40-032 Katowice, Poland     

Chlorobenzoate catabolism and interactions between Alcaligenes and Pseudomonas species from Bloody Run Creek

A mixed community of bacteria from surface runoff waters of the Hyde Park industrial landfill was enriched on 3-chlorobenzoate. Alcaligenes and Pseudomonas species were dominant in the community. Alcaligenes sp. BR60 carried an unstable plasmid specifying 3-chlorobenzoate catabolism. Metabolites detected in culture supernatants included chlorocatechol and chloro-cis,cis-muconic acid. Oxygen uptake in the presence of 3- and 4-substituted methyl-catechols revealed a catechol-1,2-oxygenase activity specific for substituted catechols with very limited activity for catechol. The isolate grew very slowly on benzoate. Alcaligenes sp. BR60 was isolated in co-culture with Pseudomonas fluorescens NR52. The latter contained no detectable plasmids and did not grow on benzoate or any of the chlorobenzoates in pure culture. Growth of the co-culture in Bloody Run Creek water supplemented with 3-chlorobenzoate indicated that phosphate concentrations in the water severely limited biodegradation. Under phosphate limited conditions in continuous culture, Pseudomonas fluorescens NR52 effectively scavenged available phosphate when it was present at a ratio of 1 cell to 20 of Alcaligenes sp. BR60. Under these conditions the growth of Alcaligenes sp. BR60 on 3-chlorobenzoate was reduced 5 fold, the frequency of plasmid deletion mutants increased, and 96% of the contaminant remained in the outflow in the form of the starting material or metabolites. No evidence was found for conjugation of the plasmid determining chlorobenzoate catabolism in Alcaligenes sp. BR60 to P. fluorescens NR52.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - Ba benzoic acid     

Pyrimidine catabolism in Pseudomonas aeruginosa

Pyrimidine catabolism in Pseudomonas aeruginosa was investigated. It was found that the pyrimidine bases uracil and thymidine as well as their respective reductive catabolic products could be utilized as sole sources of nitrogen. Reductive degradation of the pyrimidine bases was noted. The reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl-beta-alanine amidohydrolase were all detected in minimal medium grown cells. Induction of pyrimidine catabolism by uracil was observed in this pseudomonad. Pyrimidine degradation in P. aeruginosa was not subject to catabolite repression.     

A novel EDTA-degrading Pseudomonas sp.

A bacterial strain LPM-410 capable of utilizing ethylenediaminetetraacetate (EDTA) as the sole source of energy, carbon, and nitrogen was isolated from sewage sludge and identified as a Pseudomonas sp. on the basis of its phenotypic characteristics. Suspensions of exponential-phase cells degraded EDTA, Mg–, Ca–, Ba–, and Mn–EDTA at constant specific rates ranging from 0.363 to 0.525 mmol EDTA/(g cells h). The more stable chelate, Zn–EDTA, was degraded at a lower rate (0.195 ± 0.030 mmol EDTA/(g cells h)), and here was no degradation of Co–, Cu–, Pb–, and Fe(III)–EDTA.     

Pathways of d-fructose catabolism in species of Pseudomonas

Cell-free extracts of d-fructose grown cells of Pseudomonas putida, P. fluorescens, P. aeruginosa, P. stutzeri, P. mendocina, P. acidovorans and P. maltophila catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose and contained 1-P-fructokinase activity suggesting that in these species fructuse-1-P and fructose-1,6-P₂ were intermediates of d-fructose catabolism. Neither the 1-P-fructokinase nor the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose was present in significant amounts in succinate-grown cells indicating that both activities were inducible. Cell-free extracts also contained activities of fructose-1,6-P₂ aldolase, fructose-1,6-P₂ phosphatase, and P-hexose isomerase which could convert fructose-1,6-P₂ to intermediates of either the Embden-Meyerhof pathway or Entner-Doudoroff pathway. Radiolabeling experiments with 1-¹⁴C-d-fructose suggested that in P. putida, P. aeruginosa, P. stutzeri, and P. acidovorans most of the alanine was made via the Entner-Doudoroff pathway with a minor portion being made via the Embden-meyerhof pathway. An edd ^- mutant of P. putida which lacked a functional Entner-Doudoroff pathway but was able to grow on d-fructose appeared to make alanine solely via the Embden-Meyerhof pathway.Non-Standard Abbreviations cpm counts per min - edd ^- mutant lacking Entner-Doudoroff dehydrase (6-PGA dehydrase) - EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P₂ - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTs PEP: d-fructose phosphotransferase system - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate     

Pyrimidine ribonucleoside monophosphokinase and the mode of RNA turnover in Bacillus subtilis

A protein catalyzing the phosphorylation of CMP to CDP was purified and characterized. Kinase activity for UMP copurified during ammonium sulfate fractionation, DEAE-cellulose and hydroxylapatite chromatography, and gel filtration on Sephadex G-75, the ratios of activities for the two substrates remaining constant. The purified product, possessing both activities was homogeneous as judged by the single band following polyacrylamide gel electrophoresis. The protein showed no kinase activity against purine nucleoside monophosphates or the other pyrimidine nucleoside monophosphates: dCMP, dUMP, and dTMP. Thus unlike the enteric bacteria, Escherichia coli and Salmonella typhimurium which have distinct enzymes which phosphorylate UMP and CMP, Bacillus subtilis produces a single pyrimidine ribonucleoside monophosphokinase. The K _mvalues of this enzyme from B. subtilis are 0.04 and 0.25 mM for CMP and UMP, respectively, and 0.04 and 0.4 mM for ATP at saturating concentrations of CMP and UMP, respectively. The properties of this enzyme and the differences between enteric bacteria and B. subtilis with respect to the enzymes which phosphorylate CMP are consistent with the measurements which indicate that turnover of messenger RNA is largely hydrolytic in E. coli but largely phosphorolytic in B. subtilis.Non-Standard Abbreviations PRMK Pyridine ribonucleoside monophosphokinase This paper is affectionately dedicated to Professor R. Y. Stanier     

l-Lysine-2-monooxygenase production by strains of Pseudomonas fluorescens and P. putida

Twelve strains of Pseudomonas fluorescens and P. putida were grown in a synthetic medium that contained l-lysine as the only source of carbon and nitrogen, and screened for l-lysine-2-monooxygenase production. Best production was by P. putida BKM B-1458 at 30 IU/g wet wt biomass when grown in a shake-flask but 25 IU/g in a 250-l fermenter.     

Fluorene Cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens Cultures

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.     

Chemical modification of intracellular cytosine deaminase fromChromobacterium violaceum YK 391

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme.     

Pyrimidine biosynthesis in Pseudomonas stutzeri ATCC 17588

The de novo pyrimidine biosynthetic enzymes in the denitrifying bacterium Pseudomonas stutzeri ATCC 17588 were assayed and their activities were lower in glucose-grown cells than in succinate-grown cells. When P. stutzeri was grown in the presence of uracil, the de novo enzyme activities in succinate-grown cells were lowered while they remained largely unchanged in glucose-grown cells. A uracil auxotroph of P. stutzeri, deficient for aspartate transcarbamoylase activity, was isolated and its auxotrophic requirement was met by only uracil and cytosine. The inability of pyrimidine ribonucleosides to meet the auxotrophic requirement was related to the limited ability of P. stutzeri to transport uridine and cytidine. Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be repressible by a uracil-related compound in succinate-grown P. stutzeri cells. Regulation of pyrimidine synthesis in P. stutzeri was similar to that observed for other pseudomonads classified within rRNA homology group I.